Biology 1 Final

Transcription

(genetics) the organic process whereby the DNA sequence in a gene is copied into mRNA

Centimorgans (cM)

(same as a map unit) a unit of map distance obtained from genetic crosses. Named in honor of Thomas Hunt Morgan

Given 1000 offspring, 180 are recombinant What does this tell us about the chromosome map?

180/1000 = 0.18, or 18% recombinant frequency. From this information, we know that there are 18 cM between the two loci of the genes

How many ATP per glucose in aerobic respiration? How about anaerobic?

30 vs 2

How many ATP are made in oxidative phosphorylation vs fermentation?

32 ATP in oxidative phosphorylation of one glucose molecule vs 2 ATP in fermentation of one glucose molecule

How does 32P accomplish end and body labels?

32P end label DNA using polynucleotide kinase and gamma-labeled 32P-ATP. Body label with DNA polymerase and alpha-labeled dNTPs.

3 categories of modulating enzyme activity

1. Reversible inhibition (many of these are created synthetically in the lab,and are used as drugs) (Competitive inhibition is a subcategory of this) 2. Allosteric control (happens in cells mostly) 3. Phosphorylation (happens in cells mostly)

Telophase

5. Telophase · Nuclear membranes reappear around the two sets of chromosomes. Mitosis is complete Components of the new cells begin to appear. At this point, the spindle fibers are broken up. A new nuclear membrane surrounds the chromosomes at the end of each cell, and the chromosomes uncoil and return to an uncondensed state. Mitosis is now complete. The formation of two cells is all that remains.

At what point of recombination frequency does it resemble independent assortment?

50%

Arrangement of cilia and flagella

9+2 9 pairs on the outside, 2 individual tubules on the inside (2 on the inside add extra strength)

Lagging strand

A discontinuously synthesized DNA strand that elongates by means of Okazaki fragments, each synthesized in a 5' to 3' direction away from the replication fork

DNA ligase (exam def)

A linking enzyme essential for DNA replication; catalyzes the covalent bonding of the 3' end of a new DNA fragment to the 5' end of a growing chain.

Microtubules

A microtubule is a hollow tube formed from tubulin dimers (a dimer is one a-tubulin and one b-tubulin) Support (transportation), protection, some movement.

Nuclear lamina

A netlike array of protein filaments lining the inner surface of the nuclear envelope; it helps maintain the shape of the nucleus. Involved in deconstruction and reconstruction of the nuclear envelope that accompanies cell division.

Cytoskeleton

A network of protein fibers that holds the cell together, helps the cell to keep its shape, and aids in movement.

Endoplasmic reticulum (general)

A network that weaves throughout the cell, composed of phospholipid bilayers embedded with proteins

What is a regulon?

A regulon is a collection of operons that are all under similar regulation

Pinocytosis

A type of endocytosis in which the cell ingests extracellular fluid and its dissolved solutes (cell drinking) Grabbing some amount of extracellular fluid, and pinching it into its own vesicle. Just grabbing whatever's there (in the area). Common for recycling of neurotransmitters (some type of molecule released by a neuron for communication). Cell will want to recuperate and recollect some of these neurotransmitters, so it will grab some amount of extracellular fluid with some neurotransmitters and bring back into cell to be reused by cell

Operon

A unit of genetic function common in bacteria and phages, consisting of coordinately regulated clusters of genes with related functions (found in prokaryotes)

How can you see if a DNA molecule is linear or circular?

A way to tell if a DNA molecule is linear or circular is to see if it is susceptible to exonucleases

Hammerling Experiment

Acetabularia Experiment (1943) Main point: nucleus contains the genetic information in a cell Summary: In his experiments, Hammerling grafted the stalk of one species of Acetabularia onto the foot of another species. In all cases, the cap that eventually developed on the grafted cell matched the species of the foot rather than that of the stalk. This experiment shows that the base is responsible for the type of cap that grows. The nucleus that contains genetic information is in the base, so the nucleus directs cellular development.

Actin in the extracellular matrix?

Actin connects the inside of cells to the extracellular matrix. Ex: collagen connects liver cells, but what connects collagen to the cells? Actin

How does actin work in microvilli?

Actin prevents movement in microvilli, in order to ensure maximum surface area for absorption of food.

What is a CAP protein?

Activator protein catabolite activator protein

What modifications happen at the end of the mRNA?

Addition of a 'cap' at the 5' end, Addition of a polyA-tail at the 3' end

Smooth Endoplasmic Reticulum (SER)

An endomembrane system where lipids (steroids, etc) are synthesized, calcium levels are regulated, and toxic substances are broken down. No ribosomes, tubular structure Ex: muscle contraction (calcium ions)

What would we see in terms of ratios if Meselson & Stahl's experiment was allowed to continue for generations?

As generations go on, there will be an infinitely high ratio between fully light DNA and small, remote remnant of intermediate density DNA that still has a strand of the original heavy parental DNA

Hemidesmosome

Anchors intermediate filaments in a cell to the basal lamina. Connect one cell to a basement membrane instead of another cell directly. Basement membrane = variety of different proteins. Lets cell slide around a little bit (as opposed to desmosomes) Ex: skin. Not stuck to one spot! This is because epithelial cells are attached at basement membrane, and this attachment allows a little extra movement (versus desmosomes, for example, which don't allow for a lot of movement. Think of hair)

What information do bioinformatics databases contain?

Annotations and correlations with phenotypic info

Endosomes

Another name for vesicles, especially in terms of endocytosis. Basically, what happens to a particle before it is lysed.

Autosomes

Any chromosome that is not a sex chromosome

On epithelial cells in the gut, what's the apical surface? What's the basal surface?

Apical surface (closer to intestinal lumen), basal surface (closer to blood/extracellular fluid)

Transport carriers

Cannot work without concentration gradient (usually created by ATP-driven pump) Two items, transported at the same time

Uniport

Carries one molecule at a time Passive (only going one direction, and no ATP contribution)

What type of reactions are typically involved in catabolic pathways?

Catabolic pathways that break down food molecules to harvest energy from them typically involve redox reactions

Why do cells divide?

Cell division is required for an organism to grow, mature, and maintain tissues

Describe all steps in this image. What does this tell us?

Cellular respiration is a hub of catabolism and anabolism

How does stuff actually get across a membrane? (general)

Charge and size are determining factors. Size is the biggest deal! But you have to hit a balance of small and uncharged

What is a chi-square test and what does it tell you?

Checks whether a distribution is significantly different than an expected or theoretical distribution. The Chi-square test is intended to test how likely it is that an observed distribution is due to chance. It is also called a "goodness of fit" statistic, because it measures how well the observed distribution of data fits with the distribution that is expected if the variables are independent.

Exergonic

Chemical reactions that release energy (negative delta G)

What happens to chromatin during cell division?

Chromatin is further compacted during cell division

Chromatin

Clusters of DNA, RNA, and proteins in the nucleus of a cell

Chromosomes

Compact units within the nucleus that contain DNA wound tightly around proteins

Why should we care about plant photosynthesis?

Comparing/contrasting photosynthesis with cellular respiration helps understand cellular respiration

Why is chromatin necessary? How is it formed?

DNA packing allows these long ass chromosomes to fit inside the cell Positively charged histone proteins neutralize the negative charge on DNA (from the phosphate group) so that it can be compressed into a really tiny space

What do we mean when we say DNAP has the ability to 'proofread'?

DNA polymerase has an ability to proofread: if the last nucleotide or two that it added to the chain turn out to have been wrong and they're not staying very well base paired to the template strand, DNA polymerase will chop out that last one or two nucleotides that it added, and fix itself

What does the electron transport chain in oxidative phosphorylation do? What is produced?

Electron transport chain carries out redox reactions to convert energy stored as "reducing power" of NADH and FADH2 to energy stored in an electrochemical gradient of protons Know number of FADH2 and NADH produced in previous stages (from single glucose molecule), and how much each unit of each produces. For each NADH that goes through ETC, ~2.5 ATP For each FADH2 that goes through ETC, ~1.5 ATP ATP Synthase ~28 ATPs produced via chemiosmosis Compared to/in addition to 2 ATP in glycolysis (via substrate level phosphorylation), 1 ATP made in TCA (per cycle, 2 runs per glucose molecule) so 2 total Total of 32 ATP per oxidative phosphorylation per one glucose molecule (vs 2 ATP per glucose in fermentation)

What is peptidyl transferase?

Enzymatic component of the ribosome Forms peptide bonds between amino acids

What are restriction enzymes? Why do we care?

Enzymes that cut DNA molecules Allowed cloning to be done

Flagellum (eukaryotic)

Eukaryotic flagella exhibit a beating (wave-like) movement

Can you describe the redox reaction that takes place as glucose is oxidized to CO2?

As glucose is oxidized to CO2, O2 is reduced to H2O. As with all redox reactions, what's being transferred is electrons (something is being oxidized, something is being reduced). But in cells, electrons are disguised as hydrogen atoms (Electrons don't move as just electrons alone, as with metal redox reactions!). They are most often moved two at a time (taken off of a molecule, and put onto a receiving molecule). And they are not put directly onto O2. Instead, they are put onto certain carriers (these carriers tend to be particular co-enzymes)

Light-driven pump

Bacterial usually. Used in a lot of research (ex: optogenetics)

How does DNA gel electrophoresis work? What's the underlying thought, and what are the underlying steps?

Basic thought: You can separate DNA molecules by size. You can do this because DNA is negatively charged, so when you apply the electric field, they move. The smallest molecules move the fastest, because they are less massive 1. Load samples 2. Apply electric field 3. Since DNA is negatively charged, the DNA moves away 4. Smallest fragments move the farthest (move faster) 5. Use ethidium bromide to visualize where the DNA fragments are in the gel

What's the fundamental question of Hardy-Weinberg? How does this relate to evolution?

Basically: what does a population look like if no evolution happening? If no change going on, if nobody is going or going, if everyone is more or less equal in how well they mate and survive, what does the population look like? These are all the things we look at when we study evolution

Why is it called the 'lagging' strand?

Because it's growth lags behind the growth of new DNA, so there are moments when the single stranded DNA is 'naked'/exposed

Why are substrates able to float in and out of the active site? Why is the interaction between an enzyme/substrate reversible?

Because non-covalent bonds, the substrate can float in and out of the active site (ionic bonds are present, but these have already dissociated in solution) Interaction of enzyme with substrate is reversible since it involves non-covalent interactions

Compare allosteric control and phosphorylation

Both allosteric control and phosphorylation act like "molecule switches" by changing the overall shape of the enzyme. Enzymes that are regulated in this way tend to have two possible native structures that they can be convinced to convert between. One of these is generally more active than the other, so allosteric control or phosphorylation basically regulate the switching back and forth between these shapes

What's been produced by the end of glycolysis and the citric acid cycle? Where is energy stored? By what process is ATP made in these steps?

By the end of glycolysis pathway and citric acid cycle, glucose has been completely converted to CO2, but only a small amount of energy has been harvested as ATP. Most of the energy released is stored as the "reducing power" of NADH and FADH2 ATP that's made in glycolysis and citric acid cycle is made by "substrate-level phosphorylation"

What is at the end of every tRNA molecule? Why?

CCA (ACC moving from very end of 3' inward) is at the end of every tRNA molecule. The aminoacyl-tRNA synthetase actually needs to recognize that CCA sequence at the 3' end in order to attach the amino acid to it. It's going to recognize that and other features of the entire tRNA, including the anti-codon, so that it makes sure that it connects the right amino acid to that 3' end

Cadherins proteins

Cadherins proteins are connected by adherens junction. Cadherins span the membrane (transmembrane proteins) that link to actin on the inside and to each other on the outside. So actin in each cell is connected by these cadherin proteins, with a nice big space in between so things can still float around between them

Why is factor-independent transcription termination called 'factor-independent'?

Called 'factor-independent' because it only involves the RNAP recognizing signals in the DNA itself that it's transcribing, and in the RNA that it is making. There are no additional protein factors required for this type of termination

Liposugars

Ex: galactocerebroside. Not a phospholipid. Has a lipid end and a galactose end (galactose has the same structure as glucose with just an OH flipped at 4' C). Protective layer around the membrane. (GLYCOCALYX)

Multisubunit enzyme complex Describe what this is and give an example

Ex: pyruvate dehydrogenase This particular enzyme is regulated both allosterically and by phosphorylation. Large, multi-subunit enzyme. Different colors in image represent different subunits that are assembled together into a huge complex. (Multiple copies of each kind - each dot = one subunit.) The interactions between these are all regulated and the whole complex can shift regulation. Basically, this is a subunit composed of multiple kinds of protein (enzyme). The interactions between these are all regulated, and the whole complex can shift regulation

What is the turnover rate of a typical eukaryotic cell?

For many eukaryotic cells, a cell is duplicated every 24 hours

Lysozyme Describe the substrate/active site interaction?

Found in tears, saliva, mucus, human milk. Protects us from bacterial infection by breaking down the polysaccharide part of bacterial cell walls. Focus on the idea that this enzyme causes distortion of the substrate, and uses charges to encourage rearrangement of electrons within the substrate molecule. This magic that happens in the enzyme active site takes some time to happen.

Oxidative phosphorylation (overview + location/function details)

Fourth step in cellular respiration, aka the aerobic catabolism of glucose. Electron transport chain: uses reducing power of NADH & FADH2 to reduce O2 to H2O. Energy released by this is used by ATP synthase to make ATP Basically: the use of the reducing power in NADH and FADH2 to drive ATP synthesis Location: mitochondrial innner membrane (eukaryotic cells), plasma membrane (prokaryotic cells). Two parts to this process.

Dizygotic

Fraternal twins - sex may be the same or different

Describe the structure of epithelium cells

Free surface (faces lumen) called the apical side, with another face that faces the inside of your body (basal lamina/connective tissue) called the basal side. Tend to be held tightly together by cell junctions and rest upon a basal lamina (basement membrane). Without cell junctions, skin would not be able to protect properly. Without these junctions, your skin could not protect you from elements

G-actin

Globular actin. This is when actin is folded up into shape, before it assembles into a higher order fiber

Enzyme affinity

How tightly an enzyme is bound to a substrate. affinity is affected by both shape and charge. complimentary shapes and opposite charges provide the highest affinity between and enzyme and ligand. The higher the affinity of an enzyme for its substrate (due to many non covalent interactions), the greater the proportion of enzyme that will be occupied at equilibrium. The more non-covalent interactions that there are in an active site, the stronger/tighter the bonding will be, and the greater the proportion of the enzyme-substrate complex relative to free E and S floating around at equilibrium.

When could we say that a population is in HW equilibrium?

If we are assuming that we are looking at a population that isn't changing, we would say that that population is in HW equilibrium

What's happening in the interphase nucleus? What does it look like?

In cells that are not dividing ('interphase'), you can't see a lot of the chromatin because it's in the form of euchromatin, which is less-tightly compacted than some of the other chromatin that is more tightly compacted and clustered near the periphery of the nucleus (this is called heterochromatin) Only during the actual cell division process are actual individual chromosomes visible, because at that point, each chromosome has been compacted so much that it's actually visible as a separate entity from other chromosomes

Describe the process of chromatin synthesis

In eukaryotes, DNA is divided into multiple linear chromosomes. Chromosomes are then organized with protein into chromatin. This allows regulatory proteins to attach to specific nucleotide sequences along the DNA and regulate gene expression.

How many types of RNAP are at work in prokaryotes? Eukaryotes?

In prokaryotes, all RNA is synthesized by the same type of RNAP. Synthesizes all classes of RNA, able to recognize promoters for protein-coding genes, promoters for tRNA coding genes, and promoters for ribosomal RNA genes · BUT... eukaryotes are more complicated, and they have have 3 types of RNAP, and each one makes a different category of RNA

Structure of lysosome

Inside: Nucleases, proteases, glycosidases, lipases, phosphatases, sulfatases, phospholipases Proton pump Metabolite transporter

Actin vs. intermediate filament (in terms of cell structure)

Intermediate filaments criss-cross inside of cell (such that cell can't be smashed easily). Actin sits on the outside, enabling the cell to be shaped in a certain way. Intermediate filament provides the central support. Actin provides shape of the membrane itself.

Cells that are not currently in the process of dividing are in...

Interphase

Why is it called a "Southern blot"?

Invented by EM Southern

Summarize the stages of transcription, and whether or not they're the same in proks/euks

Initiation: A bit different in euks! A polymerase recognizes a promoter, and starts making an RNA strand Elongation: Same in proks and euks! Elongating that RNA strand, and moving along the template DNA, and moving the transcription bubble with it Termination: Different in proks and euks! Recognizes a terminator codon

Describe the inputs, outputs and location of Krebs Cycle

Input: 2 acetyl CoA (Nad+, FAD, ADP, P, oxaloacetate) Output: 2 ATP, 6 NADH, 2 FADH2, CO2 Takes place in the mitochondrial matrix

Where do tight junctions and adherens junctions (adherens belts) generally occur?

Just below an apical surface (a surface that faces a void or lumen)

Why is kinase relevant to Sanger?

Let's say we want to sequence a large restriction fragment that we isolated from a clone that we made. The fragment has 5' and 3' ends. So what we're going to do is use a kinase enzyme to put a radioactive label at the 5' ends of this DNA. Then, I'm going to take some of this DNA, and cut it with a restriction enzyme (a different one than I had used to create the fragment). Once it's been cut - so we have 2 strands - I'm going to denature the strands, and purify the labelled strand (the upper strand, in this example, that has the radioactive label on it). So now we have 4 fragments.

Induced fit

The change in shape of the active site of an enzyme so that it binds more snugly to the substrate, induced by entry of the substrate

Monocistronic

The coding pattern of eukaryotes in which one mRNA molecule codes for only one protein

Polycistronic

The coding pattern of prokaryotes, in which one mRNA may code for multiple proteins

How do amino acids attach to aa-tRNA?

The enzyme uses energy from ATP to create this high energy bond... in fact, it uses the equivalent of two ATPs, because it splits that ATP down to AMP, and releases 2 inorganic phosphates (using quite a bit of energy to make this high energy bond!) The amino acid is attached to the tRNA by its carboxyl group, or the C terminus. This is important!

What is the 1000 genomes project?

The goal of the 1000 Genomes Project was to find most genetic variants with frequencies of at least 1% in the populations studied. The 1000 Genomes Project took advantage of developments in sequencing technology, which sharply reduced the cost of sequencing. It was the first project to sequence the genomes of a large number of people, to provide a comprehensive resource on human genetic variation.

Perinuclear space

The space between the two layers of the nuclear envelope

OVERVIEW: how is DNA replicated in living cells? (voiceover)

Think of it as an assembly line of miniature biochemical machines that are pulling apart the DNA double helix and cranking out a copy of each strand. The DNA to be copied enters the production line from bottom left. The whirling blue molecular machine is called a helicase. It spins the DNA as fast as a jet engine as it unwinds the double helix into two strands. One strand is copied continuously, and can be seen spooling off to the right. Things are not so simple for the other strand, because it must be copied backwards. It is drawn out repeatedly in loops, and copied one section at a time. The end result is two new DNA molecules Sometimes, that imagery of how the lagging strand is looped back and grows a new loop each time an Okazaki fragment is being made and then the polymerase jumps back and a new loop is started, sometimes that's referred to as the 'trombone model' of DNA synthesis (because it's like the slide of a trombone going in and out)

What is three point mapping? Why do we care?

Three point mapping allows us to figure out the actual order of genes on a chromosome

Topsoisomerase

Topoisomerases interconvert supercoiled and relaxed forms of circular DNA

What conditions must be met to study a DCO?

To study double exchanges, three pairs of genes must be investigated, each heterozygous for alleles

What must be true about the trait that is changed for the DCO? Why can't we just look at the SCO?

Trait that is changed for the double crossover must be in the middle If we were only to look at the SCO, we couldn't tell the order. So whatever trait you see that is the smallest number, that has to be the one that's in the middle

How do biological catalysts work?

Lots of reactions that need to happen in cells are actually unfavorable reactions, and enzymes (catalysts) help these along. Enzymes couple exergonic reactions with endergonic processes to power them

RNAP I

Makes most rRNA. Recognizes promoters for ribosomal RNA genes (should make sense that promoters for these different classes of genes have slightly different DNA sequences in order to be recognized by these different RNA polymerases)

What's up with the two ends of microtubules?

Microtubules have a plus (beta) and minus (alpha) end. The plus end grows faster, and is further from the nucleus.

Compare the sizes of actin microfilaments, microtubules, and intermediate filaments

Microtubules: 25 nm Intermediate filaments: 10nm Actin microfilaments: 7 nm

How does RNAP know where to start and stop transcribing?

Nucleotide sequences within the DNA itself, flanking the coding information for a protein, tell RNAP where to start and stop transcribing Every gene has a transcription start site called a 'promoter' and a transcription stop site called a 'terminator'

Relate mRNA, tRNA, and rRNA in a sentence

ONLY mRNA GOES ON TO BE TRANSLATED; rRNA and tRNA help with the translation of mRNAs

ORF

OPEN READING FRAME Region from AUG through the stop codon (inclusive on both ends) An ORF is a region of a mRNA that encodes a polypeptide. It starts with the "start codon" AUG. (For this reason, the first amino acid in a polypeptide is always methionine when the polypeptide is first synthesized, although the methionine may be eventually be removed through post-translational processing, which we will talk about next time.) The start codon defines the "frame" or register in which the subsequent bases will be read. The ribosome will move along the mRNA reading the amino acid-encoding codons until it reaches one of 3 possible "stop codons" (UAA, UAG, or UGA), which signify that the ribosome should stop adding amino acids.

What happens once the small subunit has located the start codon? Is this the same or diff in proks/euks?

Once the small subunit has located the start codon (whether it be in prokaryotes or eukaryotes), the large subunit joins and the next aa-tRNA can enter to allow formation of the 1st peptide bond The ensuing events are the same! In prokaryotes and eukaryotes, after the small subunit has located the start codon

Which equation is genotype frequency in HW? Which is allele frequency?

P + q = 1 This is just the allele frequency P^2 + 2pq + q^2 = 1 This is the genotype frequency

T/F: Natural populations contain substantial genetic variation

True! Natural populations contain substantial genetic variation

T/F: there is always fluid outside of the cell

True! The cell is submerged at all times

Do all snRNPs recognize all parts of pre-mRNA?

Multiple snRNPs have different jobs of recognizing different parts of a pre-mRNA. Some are specialized for recognizing the sequences at the junctions between introns and flanking exons, others are designed to recognize a particular sequence within the intron sequence... once they've all recognized the thing they're supposed to recognize, they all get together and assemble together to form the spliceosome, which then clips out the intron and splices the flanking exons together covalently with a phosphodiester bond connecting them, so it's continuous RNA

Relative proportions of NCO, SCO, DCO?

NCO phenotype · Greatest proportion SCO phenotype · Two possibilities DCO phenotype · Smallest proportion, because there is the least probability of these two independent events both occurring

Do catalysts impact energy of products or reactions?

NO. Catalysts do not change the relative energies of the reactants and products. ONLY changes how fast the molecules (the reaction) reach/reaches equilibrium (the rate of reaction)

Does DNA polymerase need a promoter?

NO. needs a primer though

What's the name of the enzyme that creates peptide bonds? Where does this reaction take place?

Peptidyl transferase is the name of the enzyme activity in the ribosome that carries out this reaction. The activity is actually contained in the RNA part of the ribosome!

What is percentage of recombinant gametes dependent upon?

Percentage of recombinant gametes is dependent on the distance between the genes

What's happening on the ETC of chloroplasts?

Water comes in at the beginning, and comes out as oxygen. At the end, NADP coming in, and going out as NADPH. Along the way, some protons being pumped, and light coming in as energy.

How does water get through the cell membrane?

Water has its own channel to get through cell membranes, called Aquaporin (Nobel Prize, P. Agre, 2004)

What is PCR and what is it used for? Describe the steps.

Polymerase Chain Reaction; used to amplify DNA 1. Denaturation (high heat) 2. Annealing (cooler, 50-60 C) 3. Extension (72 C)

Polymerase

Polymerases catalyze the template-directed synthesis of new DNA (or RNA) strands

What sort of process is polypeptide chain elongation? Why?

Polypeptide chain elongation is a cyclic process 1)Peptidyl-tRNA is in P site 2)aa-tRNA enters A site (anticodon must match codon) 3)Peptidyl transferase activity cleaves existing peptide from its tRNA; the energy released allows creation of a peptide bond. Peptidyl-tRNA is now in A-site, and P-site contains an "uncharged" tRNA 4)Ribosome translocates 3 nucleotides along the mRNA. This puts the peptidyl-RNA in the P-site, the uncharged tRNA in the E-site (where it will eventually float away from the ribosome), and the next codon in the A-site, waiting for the next aa-tRNA. To extend a polypeptide chain, the ribosome must select the correct amino acids that are specified by the mRNA. An amino-acyl tRNA bound to an elongation factor-Tu (EF-Tu for short) enters the free A site on the ribosome. If the anti-codon of the charged tRNA does not match the codon in the messenger RNA, the tRNA is rejected. The process of trial and error repeats, until the correct tRNA is identified. Elongation factor TU hydrolyzes its bound GTP, and dissociates. If the tRNA is correctly matched, and remains bound for a long enough time, it is committed to be used in protein synthesis. The ribosome catalyzes the formation of a new peptide bond, and undergoes a dramatic conformational change. Elongation factor G binds the ribosome. Hydrolysis of GTP by elongation factor G switches the ribosome back to the state in which it can accept the next incoming tRNA

Cross-linking proteins

Possess 2 or more actin binding sites and join microfilaments into three-dimensional networks. Present in cell cortex

Mitochondria

Powerhouse of the cell, organelle that is the site of ATP (energy) production Carries out cellular/aerobic respiration (metabolic processes) to generate energy for the cell

What is pre-mRNA?

Pre-mRNA includes not only coding information, but also the RNA sequence that was specified by the template strand in these introns. So that non-coding information needs to get removed, in a process called splicing

What is the primary use of the HW equilibrium equation?

Primary use of the equation is to determine whether evolutionary processes are operating in a population and hypothesize what they are

How are substrates for DNAP and RNAP similar/different?

We discussed that the substrates for DNA polymerase are dntps, and that as each new nucleotide comes in, it's selected by the ability of its nitrogenous base to base pair with the next exposed base on the template strand, but then the actual bond formation occurs by clipping off the outer two phosphates, which basically provides the energy required for the actually unfavorable process of creating a phosphodiester bond The story is exactly the same for RNA polymerase, except for now we're talking about ribonucleotides. They have ribose as their sugar, instead of deoxyribose. Will always have U (not T), which will be selected anytime there's an A in the template strand of DNA

How do we get the genotype frequency from the phenotype frequency in HW?

We get the genotype frequency from the phenotype frequency by first looking at the recessive percentage in a population

Sum law

Probability of obtaining any single outcome, where that outcome can be achieved in two or more events, is equal to the sum of all such events

Histones (structure, function)

Protein molecules around which DNA is tightly coiled in chromatin Rich in basic (positively charged) R groups like lysine & arginine. Basic, so at neutral pH, tend to have positive charges, and are therefore attracted to the DNA backbone, and end up wrapping the DNA around the surface of a complex of histone proteins

In what direction are proteins synthesized?

Proteins are synthesized from their N terminus to their C terminus. So it's the N terminus sticking out of these ribosomes, and the growing C terminus is within the ribosome, having amino acids added to it. It's the 5' end of the messenger RNA that's sticking out, because RNA is synthesized 5' to 3' so it's the 3' end that's in the active site with RNA polymerase that's being extended by adding additional nucleotides to it

In what direction are proteins synthesized?

Proteins are synthesized starting at their N terminus, working towards their C terminus. So the N terminal amino acid is always encoded by AUG and is therefore methionine

Do exergonic reactions necessarily go 100% to products?

No! The product/reactant ratio is dependent upon the equilibrium constant

Do enzymes always change shape when a substrate enters their active sites?

No, sometimes they stay the same

Walk me through the structure of prokaryotic RNAP

RNAP is composed of multiple subunits, and one of the subunits that it has is called sigma. [see: diagram - the red and pink is DNA, and the green/blue is RNAP holoenzyme] the holoenzyme binds to the promoter, in the beginning, and does that because this sigma subunit [blue] has amino acid residues that recognize the landscape of chemical groups that are exposed in the DNA within the promoter region

What's an alternative to ethidium bromide? What are the pros and cons? How does this work?

Radioactive labeling (32P) (increasingly used instead of ethidium bromide) Pros - More sensitive (than ethidium bromide, for example, or other things than what you can see by eye/visible light) - Can detect double or single strands. Ethidium bromide isn't good at this because single strands aren't base paired, so it's harder for it to bind - End or body label Cons This is still pretty commonly used, but because of the dangers of radioactivity, people try to get away from this, to some extent. Still, this is necessary for some types of experiments

How is transcription initiation regulated in prokaryotes?

Rate of mRNA production is largely controlled by the ability of RNAP to bind to a gene's promoter and initiate transcription Not all promoters have exactly the same nucleotide sequence Some have higher affinity for RNAP holoenzyme than others. ("Strong" vs. "weak" promoters) These differences cause different "basal" levels of expression, determined by the strength of the promoter itself

How does this idea of "strong" and "weak" promoters relate to basal levels of expression and simultaneous transcription?

Recall - multiple RNAP can be transcribing a gene at the same time... once one of them clears the promoter area, another can hop on. Genes that have very strong promoters (ones whose sequences are identical to that consensus sequence) are so good at attracting RNAP that they most likely will have RNAP molecules following, one right after another, closely in succession (not a long period of time after one clears the promoter til another binds to the strong promoters). Genes with weaker promoters may rarely, if ever, have a single RNAP in the process of transcribing them. TL;DR: there can be a range of levels of expression that we refer to as the "basal" level of expression

What defines chromosomes?

Recall that chromosomes always need to have centromeres, so we always have to start at the side that holds the centromere. That will define the chromosome (this is just referencing the sister chromatid)

RNAP II

Recognizes promoters for protein coding genes, makes pre-mRNA/primary RNAs (and some minor RNA classes)

Nuclease

Refers to an enzyme that hydrolyzes phosphodiester bonds in nucleic acids

What do we mean when we say downstream and upstream in the context of RNAP?

Regions that the RNA polymerase is moving towards, but hasn't gotten to yet, are downstream Regions that are behind the RNA polymerase are upstream

Can you describe central dogma in terms of synthesis at each step?

Replication: synthesis by DNA polymerase Transcription: synthesis by RNA polymerase Translation: synthesis by a ribosome

Cholesterol

Required molecule for lipid bilayers. Stabilizes the membrane, also amphipathic. Fits in next to unsaturated bilipids, adds additional cohesion, stabilizes the membrane

How is amino acid sequence encoded in mRNA?

Ribosomes read the bases in a mRNA in 3-letter words called "codons" and translate them using the Genetic Code Notice that the code is "degenerate" - in many cases, more than one codon specifies the same amino acid

Basic (very basic) breakdown of the phases of the cell cycle

S: DNA replication and growth G2: Growth and preparation for cell division M phase: Mitosis - splitting chromosomes, Cytokinesis - splitting cell [G0]: Non-dividing cells G1: Rapid growth and metabolic activity

Is PCR, in its original form, a qualitative or quantitative process?

Should realize that PCR itself, in its original form, is kind of a qualitative procedure. It lets you amplify particular regions of DNA and get large amounts of them

Prokaryote

Single celled organisms that evolved before eukaryotes No interior compartments (no organelles, no nucleus) DNA stored in nuclear region

Differentiate between SCO and DCO. What are these good for, respectively?

Single crossovers (SCO) can be used to determine the distance between two linked genes, but double crossovers (DCOs) can be used to determine the order of three genes on the chromosome Double crossover is when you have two crossovers that occur

Describe the Na+/K+ ATPase pump

Single most important transmembrane protein in the body (according to Dr. G), because this moves Na+ from the inside of the cell out, and K+ from the outside of the cell in. This pump creates the Na+/K+ gradient mentioned previously. This pump is constantly going. Theorized that 50% of all cellular energy, in all cells, is keeping this pump going. Uneven exchange (2 K+ pumped out for 3 Na+ pumped in). Binding sites for Na+, K+, and ATP: energy from ATP hydrolysis drives movement of 3 Na+ out, and 2 K+ in, both against their concentration gradients. Covalent attachment of phosphate to the protein (and subsequent removal) cause conformational changes that drive the process. phosphorylation!!!

Origin of replication

Site where the replication of a DNA molecule begins, consisting of a specific sequence of nucleotides.

DNA helicase

Sitting right at the fork, because it's using energy from DNA to help pry apart the parental DNA strands to make the bubble grow

How do small molecules get through the cell membrane?

Small molecules are small enough to diffuse past the hydrophilic heads of the lipid bilayer

Vesicle

Small sacs that store and transport a variety of materials

Compare ribosomes and snRNPs

SnRNPs ("snurps") are small, nuclear, riboucleoprotein complexes that have the enzymatic activity to do the splicing Like a ribosome, they are complexes of RNA and protein, and like the ribosome, the enzymatic activity that they have for doing the splicing is in the RNA part

Describe how Sanger sequencing works (short version)

Some sort of primer DNA is base paired to a template DNA that we want to sequence. DNAP is adding nucleotides to this primer, and every so often the DNAP adds a ddntp, instead of a dntp, and the strand is terminated

Can you voice over this bacterial transformation graphic?

Stage 3: Plasmids are inserted into bacterial cells by transformation; bacterial cells reproduce and form clones. To stage 4: Clones are screened for gene of interest.

Telomeres: structure and function

Structure: DNA found at the ends of eukaryotic chromosomes. Noncoding. Consists of multiple repeats of a short genetic sequence (in humans, up to 2500 repeats of TTAGGG!) Functions as a protective cap: Since noncoding, no damage to genome as long as shortening doesn't extend beyond telomere into coding region

Why do we use the terms primary, secondary, tertiary to refer to only some kinds of RNAs and not others?

We use the terms primary, secondary, and tertiary structure with regard to tRNAs and ribosomal RNAs because unlike messenger RNAs, which are just read like tapes and we just need to consider their order of nucleotides, tRNAs and ribosomal RNAs fold up into specific structures in order to carry out their functions, so we can describe their structure in a similar fashion to how we describe protein structure

Describe the components of a ribosome?

Ribosome is large complex of proteins and RNAs, but it's actually assembled in two smaller parts called the large subunit and the small subunit Large: has close to 50 different ribosomal proteins (each of which is encoded by a gene in the nucleus) and 3rRNA molecules Small: 33 ribosomal proteins and 1 rRNA molecule. Proteins here are different from the ones that are in the large subunit The large and small subunits come together during the process of translation to make the full ribosome

How are the Southern blot, Northern blot, DNA hybridization, restriction enzymes - all related to recombinant DNA?

Taken together, the above techniques: the ability to cut DNA with restriction enzymes, the ability of DNA ligase to join DNA segments together, the ability to put DNA into bacteria and transform them to a different phenotype based on the DNA molecule that you put into them (see: Griffith, transforming principle!)... all of these things together is what led to the ability to clone DNA (to create recombinant DNA)

Endocytosis (what is it, how does it work, types)

Taking it from outside the cytoplasm and moving it in Types (3): Phagocytosis, Pinocytosis, Receptor-mediated endocytosis

Why is it important that telomeres don't code?

Since no coding, no damage to genome as long as shortening doesn't extend beyond telomere into coding region

What does it mean for a reaction to be energetically favorable, aka spontaneous? What can we say about energetic favorability in (most) reactions in living cells? Why does this matter?

Note: spontaneous doesn't mean fast! Spontaneous reactions still have to overcome the required activation energy barrier to happen. In living cells, the activation energy barrier is so high for most reactions that they will not proceed at any appreciable rate, even though they are spontaneous (have a negative delta G). This is good, because it gives the cell the opportunity to control which reactions will occur, and under which circumstances they will occur.

Finish this sentence: Since the two strands of a DNA molecule are complementary... Who made this observation?

Since the two strands of a DNA molecule are complementary, each can serve as a "template" for the construction of the other! This was one of the big things that Watson and Crick noticed immediately

BRIEFLY summarize the central dogma

The central dogma of molecular biology states that DNA contains instructions for making a protein, which are copied by RNA. RNA then uses the instructions to make a protein ("DNA makes RNA makes protein") Replication —> transcription —> translation

What is aa-tRNA synthetase? Why are they sometimes called 'charged'?

aa-tRNA synthetases attach amino acids to their corresponding tRNAs A tRNA with an amino acid covalently attached to it is called an aminoacyl-tRNA (aka aa-tRNA). In general they are sometimes referred to as charged tRNA ("charged" in the sense of "carrying" something - not electric charge in this case!)

Transcriptome

all the RNA molecules transcribed from a genome

What is a holoenzyme in prokaryotic transcription? What does it do?

core enzyme + sigma factor In prokaryotes, RNAP "holoenzyme" is responsible for recognizing the promoter in "naked" DNA. The holoenzyme recognizes the promoter sequence in double-stranded DNA, and unwinds the DNA to form the transcription bubble so that RNA synthesis can begin. Once the sigma factor has done this job of assisting with transcription initiation, it floats away and leaves just the core polymerase to make the RNA chain. (In euks, there is no sigma factor. Protein factors separate from the RNAP will recognize the promoter first and subsequently recruit RNAP II.)

How do restriction enzymes work?

cut DNA at specific recognition sites with sticky ends that are overhanging sequences at the cut site

"knock-out" mice

gene inactivated in entire animal

"knock-in" mice

gene replaced with a deliberate alteration of gene sequence to see what happens

GTP

guanosine triphosphate. responsible for microtubule growth/shrinking

What is a snRNP?

nsnRNPs ("snurps") are small, nuclear, ribonucleoprotein complexes that have the enzymatic activity to do the splicing

Where does transcription begin?

nucleus, if eukaryotic Cytoplasm, if prokaryotic

Are centimorgans/map units exact?

o Map units are relative distances, not exact ones

What does the P site do? What does the P stand for?

peptide holds the tRNA that carries the growing polypeptide chain

5 stages of mitosis?

prophase, prometaphase, metaphase, anaphase, telophase

Explain how DNA sequencing technology has improved cancer treatment

recording, sorry, my brain is exploding

What is a sigma factor?

a protein that associates with RNA polymerase that facilitates its binding to specific promoters PROKARYOTES

Rough endoplasmic reticulum (RER)

series of connected flattened sacs, part of a continuous membrane organelle within the cytoplasm of eukaryotic cells, that is involved in the production, folding, quality control, and dispatch of some proteins. Rough because studded with ribosomes.

Describe what's going on in this figure

the DNAP, as it's synthesizing through here, in addition to its polymerizing activity, has exonucleases activity that nibbles away - starting at the 5' end of this probe - and just starts releasing individual nucleotides from this probe. So on the right of the slide we see the products after the DNAP has forced its way through here, and chopped up the probe, in order to finish copying the strand. Notice - the cleavage products all floating around. So the quencher is no longer tethered to the fluorescent tag, and so that fluorescent tag is now detected. So as you go, you put in more and more probe molecules

What is peptidyl tRNA?

the short polypeptide that is already attached to the tRNA in the P site of the ribosome in the first step of translation

Metabolome

the total number of metabolites present within an organism, cell, or tissue

What is signal transduction?

the transmission of molecular signals from a cell's exterior to its interior

What is gene therapy?

the transplantation of normal genes into cells in place of missing or defective ones in order to correct genetic disorders example: recombinant mice that have received growth hormone gene

Translation

(genetics) the process whereby genetic information coded in messenger RNA directs the formation of a specific protein at a ribosome in the cytoplasm

How do you determine the full genome sequence with next-gen seqeuencing?

- Random fragmentation of the genome generates overlapping sequences that need to be assembled together to get the entire genome sequence - Complicated computer algorithms are used to do this. "Reads" in both directions (both strands) of any given region, as well as statistical criteria related to the number of reads in any given region are used to gain confidence in the deduced sequence's accuracy. (One of the biggest contributions to the genomics revolution was the development of such software as well as database software for organizing and analyzing all the information, and the availability of computers powerful enough to do all of this!)

How are linkers attached to the flow chip with next-gen sequencing?

- The linkers get covalently attached to the "flow chip" where the sequencing reactions will actually be done. Primers complementary to the linkers are used to amplify all of the fragments right on the chip prior to the sequencing reactions

Basic functions of cell membranes (5)

1. Boundary and permeability barrier --> protection and homeostasis 2. Organization and localization of function --> order from chaos 3. Transport processes --> homeostasis and nutrient acquisition 4. Signal detection --> response to stimuli 5. Cell-to-cell communication --> direct communication

What are the 3 ways that enzymes can be regulated?

1. Control how much of each enzyme is present 2. Modulate enzyme activity 3. Compartmentalize

Preliminary questions to ask about a pedigree problem?

1. Dominant or recessive: check if generations skipped 2. Sex linked: check if equal numbers males/females 3. Dominant: every affected individual must have an affected parent

2 types of diffusion?

1. Facilitated diffusion/passive transport 2. Active transport

How is translation terminated? (steps)

1. Release factor recognizes stop codon 2. Release factor causes peptidyl transferase enzyme to catalyze hydrolysis of bond connecting polypeptide chain to tRNA 3. Polypeptide chain, mRNA, ribosome, and termination factor all go their separate ways What is the purple amino acid? Methionine!

Components of cytoskeleton and their components (5)

1. Structure (intermediate filaments) 2. Support (microtubules) 3. Transport (microtubules) 4. Protection (microtubules) 5. Movement (actin filaments)

Discuss the interaction of sucrase (enzyme) and sucrose (substrate).

1. The substrate, sucrose, consists of glucose and fructose bonded together. 2. The substrate binds to the enzyme non-covalently, forming an enzyme-substrate complex. 3. The binding of the substrate and enzyme places stress on the glucose-fructose bond, and the bond breaks. 4. Since products are bound non-covalently, they are released and float away, and the enzyme is free to bind to other substrates Recall: catalyst is not changed in process of reaction

Griffith's experiment

1928 transformation principle Summary: Griffith's experiment was the first experiment to suggest that bacteria are capable of transferring genetic information through a process known as transformation. Griffith took two strains of bacteria - smooth (which killed mice) and rough (which didn't kill mice) - and heated them up. When the smooth strain was heated/boiled, the bacteria died and didn't kill any mice. But when the smooth bacteria were boiled and then added to the live rough strain, the mouse did die. This experiment proved that protein was not the molecule of inheritance, and that there must have been some other "transformation factor" - which we later discovered was DNA

How many MAJOR types of RNA are there? are these present in eukaryotes or prokaryotes?

3 (mRNA, tRNA, rRNA) Note there are other minor classes of RNA that are important in the gene expression process - e.g. snRNA, and SRP RNA. This slide is just showing the three major types that are involved in the actual production of a protein by a ribosome. These three major classes are present in prokaryotes and in eukaryotes.

Stop codons

3 stop codons: UAA, UAG, UGA · Do not code for any amino acid, but signal for the ribosome that this is the end of the info for this protein and you should stop synthesizing the polypeptide chain

What are the components of the mitotic spindle?

3 types of spindle fibers plus the poles constitute what we call the mitotic spindle

How might active sites help the reaction reach the transition state? (3 ways)

A few ways! (Enzymes don't have to use just one of these - can often use multiple) 1. Enzyme binds to two substrate molecules and orients them precisely to encourage a reaction to occur between them 2. Binding of substrate to enzyme rearranges electrons in the substrate, creating partial negative and positive charges that favor a reaction 3. Enzyme strains the bound substrate molecule, forcing it toward a transition state to favor a reaction

Each fork has what?

A leading strand and a lagging strand

Ligand

A ligand is a molecule that binds another specific molecule, in some cases, delivering a signal in the process. Ligands interact with proteins in target cells, which are cells that are affected by chemical signals; these proteins are also called receptors.

DNA ligase

A linking enzyme essential for DNA replication; catalyzes the covalent bonding of the 3' end of a new DNA fragment to the 5' end of a growing chain. Joins adjacent okazaki fragments using energy from ATP

Channel protein

A membrane protein, specifically a transport protein, that has a hydrophilic channel that certain molecules or atomic ions use as a tunnel. Less specific than carrier protein. Creates a channel, and anything that can fit through will go through Most often seen with small ions (K+, Na+, Ca2+) but there are also channel proteins for larger things like glucose, etc

Carrier protein

A membrane protein, specifically a transport protein, that holds onto molecules and changes their shapes in a way that shuttles them across the membrane. Grabs solute and moves it in/out of the cell. Very specific to a given solute. Which solute is moved is dependent upon the structure of the carrier protein itself Can be one directional, or go both ways. Depends on structure Can transfer one or multiple at once. Can transfer one or class of proteins. Pretty much anything the cell wants, the cell will have multiple of these transport proteins embedded in the membrane

What is a polylinker region?

A multiple cloning site (MCS, or Polylinker region) is a DNA region within a Plasmid that contains multiple unique Restriction enzyme cut sites. Plasmids are very useful in biotechnology and one key feature of their use is the multiple cloning site, which allows for foreign DNA to be inserted into the plasmid

What's a repressor protein? What happens when it's at work?

A repressor protein typically binds to a sequence called the "operator," usually within or overlapping the promoter When repressor is bound at operator, RNAP is physically blocked from binding to the promoter

How are ribosomes different from most enzymes? Can you think of any other kinds of snRNP's?

A ribonucleoprotein complex is made of RNA and protein (unlike most enzymes, which are proteins). In addition to ribosomes, other examples of ribonucleoproteins (RNP's) are "snurps" (snRNP's), which are involved in RNA splicing, and SRP, which is involved in targeting proteins for secretion.

Describe the structure of a ribosome. Are they the same in prokaryotes and eukaryotes?

A ribosome is composed of a "large" subunit and a "small" subunit - each of which is a ribonucleoprotein complex composed of many specific protein subunits (called ribosomal proteins) and a small number of specific RNA molecules (called rRNAs). The ribosomes in eukaryotes and prokaryotes are very similar to each other, but differ slightly in size (eukaryotic are bigger).

What's the purpose of a Southern blot? (voiceover)

A way to use gel electrophoresis to separate out pieces of DNA by size, and then to probe that two dimensional pattern of pieces of DNA to find out whether there are any pieces of DNA that have sequences complimentary to a particular probe molecule... and if so, which ones?

Start codon

AUG (methionine)

Of these four categories, which types are able to get into the cell unaided? Which require assistance from transmembrane proteins? Small hydrophobic molecules Small uncharged polar molecules Larger uncharged polar molecules Ions

Able to get in unaided: Small hydrophobic molecules (O2, CO2, N2, benzene), small uncharged polar molecules (H2O, glycerol, ethanol - all through diffusion) Require assistance from transmembrane proteins: Larger uncharged polar molecules (amino acids, glucose, nucleotides) Ions (H+, Na+, HCO3-, K+)

Finish this sentence: Adjacent expanding replication bubbles on eukaryotic chromsomes...

Adjacent expanding replication bubbles on eukaryotic chromosomes eventually fuse

Why are allosteric enzymes particularly amenable to regulation?

Allosteric enzymes are particularly amenable to regulation because allosteric enzymes have effector sites and they have multiple subunits, that's what gives them the sigmoidal rate curve. The sigmoidal curve means that they have this range of substrate concentration over which very small substrate concentrations can cause really big changes in reaction rate ("get a lot of bang for your buck")

How do amino acids attach to tRNA? Why do we care?

Amino acid is attached to tRNA by a covalent "high-energy" bond (made using energy from ATP hydrolysis). Hydrolysis of this bond connecting the amino acid to the tRNA is very favorable and will provide the energy needed to make peptide bond formation favorable.

What is an anticodon?

An "anticodon" is a sequence of 3 nucleotides that is complementary to a codon for a particular amino acid

What's an activator protein? What happens when it's at work?

An activator protein binds to an activator binding sequence upstream and adjacent to a relatively weak promoter, and increases the ability of RNAP bind to the promoter Because the promoter is relatively weak, the gene is expressed at a very low level unless the activator is bound

Recombination frequency (what is it, equation, used for, units, etc)

An estimate of the relative distance between two genes along the chromosome Can be used to "map" the genes on a chromosome o = # recombinants/total Unit: cM (map unit) Mathematically, this is just number of recombinants divided by the total ^

Anaphase (detailed)

Anaphase Sister chromatids split at the centromere and start to move toward their respective poles Poles move apart from each other. This motion happens because of enzymes that clip the proteins that are holding the sister chromatids to each other. The microtubules can now pull them towards the respective poles Two aspects of anaphase (two types of movement - both occur simultaneously): Anaphase A, Anaphase B

Anaphase

Anaphase · Chromosomes separate · Occurs through a shortening of the microtubules attached to the kinetochores. Also, the poles of the cell move farther apart, causing increased separation of the sister chromatids. At the end of anaphase, sister chromatids have moved to two ends of the cell

Differentiate between Anaphase A and Anaphase B

Anaphase A Individual chromosomes are now being pulled towards the poles. Kinetochore microtubules are depolymerizing at the end with the kinetochore, and yet maintaining interactions that allow it to be attached to the kinetochore and pulling it towards the pole as it shortens Anaphase B There's a pushing force due to polar microtubules lengthening and sliding relative to each other, and pulling forces that are pulling the two poles farther apart, towards the cell membrane

What is Antibodies to detect specific proteins? Why do we care?

Antibodies are proteins that are made by cells in your immune system, in order to recognize foreign materials and help your immune system destroy those foreign materials You can deliberately generate antibodies to proteins that are interesting to you by purifying the protein and injecting it into a mouse or rabbit. The mouse or rabbit will mount an immune response, and you can isolate the antibodies from their blood. You can then use those antibodies to detect the presence of a protein that you'd like to study

Where are epithelium cells found?

Any tissues that line a void in the body - skin, intestines, etc

Centromere (structure, function)

Area where the chromatids of a chromosome are attached. Sequence in every chromosome Has a very repetitive DNA base pair sequence to it (unlike the types of sequences that code for proteins, because it serves a very different function) Does not code for proteins! Instead, it's the place on the condensed sister-chromatid pairs where a group of proteins called kinetochore proteins assemble to form kinetochore complexes. This region where the kinetochore complexes form is called the centromere

Why would leaky cell membranes cause you to lose weight?

Artificially increases metabolism because your body is trying to make more ATP (burning more fats, breaking down glycogen, etc), lots of glycolysis, TCA, ETC redox reactions, and it's like a bicycle with the wheels spinning - pumping protons, but protons leaking through. Metabolism goes up in an attempt to get ATP Instead of energy being captured as ATP, energy is released as heat. So people get fevers and don't have enough ATP to provide energy for their cells

What is end product inhibition?

As a metabolic pathway occurs, the concentration of the end product gradually builds up. The cell has evolved such that an enzyme early on in the pathway is regulated by the end product, typically by allosteric inhibition. Because that's being done by the end product of a pathway feeding back to an early step in the pathway, we often refer to that as either end-product inhibition or feedback inhibition, but these are carried out by this end product acting as an allosteric effector on that enzyme (this can also be called a negative feedback loop)

Can primase synthesize primers anywher?

As long as a single-stranded DNA template is available, yes! Unlike the RNA polymerase that makes RNA copies of genes for subsequent translation, primase does not need to recognize a specific nucleotide sequence telling it where to start copying. Primase is also designed to only synthesize a short stretch of RNA before falling off the template strand, whereas the RNA polymerase for gene expression will keep going until it recognizes a signal telling it where to terminate transcription.

At a high level, why are glycocalyx important for human health?

Basically, glycocalyx are important for human health because it regulates interactions between cells and can provide information about the health of bodily systems

What does the term "looping" refer to in the context of eukaryotic transcription?

Because of the distances involved between enhancers and the promoter, there is often looping of the DNA involved these interactions in order to have all of these different proteins at their distant binding sites all interacting with the basal transcription machinery at the core promoter

How do we know if something is 'recessive' in a pedigree analysis? If we know a member of a pedigree has a recessive trait, what do we know about their parents?

Both copies, trait expressed (aa) Can skip generations Both parents can be carriers (Aa) - do not express trait - or can actually carry the diseased state

Which point in the 'flow' of a protein is gene regulation most common? Why?

By far, the most prevalent type of control is actually at mRNA synthesis When you think about it, that makes sense - why go through all of these additional processes, many of which consume energy, if you don't need the final product? (Previously, we've talked about how energetically expensive it is to actually synthesize a protein, so certainly, it's better to regulate at earlier steps than that)

What is CRISPR and why is it such a big deal?

CRISPR: a molecular scalpel for making precise, targeted edits to DNA Instead of having to rely on these time consuming and laborious approaches to making changes to genome, CRISPR allows you to go in and make a precise edit to the DNA sequence in a gene

Briefly explain CRISPR and the concept of adaptive immunity

Came from the discovery that bacteria have this machinery within them, and use it as an immune system for themselves, for recognizing foreign sequences and chopping them up Whole process of discovering immunity process in bacteria and adapting it so that it could be engineered by a scientist in a test tube and programmed to target specific places in genomes

Why use a carrier protein vs a channel protein? Provide an example

Can have both channel and carrier proteins specific to one thing (eg glucose!) If directionality matters more, carrier protein. If directionality matters less, channel protein Ex: cells in vascular system want to pull glucose out of your blood, and into the surrounding tissue (transport of energy. So one type of carrier protein on one side, and the other type of carrier protein on the other side. If directionality didn't need to be so exact, then there would just be channel proteins

Why would you want less than perfect stringency in DNA hybridization?

Can modulate temperature, salt concentration, amount of formamide (which affects the ability to base pair), so that you're demanding perfect base pairing for that hybridization to occur, or in other cases, your'e demanding less than perfect complementarity, so that you can detect a gene in another organism that's similar but not exactly the same in nucleotide sequence to the gene that you're already studying

Chloroplasts (structure + function)

Capture energy from sunlight and use it to produce food for the cell Like mitochondria, have an inner membrane and an outer membrane, and inter-membrane space between them. But none of these places is where the important action is happening in photosynthesis! Instead, it's happening inside of thylakoid discs

Cytokinesis (in animal cells)

Cell is compressed by a contractile ring that divides the cell in nearly equal halves. By now, the organelles in the cell have been replicated, and are now divided between the two halves of the cell. This includes mitochondria, Golgi bodies, and the rough ER. Plant cells also have chloroplasts. Once split, the two new cells are in the G1 stage of interphase, and are ready to begin their growth

Relationship between kinetochore proteins and centromere

Centromere is the place on the condensed sister-chromatid pairs where a group of proteins called kinetochore proteins assemble to form kinetochore complexes. This region where the kinetochore complexes form is called the centromere

What are the 'poles' of the mitotic spindle?

Centrosomes

How does temperature impact enzyme affinity?

Changes in temperature may affect shape of enzyme, which could impact affinity for substrate (see dramatic curve above - activity goes up as you increase temp initially, because increases reactant rate, because stuff bumps into each other faster and thus wedges into active sites. Then, at too high a temp, the proteins denature) Changes in temperature could also impact turnover number (number of reactions that can be carried out by an enzyme per unit time. Ex: some enzymes can carry out a bunch of reactions in a matter of nanoseconds). This figure is based onto the idea that 'as soon as one substrate floats away, another one immediately binds.' What's the max number of reactions per unit time that could be carried out if the enzyme immediately got another substrate as soon as it released a product? Impact to turnover number here exists because maybe enzyme could still bind, but maybe it's not as good at catalyzing reaction, which would decrease turnover number

How did we come up with the idea of the double helix?

Chargaff's ratios, Watson and Crick used Chargaff's rules in combo with X-ray diffraction data courtesy of Rosalind Franklin to come up with the double helix structure

What do chi squared analyses work great for? What do they work poorly for?

Chi squared analyses work great for things like fruit flies, where we are able to have thousands of results, or things like peas, where we can (again) have hundreds or thousands of results These don't work really well for humans, however, because most human matings don't result in thousands of offspring (chi squared depends heavily on sample size)

2 important participants in photosynthesis?

Chloroplasts, thylakoid discs

Competitive inhibition

Competitive inhibition is a process by which a chemical substance has a shape that fits the active site of an enzyme and competes with the substrate, effectively inhibiting the enzyme. This is a subcategory of reversible inhibition, and a common method of engineering small molecule drugs

What do we mean when we say that DNA polymerase requires a primer?

DNA polymerase requires a "primer" Can only add nucleotides to the 3' end of an existing chain Always and only at the 3' end of a primer. It is only able to grow chains in the 5' to the 3' direction as it moves along a template strand in the 3' to the 5' direction Therefore, can never start a new chain "de novo" (by joining two dNTPs) A separate enzyme makes an RNA primer; DNA polymerase then adds DNA nucleotides to the 3' end of the primer Later, the RNA primer is removed

Do you add more dntp or ddntp with Sanger? Why?

DNAP sit down at the 3' OH end of the primer, and use dNTP as substrates. When the DNAP comes to a c in the template (what is the c???) they have a choice between adding a regular dNTP or a ddNTP, and you set it up so that there's a way higher concentration of the regular nucleotide than of the ddNTP, so that when the DNAP reaches each position, they have a higher probability of putting in the regular nucleotide than of putting in the ddNTP

Which prokaryotes use which kind of transcription termination mechanism?

Different genes seem to use different mechanisms. Some use one other the other. It's not well understood why there are two different mechanisms, and why some genes use one versus the other

How do reading frames work in translation?

Different reading frames are possible... but the AUG start codon sets the reading frame, so the ribosome correctly locating the start codon is key to successful gene expression. This process is different in prokaryotes and eukaryotes

DNA polymerase

Does continuous synthesis on leading strand Just finishes Okazaki fragments on lagging strand (and bumps into RNA of next Okazaki fragment)

What do we mean by 'addition of a cap' to the 5' end?

Elaborately modified methylated guanine nucleotide with phosphates... this is important for the initiation process of translation by a ribosome

How do elongation factors work?

Elongation factors use energy from GTP hydrolysis to escort aa-tRNAs to A-site and to drive translocation

Summarize stage 2 of replication

Elongation! - Helicase enzymes expand the replication bubble, revealing additional template for continued DNA synthesis - Growth of replication bubble is bidirectional (DNA synthesis occurring at both replication forks) - Adjacent expanding replication bubbles eventually fuse

What's the difference between endo/exocytosis and transmembrane protein transport?

Endocytosis/exocytosis = bulk material. If transport protein process might be too slow, so that's why cells use endocytosis/exocytosis

Endonuclease

Endonucleases hydrolyze phosphodiester bonds in DNA, hence break chain

Is the addition of dNTP monomers energetically favorable?

Energetically, this process is helped both by the cleavage of the bond between the innermost and outer two phosphates, and by a subsequent enzyme that comes along and clips that pyrophosphate to make it into two individual inorganic phosphate molecules

Active transport

Energy-requiring process that moves material across a cell membrane against a concentration difference in order to create that gradient. Provides a way for the cell to transport additional molecules against its concentration gradient

Enzymes

Enzymatic proteins help catalyze reactions

Telomerase

Enzyme responsible for adding (replenishing) telomere repeats Has reverse transcriptase activity (enzyme that uses RNA as as template to synthesize a DNA strand)

Enzymes (general definition)

Enzymes act as catalysts for chemical reactions in living systems. They decrease the activation energy so that reactions can occur at rates that are relevant to living organisms (even if reaction is spontaneous, activation energy can still be pretty high!)

Do catalysts have an impact on equilibrium constant?

Enzymes can't change whether a reaction is favorable or not. They can only increase the rate at which a reaction approaches equilibrium. Therefore, catalysts have no impact on equilibrium, or equilibrium constant

Dehydrogenases

Enzymes that catalyze the reduction NAD+ (this is the coenzyme for the malate dehydrogenase) to NADH (or in some cases FAD to FADH2) by taking hydrogen atoms off another molecule. In the reaction shown here, the COOH (group attached to whatever) is the intermediate (this is a zoom into a larger step-wise process)

Electron carrying coenzymes (definition, examples)

Enzymes whose job it is to carry the hydrogens who have taken off as intermediates along the catabolic pathway in order to oxidize those intermediates) Ex: NADH, FADH2

Why do eukaryotic chromosomes have more than one origin of replication?

Eukaryotic chromosomes are way, way large, so if a eukaryotic chromosome had only one origin of replication, it would take way too long to replicate each chromosome. So, eukaryotic chromosomes tend to have multiple origins of replication, and each one grows until eventually they all fuse together and you end up with two separate chromosomes

How do cells ensure concentration gradients? Explanation + example

Ex: Na+, K+ (arguably the two most important ions in your body. This is why you need to eat salt to survive) Na+ exists in low concentration inside of the cell, and high outside of the cell. K+ exists in high concentration inside of the cell, and low outside of the cell. The cell maintains these levels in order to sustain the concentration gradient What allows them to stay in balance? Active transport!

Is the ETC endergonic or exergonic?

Exergonic Energetically, process of oxidizing NADH or FADH2 (and taking those electrons to place them on oxygen) is a very exergonic process Electrons become lower in energy as they pass from each redox center to the next The released energy is harnessed in the form of a H+ gradient across the mitochondrial inner membrane

How does expected frequency of DCO compare to SCO?

Expected frequency of DCO gametes is much lower than SCO

Why is the 'run of U's' relevant to transcription termination?

Factor-independent transcription termination involves a sequence of nucleotides in the DNA... We are looking at an RNAP (blue blob) that's in the process of transcribing a gene... notice the mRNA that has already been produced is extruding from the RNAP, with 5' end visible... RNAP at this point has transcribed the part of the DNA, or most of the part of the DNA, that encompasses this termination signal. The sequence of the RNA that's made is such that there are two short regions of nucleotide sequence in it that are complementary to each other, so that if given the opportunity, they will be able to base pair with each other and form a stem loop structure (similar to what we've seen before with the secondary structure of tRNA). RNAP has reached this position, where it's transcribed that RNA that has those sequences, that have the potential to form a hairpin In addition to that sequence, there is a sequence of RNA that is very rich in U residues. This is still within the transcription bubble, this part is still base-paired to the DNA template, and it's very rich in U's. RNA within termination region contains 2 segments of complementary sequence followed by a run of U's (something about running into the run of U's sitting in the transcription bubble causes the RNAP to pause, momentarily. Previous to that, it's been chugging along at a constant rate, moving from one nucleotide to the next, adding nucleotides to the RNA chain, but something about the U's causes it to temporarily pause) U residues are on newly-synthesized RNA

Golgi apparatus

Flattened stacks of membranes in the cell that modifies, sorts, and packages compound proteins from the endoplasmic reticulum (glycolipids, lipoproteins, etc) Cis face: closer to nucleus Trans face: closer to outer membrane

Very broadly, what are the 3 main stages of all 3 parts of the central dogma?

For each of the polymerization processes that we will learn about (DNA replication, transcription, and translation), we will describe them in terms of how the process is initiated, how the polymer is elongated, and how the process is terminated.

Gibbs free energy (equation + definition)

G = H - TS · H = enthalpy, or total energy of the system (for chemical systems, energy stored in molecules themselves) · S = entropy, or unavailable energy (usually heat), disorder of molecules in the system (disorder happens "spontaneously"). The greater the entropy, the less energy is available to do work T = temperature in degrees Kelvin Energy available to do work

Stages of the cell cycle

G1, S, G2, M

Endomembrane system

GERL. Lysosomes, golgi, ER Synthesis, post-translational modification, and sorting of proteins that are destined for certain organelles, the plasma membrane, or even for secretion

What will the F1 genotype be if genes are completely linked? How about an F1 X F1 cross (F2)? How about an F1 X test cross parent?

Gametes will have same genotype as parent. SO, F1s will be heterozygous for both traits (assuming both parental gametes were completely linked) F1 X F1 —> F2 When this is crossed, then instead of that 9:3:3:1 you'd see with a dihybrid cross, you'll get a 1:2:1 ratio (this tells us that they're linked) in the F2 progeny Does not result in a typical 9:3:3:1 phenotypic ratio predicted for a dihybrid cross assuming independent assortment (unlinked) F1 X test cross parent Test-cross progeny will have a 1:1 ratio

What do genes that encode proteins do? What do other genes do?

Genes that encode proteins are transcribed to make mRNAs. But there are two other [major] types of RNA, which do not encode protein but are needed in order for the information in mRNA to be translated, and these RNAs are synthesized by RNAP using genes in DNA as templates.

Phosphlipid What varies between different types? Why does this matter?

Glycerol + phosphate with two fatty acid tails Amine group attached to phosphate determines what kind of phospholipid - is the only thing subject to change, and determines how proteins attach to the membrane.

Provide an example of real-world applications of biotechnology and genomics info involving crops

Golden rice! Made by introducing a number of non-rice genes (genes from beans, fungus, daffodils, etc) that gave the recipient rice plant all of these benefits, like increased iron content, destroying an enzyme that might inhibit the ability of the rice plant to accumulate iron, precursor to vitamin A content, etc (basically just extra nutritious rice)

In what direction do replication bubbles grow?

Growth of the bubble is by bi-directional Growth of replication bubble is bidirectional (DNA synthesis occurring at both replication forks)

Histones are ______________ in evolution

Histones are proteins that are highly conserved in evolution All eukaryotic cells have histomes in them whose amino acid sequences have been very highly conserved over the course of evolution (this is an indication that there's something about those sequences that are very important to function). Histomes are so fundamental to the function of eukaryotic cells that they have been preserved with very few changes to them across the eukaryotic kingdoms

How many base pairs does human DNA have?

Human DNA has ~3 x 10^9 base pairs. Total human genome is billions of base pairs, compared to millions that we talked about in prokaryotic cell (see: E. Coli)

How do conformational changes factor into the creation of polypeptide chains?

If the tRNA is correctly matched, and remains bound for a long enough time, it is committed to be used in protein synthesis. The ribosome catalyzes the formation of a new peptide bond, and undergoes a dramatic conformational change. Elongation factor G binds the ribosome. Hydrolysis of GTP by elongation factor G switches the ribosome back to the state in which it can accept the next incoming tRNA

Why is HW equilibrium important

Important because if you have a population that is violating these principles - that does not meet all of the HW assumptions - then, most likely, that population is not in HW equilibrium. And if a population is not in HW equilibrium, evolution may very well be happening So there are a whole bunch of things that we call 'agents of evolutionary change'

What is the fluid mosaic model? Who discovered it? Why is it important?

In 1970, Frye and Edinin demonstrated protein fluidity within the membrane. They forced two cells to fuse, and found that after 40 minutes, the membrane proteins had mixed together (instead of staying anchored). This led to the fluid mosaic model of the membrane: a "mosaic" of proteins floating in a "sea" of lipids

At a high level, how do enzymes in a metabolic pathway work?

In a metabolic pathway, enzymes act like an assembly line - the product of each enzyme is the substrate for the next, until a desired end product is formed (series of enzymes that act on each others' products. Each successive product becomes an intermediate)

Differentiate between "necessary" and "sufficient" with regard to proton gradients in the ETC

In a natural mitochondrial system, anything that destroys the ability of the ETC components in that membrane to create a gradient dissipates the gradient needed to make ATP, showing that a proton gradient is necessary for making ATP by synthase. ATP synthase doesn't care from where this proton gradient comes from, though (see: Racker and Stoeckenius), which proves that it's sufficient.

What do we mean by 'degree of freedom'? How does this relate to expectations for deviation?

In chi-squared analyses, degree of freedom (df) = n-1 Where n is the number of groups that we are observing More deviation is expected with higher degree of freedom

How were primers generated in the old days? What did you need in order to do this?

In the "old days" primers were actually generated by end-labeling restriction fragments that overlapped w/the fragment to be sequenced! You needed lots of a fragment to be able to do sequencing. Large amounts of DNA were obtained by growing up large cultures of bacteria containing the cloned DNA and purifying plasmid from them.

What is bi-directional replication? Why do we care?

In the context of the growing bubble: as this bubble is growing, and Okazaki fragments are accumulating on the lagging strands, those enzymes involved in degrading the RNA primers, extending the remaining DNA, and that DNA ligase that seals together - those are working on those Okazaki fragments as the process is going, so that in reality, that last part of the DNA drawing diagram (see recording) that showed whole region of lagging strand still existing as Okazaki fragments - in reality, it doesn't end up existing like that, because these enzymes are kind of keeping up as you go along. But the part nearest to the fork is still existing as Okazaki fragments

What happens after the holoenzyme recognizes the promoter? (full process)

Initiation phases - Initiation begins: sigma binds to promoter region of DNA - Initiation continues: RNA polymerase opens the DNA helix; transcription begins - Initiation is complete: sigma is released form the promoter; RNA synthesis continues from DNA Voiceover: DNA helix starts to get melted open, starting at the -10 box, to form the transcription bubble. Then, transcription can be initiated, and importantly, in the context of how prokaryotic transcription works, the sigma subunit dissociates from the holoenzyme after transcription has been initiated (sigma recycles after RNA synthesis has started). It can then go bind to another core RNA polymerase. So the RNA polymerase without the sigma subunit is called 'core' RNA polymerase, and once it has sigma bound to it, it's called holoenzyme (sigma is kind of acting like an escort to carry the RNA polymerase, recognize a promoter, and get the RNA polymerase started, and then it leaves)

Describe translation in terms of the 3 process stages we've discussed

Initiation: Ribosome locates an AUG start codon at beginning of an ORF on mRNA Elongation: Ribosome moves in 5' —> 3' direction along mRNA, making peptide bonds to grow a polypeptide in N —> C direction Termination: Ribosome reaches a stop codon and releases a completed polypeptide chain

Inputs, outputs, and location of oxidative phosphorylation

Input: NADH, FADH2, O2, ADP, P Output: 34 ATP, H2O, NAD+, FAD Location: takes place in the mitochondrial membrane

Inputs, outputs, and location of glycolysis

Input: glucose, ATP Output: 2 pyruvate, 2 ATP, NADH Takes place in cytoplasm

What is the cytoskeleton made of?

Intermediate filaments, microtubules, actin microfilaments

Desmosome

Joins the intermediate filaments in one cell to those in a neighbor. Also cadherin family proteins, but big difference is that intermediate filaments connecting these two to create a junction, not actin.

Kinase/phosphatase

Kinases transfer the gamma (outermost) phosphate of ATP onto a hydroxyl on a DNA molecule Phosphatases hydrolyze phosphomonoester bonds (release Pi; not the reverse of the kinase reaction) Voiceover: Haven't really talked about these in the context of DNA (only in proteins so far). But DNA has a phosphate group... if you have a piece of DNA that has ends to it, it typically has a phosphate group at the 5' end of any strand of DNA. And you can use a phosphatase enzyme to clip the phosphate off of the end of the DNA, and then you can use a kinase to put on a radioactive phosphate group onto that DNA, and that makes your DNA molecule radioactive and easier to detect (on a gel, etc)

How/why are hormones an example of signal transduction impacting transcription?

Learned that molecules that are hydrophobic are able to pass through membranes on their own, without the help of a protein transporter... so there are some hormones, those derived from steroids (like cortisol, shown here) that can penetrate the membrane and get into the inside of the cell, and bind to an intracellular receptor, and when steroid hormones like that bind to intracellular receptors, that actually causes the hormone receptor complex itself to go into the nucleus and influence gene expression So... cells respond to signals that may have originated far away in the body, from other cells, and travel through the bloodstream, and arrived at a cell, to trigger the cell to change its gene expression

What does it mean for genes to be linked?

Linkage: Genes on same chromosome are linked Parental gametes: Gamete that looks like same genotype as you would see in the parent (note: NOT fertilized zygote. Just gametes) · Ex: Meiosis. Those that separate in meiosis are parental gametes. These are gametes that look like the same genotype that you would see in the parent

What would happen to cells without the glycocalyx?

Lipid membranes of cells would merge, and cells would lose themselves to other cells (among other things)

Cytokinesis in animal cells (how does it happen? What's the purpose?)

Literally caused by pinching a cell into two. Caused by the actin cortex from the cytoskeleton. Towards the end of mitosis, there's a rearrangement in the actin cortex of the cell that forms a belt of actin filaments (contractile ring) around the waist of the cell (the equator of the cell at the same location as that old metaphase plate). For cytokinesis, motor proteins (myosin motors) interacting with actin filaments cause them to slide relative to each other, and to contract that belt around the waist of the cell, until it literally pinches into two cells (cleavage furrow)

Thylakoid discs

Located in chloroplast, associated in the light reaction part of photosynthesis, round-circle like membranes, chlorophyll is located here Compartments within inner membrane, surrounded by a membrane, space inside called thylakoid space (contains chloroplasts, ETC, proton gradient, etc) Action in photosynthesis happens inside of the thylakoid membranes Contain chlorophyll and other pigments that absorb light, contain an electron transport system that does redox reactions and pumps protons, and have an F0F1 ATP synthase that uses that proton gradient to make ATP

What are the relative lengths of each stage in the cell cycle?

M phase, which includes both mitosis and cytokinesis, is relatively short. The rest of the time is interphase, and within that time, there's S, G1, and G2

Michaelis-Menten Kinetics (what did they propose, and what's the equation?)

Michaelis and Menten proposed that the number of substrate molecules that can be processed per unit time is limited by the length of time the catalysis step takes The Michaelis-Menten equation is the rate equation for a one-substrate enzyme-catalyzed reaction. This equation relates the initial reaction rate (v0), the maximum reaction rate (Vmax), and the initial substrate concentration [S] through the Michaelis constant KM—a measure of the substrate-binding affinity.

Can you provide an example of the applications of DNA hybridization?

Microarrays! You're using those individual spots that each has a different gene on it... each of those is basically being used as a probe to look for whether, in the microarray example, there is messenger RNA that is complementary to that gene in the complex RNA sample that you happen to be putting on the microarray

Are microtubules stable structures? Describe why/why not and any related processes.

Microtubules exhibit dynamic instability. At any given moment, they may be growing or shrinking, constantly. This helps establish cell shape and move things around - things tend to "walk" along the microtubule. Microtubules can assemble due to GTP (dimers bind to GTP). Then the reaction slows, GTPs are hydrolyzed, and become GDP plus phosphate - GDPs begin to fall off. If the process goes in reverse (ex: GTPs hydrolyze), then we have dynamic instability.

Mitosis

Mitosis is the process of dividing the duplicated DNA of a cell into two new nuclei

Why is splitting a eukaryotic cell more complicated than splitting a prokaryotic cell? Which process(es) ensure(s) that eukaryotic cells split properly?

Much more complicated to divide a eukaryotic cell, because you need to ensure an organized way to ensure all chromosomes are duplicated and each daughter nucleus gets one and only one copy of each of the duplicated chromosomes when the cell divides Mitosis (splitting of chromosomes) is the chromosomal process that ensures this (carefully distributing duplicated chromosomes to separate areas that become 2 new nuclei) Cytokinesis (splitting of the cell that divides cell in two so that one of those nuclei is in each of the two daughter cells) Mitosis is the splitting of the chromosomes. Cytokinesis is the splitting of the cell

What is a spliceosome?

Multiple snRNPs recognize splicing signals in each intron in the pre-mRNA and interact with each other to form a "spliceosome"

Mutation

Mutation (this is the mechanism of evolution!) The ultimate source of variation. Individual mutations occur so rarely that mutation alone usually does not change allele frequency much Leads to new genes, or changes in genes (so new alleles) Evolution happens because of random mutation creating new alleles

Name 5 agents of evolutionary change that makes populations vary from HW. If any one of these factors is present, what does that mean for your predictive abilities?

Mutation, gene flow, nonrandom mating, genetic drift, selection The 5 above factors would make a population change - would make a population NOT fit HW - so you wouldn't be able to use HW to predict the next generation

What is NAD+?

NAD is a coenzyme that can float in and out of enzyme active sites

What do NAD and FAD do?

NAD+ and FAD+ act as H+ ion shuttles [carriers], taking them from the Krebs cycle to ETS Further along in the respiration pathway, the electrons carried by NADH or FADH2 will be used to reduce O2 to H2O (while oxidizing the NADH or FADH2 back to its NAD+ or FAD form). We say that the NADH and FADH2 carry "reducing power" because this transfer of electrons from them onto oxygen releases a lot of energy. This transfer also makes water (remember, adding two hydrogens) and allows a lot of ATP to be created (release of energy)

Are nucleosomes the most compact form of DNA? If not, what is?

NOPE. Even this level of compaction of DNA into nucleosomes is nowhere near enough to fit the DNA into the cell, so the nucleosomes themselves then assemble with each other into higher order compacted structures. DNA is stored/packed at varying degrees of compaction Ultimately, this is called a "30 nm fiber" or a "solenoid," because of the coiled conformation of the string of nucleosomes Then, additional proteins fold that solenoid into a bunch of looped domains, and then condensed further The most condensed form of DNA is found in the "sister chromatids" seen during mitosis

Does Hardy-Weinberg equilibrium apply to most real-world populations?

No

Where does protein synthesis begin? Does the ribosome ever halt elongation?

Note that all protein synthesis starts on a free ribosome. For some proteins, after a small number of amino acids have been incorporated, the ribosome will temporarily halt elongation and attach to a membrane (RER in eukaryotes, plasma membrane in prokaryotes) before resuming elongation of the polypeptide chain. You will hear more about this next time.

After a peptide bond is formed, what happens to the tRNA that had the 1st amino acid attached to it?

Note that the tRNA to which the 1st amino acid was attached no longer has an amino acid attached to it.

How do things get started during the initiation phase of translation?

Note that to get things started in the initiation stage, the initiation factors delivered the met-tRNA to the P-site so the 2nd aa-tRNA would be able to enter the A-site. Thereafter, aa-tRNAs are delivered to the A-site by proteins called elongation factors. The elongation factors use energy from GTP hydrolysis to deliver the incoming aa-tRNA, and also use energy from GTP to push the ribosome over to the next codon (this process is called translocation).

Function of telomerase (how does it work?)

Notice - 3' overhang on end of chromosome. This hasn't yet been able to have that other strand filled in with DNA. And here's telomerase, carrying an RNA template within it that's complimentary to the repeating sequence that's in that telomere DNA. Base pairs to overhang and uses reverse transcriptase activity to add DNA nucleotides the 3' end of that 3' overhang. Once it synthesizes that whole sequence that's complimentary to its RNA, its RNA overlaps onto the next repeat and it can jump over and pair more and more DNA. 1. End is unreplicated 2. Telomerase extends unreplicated end 3. Telomerase repeats activity

How does radioactivity factor into Sanger?

Now I have single stranded pieces of DNA that are radioactive because the primer was radioactive, and whose lengths represent all of the positions where DNA had to stop because it incorporated a dideoxyA instead of a regular A. And then in the next tube, analogous thing, except all lengths where it had to stop because dideoxyC. And so on And then when I run these out onto my denaturing electrophoresis gel, that's separating single stranded DNAs, with single nucleotide resolution, and run each in 4 lanes, I can actually read up the ladder! Read right up the gel to get the nucleotide sequence of the DNA

Why does it help to extend the 3' overhang if that's what we wanted to get rid of?

Now that we have more telomere repeats, the normal replication machinery, or other enzymes that are designed to deal with these long overhangs, can come in and put a new RNA primer in and have new DNA polymerase extend the RNA primer so that we have extended the double stranded telomere repeat DNA

Nucleus (structure)

Nuclear envelope (continous with ER), nuclear lamina, nuclear pores, heterochromatin/euchromatin, ribosomes

Describe nuclear pores

Nuclear pores are quite large, as large molecules must be able to pass through. However, transport through these pores is highly regulated.

Name 5 digestive enzymes

Nucleases, proteases, glycosidases, lipases, phosphatases, sulfatases, phospholipase

How does RNAP know where to start and stop transcribing?

Nucleotide sequences called "promoters" and "terminators" in the DNA tell RNAP where to start and stop transcribing This is showing you that along a chromosome, there are many genes and each gene will have a transcription start site, but not all genes are oriented the same way. Some are oriented to have RNA polymerase travel to the right, which means that it will use the bottom strand as the template strand and create a strand that is identical to the non-template strand, except for it will have U's in it, while another gene might have its promoter oriented to direct the RNA polymerase in the other direction

Does a double crossover mean that single crossovers haven't happened?

Observing a double crossover doesn't mean that single crossovers haven't happened - you could imagine that there might be two separate single crossover events, and then a double crossover event Basically, this means that we get a ton of information. Many kinds of gametes, more different kinds of offspring

If we are certain about something, what's the probability of that event?

Once we know for certain, we can say that the probability is 1

What's the biggest difference between RNAP and DNAP?

One important difference between RNA polymerase and DNA polymerase: RNA polymerase can start RNA chains de novo (from scratch). RNA polymerase can start de novo, DNA polymerase cannot So whatever base the first base that is specified by the template strand, after the RNA polymerase gets oriented on the promoter, it can take that base and it can take the next individual base that is specified by the template, and it can join them together to make a dinucleotide, and then it can add another to make a trinucleotide. So it does not need a primer

Map unit (mu)

One map unit (mu) is defined as 1% recombination between two genes on a chromosome

Glycolysis pathway

One molecule of glucose —> energy investment to be recouped later —> fructose 1,6-biphosphate —> cleavage of six-carbon sugar to two three carbon sugars—> two molecules of glyceraldehyde 3-phosphate —> two molecules of pyruvate —> energy generation Energy generation occurs in the process of turning the glyceraldehyde 3-phosphate into pyruvate (there are two "substrate-level phosphorylation" steps in this part of the pathway)

How are operons for alternative energy sources regulated?

Operons for catabolism of other alternative energy sources also are regulated by CAP ("catabolite activator protein) so that they are not expressed unless glucose is unavailable. Each of these operons has its own promoter and its own specific repressor protein that ensures it cannot be expressed unless its particular alternative energy source is present. These operons are scattered throughout the E. coli chromosome. A collection of operons that is coordinately regulated is called a regulon.

What's the difference between NADH and NADPH? In what organisms is NADPH found?

P stands for phosphate. Similar to NAD and NADH, but with a phosphate. This group allows cells to differentiate, and have two separate pools of these electron carriers. NAD and NADH used for catabolic pathways, NADP and NADPH used for anabolic pathways) (it's not just choloroplasts that differentiate this way - humans use NAD and NADP as well)

How long are promoters? Do promoters only tell RNAP where to start?

Particular sequences of DNA... orders of nucleotides, in short regions of around 25 or 30 base pairs in the DNA, can be recognized by RNA polymerase as the place to start. These sequences are called "promoters." Promoters not only tell it where to start, but also orient RNA polymerase (of course, this also defines which strand is going to be the template strand, and which is going to be the non-template strand

Can polyribosomes exist in eukaryotes?

Polyribosomes exist in eukaryotes too, but only outside of the nucleus! Because the rNA gets made in the nucleus (as a pre mRNA, gets spliced and has the poly A tail added, and only then is allowed out, but still, once it's out there and ribosomes have access to it, as each ribosome gets started and moves away from the start codon and starts to move along the messenger RNA, another ribosome can get on and follow behind it

How does RNAP II know where to bind?

Prior to RNAP II binding, accessory proteins called basal (aka general) transcription factors first bind to the core promoter and subsequently recruit RNAP II

How do prokaryotes divide? What other organelles divide this way?

Prokaryotes divide by binary fission Single, circular chromosome attached to plasma membrane, with no exposed 5' or 3' ends. Each strand has its 5' 3' ends joined together in one phosphodiester bond. Replicate DNA from single origin of replication (bi-directionally), double cell contents, split into two between attachment sites (involves forming a septum and pinching-in). Mitochondria and chloroplasts also divide this way

Gene expression in prokaryotes vs eukaryotes?

Prokaryotes: single circular chromosome, located in cytoplasm (may also have plasmids) Eukaryotes: many linear chromosomes contained in nucleus ("nuclear genome"); mitochondria and chloroplasts also contain DNA (circular like that of prokaryotes)

How do cells divide?

Prokaryotic cells divide by binary fission. Eukaryotic cells divide by a process that includes mitosis, division of the nucleus, and cytokinesis, division of the cytoplasm.

Flagellum (prokaryotic)

Prokaryotic flagella use rotary motor to drive movement - locomotion of the cell itself

What's the main difference in origin of replication between prokaryotes and eukaryotes?

Proks: Circular genome; one replication origin Euks: Multiple "linear" chromosomes; each typically containing multiple replication origins

Prometaphase (basic)

Prometaphase · Microtubules attach to the chromosomes · Begins when the nuclear membrane is broken down. At the same time, microtubule strands (spindle fibers) are growing from the centrosomes. These strands attach to a protein structure called the kinetochore. One kinetochore is attached to the centromere of each sister chromatid.

In what direction do protein chains grow?

Protein chain grows from its N terminus towards the C terminus

Signal hypothesis

Protein synthesis starts out on a free ribosome, and if proper signal is there as part of the protein, then ribosome can land on ER membrane, and ER will continue processing that protein (ribosome also participates)

Describe the process of RNAP at work

RNA polymerase locates a "promoter" sequence in double-stranded DNA, and then unwinds the DNA in that region to form a "transcription bubble". RNA synthesis can then begin. As the RNA polymerase moves along the DNA, the RNA product is displaced from the template starting at its 5' end, and the DNA duplex is allowed to reclose, so the transcription bubble stays constant in size and moves along with the polymerase.

Give a brief overview of how transcription begins and progresses (in eukaryotes)

RNA polymerase locates a "promoter" sequence in double-stranded DNA, and then unwinds the DNA in that region to form a "transcription bubble". RNA synthesis can then begin. As the RNA polymerase moves along the DNA, the RNA product is displaced from the template starting at its 5' end, and the DNA duplex is allowed to reclose, so the transcription bubble stays constant in size and moves along with the polymerase.

Receptors

Receptor proteins enable the cell to signal and receive signals (ligands)

What is stringency modulation?

Refers to DNA hybridization. Can modulate the "stringency" of the annealing process to demand perfect or less-than-perfect complementarity of sequence Can modulate temperature, salt concentration, amount of formamide (which affects the ability to base pair), so that you're demanding perfect base pairing for that hybridization to occur, or in other cases, your'e demanding less than perfect complementarity, so that you can detect a gene in another organism that's similar but not exactly the same in nucleotide sequence to the gene that you're already studying

How is eukaryotic nuclear DNA packaged and why is that relevant here?

Remember also that eukaryotic nuclear DNA is packaged with histones, which impacts accessibility!

What is the overall difference in the physical and temporal relationship of transcription and translation in prokaryotes vs eukaryotes?

Reminding us that the overall difference between the physical and temporal relationship of transcription and translation in prokaryotes vs eukaryotes Because there's just a single compartment in prokaryotes, the 5' end of the messenger RNA is being extruded from the RNA polymerase as its being synthesized, and it's extruded into the cytoplasm. Ribosomes are able to grab on to it and and able to work simultaneously alongside the transcription process In eukaryotes, though, the pre-mRNA is made in the nucleus, and processed in the nucleus, and is only allowed to exit through the nuclear pore, once it has the 5' cap and the poly A tail, and introns have been spliced out. Only then can it be translated

What's the main difference between passive and active transport?

Requires input of energy. This is the big difference! "Is there energy powering the movement of that particle?" If so, then this is active transport

Where are restriction enzymes derived from?

Restriction enzymes all came from bacteria They are one of the ways that bacteria protect themselves from viruses (cut up viral DNA! And have ways of preventing their own DNA from being cut up by these enzymes) So the enzymes are named after the bacterial species from which they're derived

Name 2 major advances that led to recombinant DNA technologies (and our current "omics" world!)

Restriction enzymes and cloning

How do reversible inhibitors work?

Reversible inhibitors can modulate enzyme activity (affect Km, Vmax, or both, generally impact those parameters of typical Michaelis-Menten kinetics). Other types of reversible inhibitors bind elsewhere on the enzyme, changing its shape and rendering the enzyme less effective (more subtly than the switches)

In eukaryotes, why does RNA need to be transported out of the nucleus before it can be translated?

Ribosomes are in the cytoplasm. In prokaryotes, ribosomes have access to mRNA while it is being synthesized and can hop on and start translating it even before RNAP has reached the terminator! In eukaryotes, functional ribosomes are only found in the cytoplasm (even though they are partially assembled in the nucleolus; more on this later), so the RNA must be transported out of the nucleus before it can be translated. And, in fact, there are some extra things that are done to RNA that is destined to be translated, even before it is transported out of the nucleus. For this reason, we call the initial product of transcription of protein-coding genes by eukaryotic RNAP a "primary transcript" or "pre-mRNA," to distinguish it from the "mature mRNA" that gets transported out of the nucleus

What is a ribonucleoprotein complex and why do we care?

Ribosomes are ribonucleoprotein complexes They are large assemblies of multiple proteins and a few specific ribosomal RNA molecules (same in all of the ribosomes)

What was the Creighton McClintock experiment and why do we care? How does this relate to Mendel?

Showed that in recombination, actual pieces of chromosome structure are being swapped. You can imagine that it be possible to understand that genetic recombination is occurring, but not understand the physical mechanism. I mean, that was Mendel's whole thing: Mendel knew that inheritance was going on, but didn't know how it happened. So then, later on, when we start to see this recombination, we know that genetic recombination is happening, but how do we know it? This experiment! It was a way to see both the genetic output and the chromosomes The chromosomes they tested were unique chromosomes because upon inspection in the karyotype they looked different from one another, but they weren't genetically different. So when crossing over happened, not only was the genetics able to be visualized, in terms of the phenotype, but the chromosomes also looked different. Saw separation of cap from end piece. This was evidence that not only were genes being swapped, but it was because the actual chromosome was being swapped This was stunning and compelling evidence that what's really happening is a change in the physical rearrangement of the chromosome structure

What and where does sigma factor recognize in prokaryotic transcription?

Sigma factor recognizes primarily two little segments of DNA sequence within the promoter region, that are referred to as the '-35 box' and the '-10 box' (the minus numbers result from a numbering system that treated the place where the first nucleotide of the RNA is going to be copied from... calls that +1, and the one just upstream is -1, -2, -3, -4, etc). So there are these groups of nucleotides at around -10 and -35, relative to the start of transcription, that are recognized by this sigma factor

In one sentence, what does sigma factor do?

Sigma helps this whole enzyme bind to the DNA

Where does sigma go after RNA synthesis begins?

Sigma recycles after RNA synthesis has started.

Finish this sentence: Signal transduction pathways in eukaryotes often culminate in...

Signal transduction pathways in eukaryotes often culminate in the regulation of transcription

Can next-gen sequencing use similar tech for DNA and RNA sequencing?

Similar approaches can be used to sequence all the RNAs in a cell sample (RNA-seq)

Relate tetrads and recombination

So when we have these crossover tetrads forming, we're looking at what happens if we get just two of the non sister chromatids connecting, within the same homologous pair You should be able to identify parental and recombinant gametes (respectively)

How are plasmids related to promoters?

Some of these plasmids are also set up so that when you put a piece of DNA into this polylinker region, it ends up being positioned next to a promoter for RNAP, and may even also have a start codon positioned that has been engineered so that it will be in frame with whatever sequence you're trying to put in, so that your bacteria will actually express the protein that's encoded by then piece of DNA that you've put in

How are proteins that are destined for some location (the GERL, e.g.) marked as such?

Some proteins have amino acid sequences within them that can act as signals to show where that protein should go, if it belongs somewhere other than the cytoplasm... so proteins that are destined for that GERL pathway (the secretory pathway) have a signal near the N terminus of the protein (the first part that gets extruded from the ribosome as the protein is being synthesized), and that's the signal that that ribosome should be carried to the ER membrane and continued to synthesize its protein and extrude it into a channel that goes through the ER membrane into the lumen of the ER There are some polysomes that are bound to the ER membrane because the protein that they're making had that signal on it. But there are also polyribosomes that just float free in the cytoplasm, because they're making proteins that are either destined for the cytoplasm or destined for locations other than the GERL pathway Intention is not to focus on GERL right now! Just talking about protein synthesis. Only brought it up because animation mentioned membrane bound polyribosomes

What is the trombone model of DNA synthesis?

Sometimes, that imagery of how the lagging strand is looped back and grows a new loop each time an Okazaki fragment is being made and then the polymerase jumps back and a new loop is started, sometimes that's referred to as the 'trombone model' of DNA synthesis (because it's like the slide of a trombone going in and out)

What type of primers are needed for old fashioned sequencing? How about next-gen sequencing?

Specific primers are not needed for next-gen sequencing approach, because you take genomic DNA fragments, and you ligate on linker molecules to them, so that you've basically created a common sequence at the ends of all of your genomic fragments (see: slide, genomic fragments on the right with universe adaptor ligated onto it). These linkers then get covalently attached to a chip, which is sometimes referred to as a "flow" chip, and reagents are going to flow through and enable nucleotides to get added. Then, you use primers complimentary to these linkers, and so you're able to amplify all of these fragments that are sitting on this chip (you do PCR to amplify these fragments), and the reactions are fully automated (controlled by computer)... no electrophoresis either, because as each nucleotide is added, a flash of light is emitted, and is detected within the machine (see: colored lights at the bottom of slide) · If we were doing normal sequencing the old fashioned way, and if we head a bunch of fragments of a genome that we wanted to sequence, we would need a different specific primer for each fragment, which would be laborious, expensive, and time consuming · In this approach, we fragment the genome however you're going to do it, and then you take all of those fragments, and each one gets little adapters added to both ends, and those adapters are the same for all of the fragments, and now you can use the same primer for all of the fragments

How does DNA synthesis end? How does this differ in prokaryotes vs eukaryotes?

Stage 3: termination of DNA synthesis Synthesis at a replication fork ends when the fork meets another fork coming from the other direction At this point, there's no more DNA that needs to be copied! That happens at the end of a replication of a prokaryotic circular chromosomes (singular origin of replication, etc) Visualize two replication forks running into each other You can imagine, similarly, that when two adjacent replication bubbles... when their forks meet up with each other, this continuous strand is going to be continuously synthesized until it bumps up against an RNA primer from the lagging strand of the other, and the other one will be continuing, and you'll end up with continuing DNA on both strands But what happens when a fork reaches the end of a linear (eukaryotic) chromosome? A problem completing synthesis of the lagging strand at the end of a linear chromosome

Solenoid

String of nucleosomes. Called this because of coiled conformation of the string of nucleosomes

What is stringency controlled by?

Stringency controlled by: - temperature - salt concentration - presence of formamide

Topoisomerase (DNA gyrase)

Suppose you have a piece of rope (rope is made of a bunch of little individual strands of fiber twisted around each other) and you grabbed half of the strands with each hand hand started pulling them apart - so basically doing what an origin of replication would have done to it - and you start growing that replication bubble... the rest of the rope is getting rotated as you pull open that bubble, because those strands are wound around each other, and as you open that up, the rest of the rope is going to start to coil upon itself, because of the extra torque that's being put into it as you unwind the replication bubble. So that extra folding of the rope upon itself also happens in DNA and forms what we call supercoils Supercoiling is going on beyond the replication fork. Eventually, it will become so energetically difficult to open any more of the bubble that the helicase won't be able to act unless some of that supercoiling is relieved. So there's an enzyme called topoisomerase... Named for a whole class of enzymes that are able to interact with and modify the amount of supercoiling in DNA, and the specific one in bacteria is called DNA gyrase Acts at the parental DNA helix to relieve "supercoiling" tension Acts downstream of each of the replication forks to help relieve the tension, so that the helicase can continue to expand the bubble

What's the relationship between daughter and parent strands of DNA?

TL;DR: DNA replication is semi-conservative. New daughter DNA "conserves" one of the two original or parental strands Note: daughter strand sequence (one of the strands) is identical to parental sequence DNA replication begins at "origin of replication" To actually replicate a DNA molecule, you need to unwind it, and that happens at "origins of replication." Those replication bubbles grow as complementary DNA is added to the two template strands of the parental DNA

Where does glycolysis take place? One sentence overview of what it does

Takes place in the cytoplasm of all cells Overall pathway oxidizes glucose to pyruvate while reducing NAD+ and NADH

[linking reaction] (pathway)

Takes pyruvate —> splits off carbon and two oxygens as a CO2 molecule —> attaches remaining atoms to CoA —> Produces acetyl CoA In the process, 2 out of the 3 carbons in pyruvate are turned into a higher oxidation state than they used to have, so the reducing power ends up on NAD+, creating NADH + H+ CoA, which has the two remaining carbons that came from the pyruvate covalently attached to it, carries those two remaining carbons to the citric acid cycle

Exocytosis (what is it, how does it work, types)

Taking from inside the cytoplasm and moving it out. These processes are basically the same no matter what is being transported in or out (hormone, waste products, etc). If a cell wants to get something out, it's through exocytosis Type (only one type): Vesicle lipid fusion

Why was understanding of DNA polymerase crucial?

Technological offshoots of understanding DNA polymerase By the late 1950s, we already understood how DNA polymerase worked (had already been purified from bacteria at this point) Our understanding of this allowed for the development of these really important technologies... understanding how this worked was absolutely critical to the ability to sequence DNA, the human genome, and other genomes

Structure of telomerase

Telomerase is a reverse transcriptase type of enzyme, but it carries its own RNA folded into it (protein enzyme that has a little piece of RNA folded into it. Carries its own RNA template!

Can you name an exception to the central dogma? Explain.

Telomerase! This is one of those exceptions to the central dogma, that says that information always flows from DNA to RNA to protein. Reverse transcriptase are proteins that reverse this flow of information and use information from RNA to make DNA Reverse transcriptases exist in certain viruses (ex: HIV, retroviruses. They are so named because they have reverse transcriptase activity in them). Their genomes are made of RNA, and when they infect cells, they use their reverse transcriptase enzyme to copy their RNA genome into DNA and insert their DNA into the host cell's chromosomes

Prophase (basic)

The DNA condenses, organizes, and the classic chromosome structure appears This is when we first see the classic chromosome structure. This occurs through a condensation process. At the same time, microtubules appear from the centrosomes in animal cells. Finally, the nucleolus disappears

What are we looking at in this image?

The ethidium stained gel, and the X-RAY film that was obtained from that ethidium stained gel after the blotting procedure (of the Southern blot) Showing us which bands of DNA contain DNA molecules that have sequences complementary to a particular probe Ex: notice that you can see prominent ethidium staining in several lanes, but on the blot image, you may not see all of the same staining 'lit up' or showing after probing with the radioactive probe

Can you think of a field of study that arose out of sequencing? Explain

The field of bioinformatics (databases of gene sequences, and ultimately of whole genome sequences) started to develop from this accumulating sequence information. Through sequence comparisons, evolutionary relationships between organisms could be deduced, and highly conserved regions of DNA sequence in homologous genes identified - which made it easier than ever to sequence new genes without even cloning them - amplify a region of genomic DNA using oligo primers from conserved regions flanking the region of interest, then use one of these primers to actually sequence the amplified fragment!

What is a primary transcript?

The initial product of transcription of a protein coding gene in eukaryotes is called a "primary transcript" or a "pre mRNA," and then it goes through these processing steps that we'll talk about a bit later, and only then can it exit through a nuclear pore and give access to a ribosome in order to translate it

Generally speaking, describe the structure of an mRNA

The protein-coding information in a mRNA extends from a start codon (AUG, coding for methionine) through many consecutive codons to a stop codon (UAA, UAG, or UGA)

Equilibrium constant

The ratio of products to reactants. The greater the difference in energy between the reactants and products, the greater the value of the equilibrium constant will be Ex: The more negative the delta G, the higher the ratio products to reactants when the reaction reaches equilibrium. Just remember: a favorable reaction doesn't necessarily go 100% to products

Substrate

The reactants that enter into an active site. They need to bind to an active site, and they bond non-covalently. Each enzyme is extremely specific re: which substrates will bind to its active site and then subsequently have a reaction catalyzed. Substrates are not just 'substrates' when they fit into the active site. Can be this and floating around

What do we mean by 'upstream' and 'downstream'?

The region preceding ("upstream" of) an ORF is called the 5' untranslated region (5' UTR) Typically, messenger RNAs are not composed of just this ORF - also have 'upstream' region from the AUG (starting point of ribosome for ORF), and a 'downstream' region (past the stop codon) The region following ('downstream' of) an ORF is called the 3' untranslated region (3' UTR)

Splicing

The removal of introns from a eukaryotic pre-mRNA is referred to as "splicing."

How does a ribosome move along an mRNA in order to add more amino acids to the polypeptide chain?

The ribosome, with its channel that holds the messenger RNA, is going to be moving towards the right, which means that in the context of the ribosome itself (if we imagine the ribosome just sort of sitting still) the messenger RNA is moving in the right to left direction, through the ribosome (toward 5' end), which means that any tRNA that comes in and base pairs to the codon in the messenger RNA, takes a path from right to left through the ribosome, so it enters the A site and there are proteins called 'elongation factors' that assist with this process Elongation factor carries the aminoacyl-tRNA into the A site. Once the peptidyl transferase activity has acted to make a peptide bond between that amino acid and the C terminus of whatever peptide was currently sitting in the P site as a peptidyl tRNA, that is going to cause the ribosome to translocate exactly 3 nucleotides over to the right, towards the 3' end of the messenger RNA, with the help of an elongation factor, and that's going to move the aminoacyl-tRNA into the P site, and the now uncharged tRNA that was previously holding the peptide into the E site

What do spliceosomes do?

The spliceosome cuts out the intron and splices together the ends of the flanking exons

Stages in gene expression?

The stages in gene expression are transcription (to copy a template strand of a gene to make messenger RNA), translation (messenger RNA translated to protein) signal → chromatin modification → transcription → RNA processing of primary transcript (w/ introns/exons) → mRNA in nucleus transported to cytoplasm → Translated → polypeptide processed into protein → transported to cellular destination

What is enzyme kinetics?

The study of the rates of enzyme-catalyzed reactions

What happens if the anti-codon of the charged tRNA does not match the codon in the messenger RNA?

The tRNA is rejected

What is a tRNA with an amino acid attached called?

The tRNA with amino acid covalently attached is referred to as a "charged tRNA" or "aa-tRNA" and is named according to the name of the specific amino acid and tRNA, e.g. "trp-tRNAtrp".

What nucleotide substrates go with RNAP? Are these processes energetically favorable?

The two nucleotide substrates for RNAP are NTPs. Two of the phosphates on each NTP are clipped off as it is added to the growing RNA chain Making a phosphodiester bond is an endergonic reaction (energetically unfavorable). Clipping the phosphate groups off of the nucleotide being added to the chain is exergonic (energetically favorable) and is what makes the process energetically feasible.

Compare/contrast strand separation in replication vs transcription

The two strands of DNA need to be separated from each other, so that one of them can be used as a template to make an RNA strand. Unlike DNA replication, where the goal is to do semiconservative replication, and make complimentary copies of both parental strands along the entire length of the chromosome, in transcription, the goal is only to make an RNA copy of a particular segment that corresponds to the coding information in a gene. So the transcription bubble is only a local region of melted apart strands of DNA, and it's not going to grow, like a replication bubble. Rather, it's going to move with the RNA polymerase as it moves across the template

How many steps are there in the expression of genetic information? Describe.

There are two steps in expression of genetic information. The first step is that an enzyme called RNA polymerase makes an RNA copy of one of the strands of the DNA where the gene is located, and then ribosomes use this RNA and translate the order of nucleotides in the RNA into an order of amino acids to make a protein. That copying by RNA polymerase is called transcription, and the process of the ribosome using information in the RNA to make a protein is called translation

Is there a limit to recombination frequency? Explain

There's a limit to recombination frequency! Because at a certain point, if recombination occurs in 100% of the tetrads - so every single tetrad undergoes crossing over - what that means is that at the end, if every single one does this, we have 25% that look like noncrossover gamete (a), crossover gamete (1), crossover gamete (2), noncrossover gamete (b). And that is going to look exactly like independent assortment. And there's no way to differentiate from those, just by looking at genotypes and phenotypes. So when you have two genes that are so, so far apart on the chromosome, even though they are on the same chromosome, they behave as if they are unlinked/independently asserting, just because there is so much crossing over happening. So there's a limit! You can't actually map two things that are more than 50 cM apart, because once they get past that 50% recombination - 50% recombination means independent assortment. (50 + 50 = 100)

How do you acquire recombinant DNA components?

These days, all recombinant DNA components/reagents can be custom designed and rapidly obtained from a commercial supplier, designed with different antibiotic resistant genes based on the bacterium that you're going to be putting them into, etc...

Describe the structure of a typical plasmid

These vectors typically have a polylinker region, which is a short region of typically between 50-100 bp (out of thousands total of the plasmid) that have one restriction recognition site after another, so gives you a lot of choices of where you could cut open this vector, and seal in some other fragment of DNA to make a recombinant plasmid that when you put it into bacteria will replicate and basically clone itself inside these bacteria and make many many copies of this recombinant plasmid

Describe some of the 'additional' experiments that AMM did. Why were these important?

They also did further experiments... Tested the elemental composition of the transforming substance, once they'd purified it even further from the fraction, and showed that its elemental composition resembled that of DNA Tested density of purified transforming substance, and showed that it had the same density of DNA Treated the original cell-free extract with a DNA digesting enzyme, and showed that that destroyed the transforming activity This combination of experiments really was what convinced the scientific community that at least in the case of bacteria, the genetic information that could be passed from one cell to another is contained in DNA

Can you provide an example of alternative splicing related to tropomyosin?

This is a gene that encodes tropomyosins. Look at all the different exons it has, and all the different alternatively spliced mature mRNAs it has. So this one gene is encoding a whole bunch of different Proteins encoded by this gene are members of the tropomyosin family - proteins that serve important functions in muscle contraction and in the cytoskeleton of many nonmuscle cells.

How do basal transcription factors find eukaryotic core promoters if they're relatively weak?

This is where the regulatory region, and regulatory transcription factors that are considered regulatory to modulate the level of basal transcription, come in Activator proteins bind to DNA sequences called "enhancers" and help the basal factors find the promoter, with the help of a bridging protein complex called the "mediator" Some enhancers are quite close to the promoter ("promoter proximal") Others may be quite distant from the promoter (and even downstream)

Why was PCR a revolution in forensics?

This meant you could quickly amplify a fragment of interest (rather than growing up huge quantities of bacteria and doing plasmid preps). You could label an amplified fragment from one organism and use it as a probe to detect a clone of a related gene in another organism (through Southern blotting). Then you could grow up lots of that clone to isolate enough DNA to sequence that gene using the old-fashioned approach. PCR can also be used to detect specific mRNA molecules in samples, by preceding the amplification step by a reaction that uses reverse transcriptase to make a complementary DNA strand to the mRNA (hence the name "RT-PCR" for the technique.)

How do the poles move apart in anaphase?

This motion happens because of enzymes that clip the proteins that are holding the sister chromatids to each other. The microtubules can now pull them towards the respective poles

How do you determine Km from a Michaelis-Menten curve?

To figure out the Km, you have to know the Vmax, then figure out ½ Vmax. Then read across, and look down to the x-axis value. Km is always a substrate concentration value, while Vmax has units of reactions/unit time Km is the value on the x-axis that corresponds

What's the function of transmembrane proteins?

To move large, charged molecules into the cell

What does circadian rhythms have to do with gene regulation? Why is this relevant to our current unit?

Today, we're focusing on what kinds of things have to happen at the gene in order to change its expression Example: light can cause changes in certain genes, and the products of those genes can inhibit or activate the expression of other genes, which can go back and inhibit the initial genes... this is how circadian rhythms are regulated. DO NOT need to memorize this diagram "Elucidating regulation of circadian clock genes" In order to be able to figure this out, the scientists needed to be able to detect what messenger RNAs were present in the cell at any given moment... using technologies below!

Transporters

Transport proteins that move things in and out of the cell

What's the structure of eukaryotic protein coding genes?

Typical eukaryotic protein coding genes have this really weird structure to them where their coding information is actually interspersed with segments of non coding DNA Exons are the coding sequence, introns are non-coding information

Allosteric enzymes

Typically have more than one subunit (aka quaternary structure) and the binding of substrate to one active site influences the substrate affinity of active sites in other subunits, and that's why the S shape of graph is caused

What is the end replication problem and how do eukaryotes deal with it?

Unlike bacterial chromosomes, the chromosomes of eukaryotes are linear (rod-shaped), meaning that they have ends. These ends pose a problem for DNA replication. The DNA at the very end of the chromosome cannot be fully copied in each round of replication, resulting in a slow, gradual shortening of the chromosome. Why is this the case? When DNA is being copied, one of the two new strands of DNA at a replication fork is made continuously and is called the leading strand. The other strand is produced in many small pieces called Okazaki fragments, each of which begins with its own RNA primer, and is known as the lagging strand. (See the article on DNA replication for more details.) In most cases, the primers of the Okazaki fragments can be easily replaced with DNA and the fragments connected to form an unbroken strand. When the replication fork reaches the end of the chromosome, however, there is (in many species, including humans) a short stretch of DNA that does not get covered by an Okazaki fragment—essentially, there's no way to get the fragment started because the primer would fall beyond the chromosome end^11start superscript, 1, end superscript. Also, the primer of the last Okazaki fragment that does get made can't be replaced with DNA like other primers. [Why is that the case?] Thanks to these problems, part of the DNA at the end of a eukaryotic chromosome goes uncopied in each round of replication, leaving a single-stranded overhang. Over multiple rounds of cell division, the chromosome will get shorter and shorter as this process repeats. Solution: telomeres!

What is PCR?

Uses thermostable polymerases Forensic applications Detect mRNA (RT-PCR) Quantitative measurement of genes or mRNA (qPCR; also known as "real-time PCR") (how qPCR data is analyzed)

Summarize the 3 prevalent models for DNA replication that Watson and Crick proposed

Watson and Crick led to 3 prevalent models for DNA replication in the late 1950s... (all very abstract models, not referencing kinds of enzymes, etc - just thinking about how could you go from parent DNA duplex to two daughter duplexes that are identical to the parent, in terms of DNA base pair sequence) (in these diagrams, white = original DNA, and after replication the white are the parts of the original DNA that are still existing, and anything pink is newly synthesized DNA) 1. The semi conservative model This is basically what Watson and Crick alluded to - two parental strands would be separated, each one would serve as a template for the other. Called 'semi-conservative model' because each daughter DNA duplex conserves one of the two strands of the parental DNA, and has another strand that has been newly synthesized in the process of replicating the DNA 2. The conservative model Proposed that DNA is actually replicated by... somehow, the parental DNA is completely conserved, even though its sequence of base pairs has somehow been used to direct the sequence of base pairs of another duplex of DNA that is entirely newly synthesized 3. The dispersive model Proposed that after replication, both of the daughter DNA duplexes contain some of the parental DNA, but that parental DNA is dispersed between the two strands of both of the daughter DNA duplexes

How is a "pre-mRNA" turned into a "mature" mRNA?

We've already talked about how RNA polymerase does transcription. It needs to recognize a promoter, it creates a bubble, it moves along synthesizing RNA, the 5' end of the RNA is sticking out of the transcription bubble as it goes along, and eventually, some sort of signal tells it to stop transcribing. So then we've made a pre-mRNA But, it turns out... The typical eukaryotic protein-coding gene has coding sequence ("Exon's") interspersed with no coding DNA ("introns")

What is a 'null hypothesis'? Why do we care?

What is being claimed or assumed. Statistical test designed to assess strength of evidence against null hypothesis. No real difference between the measured values (observed) and the predicted values (expected) We care because an acceptance of the null hypothesis means that any apparent difference can be attributed purely to chance. Ex: Mendel's 3:1 ratio. The variations in 3:1 were due to the small amount of chance (which had variable effects in small sample size)

What type of bonding is occurring between RNAP and a promoter? What do these bonds lead to?

When we say that RNAP "binds" to a promoter, we mean that noncovalent interactions between R-groups on the enzyme and chemical groups in the groove cause the enzyme to poise itself at a particular position on the DNA molecule. Those interactions in turn lead to a conformational change in the DNA that melts open a small region (~15-20 bp) of the DNA to form the "transcription bubble." This reveals single-stranded RNA that RNAP can use as a template to make a complementary RNA chain. The interactions with the promoter orient the RNA polymerase so that it will move from the promoter toward the terminator as it creates the RNA.

Describe the differences in the 'initiation' phase of transcription vs replication

When we talked about DNA replication, we talked about initiator proteins that recognize the origin of replication, and recruit helicases to peel apart the DNA and create a replication bubble. RNA polymerase does that itself, and doesn't need accessory proteins to do that

DNA ligase

Will act once that enzyme has come in and removed the RNA at the beginning of the next Okazaki fragment, and a separate DNA polymerase enzyme has come in and extended the DNA right up to the beginning of the next fragment. Then DNA ligase will act

Can you tell which genes are farther apart from each other just by looking at percentage recombination?

Yes! That's like the whole pointl Ex: You can ascertain that the genes in cross B are way farther apart than those in cross A, just because the recombinant rate is so much higher

Can an enzyme have more than one substrate?

Yes, an enzyme can have more than one substrate

Why are concentrations so important in Sanger sequencing?

You figure out what relative concentrations of the dideoxy and the regular nucleotides you need in each of your 4 tubes so that among the many DNA polymerases that are busy making DNA chains through random grabbing of dideoxy instead of regular one, that all possible positions at which an A in the first tube needed to be added get represented in the prematurely terminated DNA products

What's the setup for Sanger sequencing? How does it work? (long version)

You set up 4 different test tubes In each of the 4 test tubes, you put all 4 of the regular dDNTPs DNA polymerase will go about its business making DNA from this primer (and by the way, you radioactively label your primer, using kinase and radioactive phosphorus), and into each of your 4 test tubes, you put 1 of the 4 possible dideoxynucleotides So the first tube has all of the regular nucleotides, but it also has some ddATP in it, along with many copies of the template sequence, many copies of the primer, many copies of DNAP. So all busy, trying to extend these primers. But every so often, when they reach a position when they need to add an 'a' as the next nucleotide, they grab onto a dideoxy a instead of a datp. So now, when that happens, that chain, that the DNAP was making, is basically terminated, because it does not have a 3' hydroxyl that DNAP can add to

How did XRAY film (used to) factor into DNA imaging?

You used to detect radioactive DNA by using X-RAY film. Literally take gel, go into a dark room, put a piece of X-RAY film on the surface of the gel, and leave it in the dark room for awhile, and the radioactive emissions would expose the film. So you'd see black spots on the film wherever there was DNA!

Describe how oxygen moves into cells via diffusion

Your cells use oxygen to produce energy. The byproduct is carbon dioxide. As your cell uses more oxygen, it lowers the concentration of oxygen inside the cell. So oxygen outside the cell will want to flow from outside, to inside

What does the E site do? What does the E stand for?

eject/exit the exit site, where discharged tRNAs leave the ribosome

Gene flow vs genetic drift vs natural selection?

https://biologydictionary.net/genetic-drift-vs-gene-flow-vs-natural-selection/ GOODNIGHT

Which types of RNA are involved in protein synthesis?

mRNA, tRNA, rRNA

What's the typical lifespan of an mRNA? Why do we care?

mRNAs have short lifetimes so that the cell can easily change what proteins are being produced by "turning on" or "turning off" the production of mRNA from individual genes It's important to note that mRNAs have short lifetimes. That's one of the reasons why we evolved to have RNA as our messenger molecule, but DNA as our genetic information storage molecule. One of the things we didn't talk about when we first discussed the chemical properties of RNA vs DNA is that the existence of that extra hydroxyl group on each sugar of an RNA molecule makes it chemically more prone to degradation (there are enzymes that take advantage of that extra hydroxyl and catalyze the reaction that breaks down RNA. Even without those enzymes, though, RNA, at least in a lab, is way more labile than DNA) But this short life span is fine, for something that's a messenger! You don't need it to stick around permanently, like DNA. In fact, it can be really useful that it doesn't hang forever, because if you want to stop expressing a gene, you can count on its mRNA being gone after a certain amount of time, and you can turn on new genes, by starting to transcribe them If every messenger RNA that you ever transcribed just hung around forever, then it would be more difficult to switch the profile of what proteins are being expressed

Can you summarize the process of dyenin walking on microtubules?

motor protein binds to ATP --> puts its foot down --> hydrolyzed, causes one of the feet to pick up and move closer --> releases ADP and phosphate --> causes one foot to be lifted up, binds with ATP and moves on

rRNA

ribosomal RNA a structural component of the ribosome. They are the same in every ribosome. All ribosomes are identical to each other. Any ribosome can synthesize any protein, as long as it gets a hold fo a messenger RNA that encodes that protein. So the ribosomal RNAs are components of the ribosome In fact, there are exceptions to the 'enzymes are proteins' rule. Some RNA molecules have enzymatic activity, and it turns out that the enzymatic activity in a ribosome is actually part of the RNA components of the ribosome, and not the polypeptide portions of it. These portions actually perform more of an architectural or structural role. They're kind of holding the RNA that are doing the catalysis in the right conformations to do their job. So that's an interesting bit of info about this ribosomal RNA Ribosomal RNAs have names to them like 16S rRNAA, 5S rRNA The S is a weird unit that has to do with how quickly the molecule sediments in a centrifuge (lol what) no need to know this, just explanation of crazy names. Eukaryotic RNAs are slightly larger, so names like 23s, 25s

Okazaki fragment

short segment of DNA synthesized discontinuously in small segments in the 5' to 3' direction by DNA polymerase

What is tRNA? What's the structure of tRNA? How/why?

tRNA are this third class of RNA. There are genes for tRNAs in the nucleus as well. tRNAs in general have sequences within them that will base pair internally to form a secondary structure that looks like a clover leaf (3 stem loop structures and a stem where the 5' and 3' ends of the RNA are). Within one of the loops is a sequence of 3 nucleotides that is going to be complementary to a codon in the messenger RNA Not only does the tRNA fold into this secondary structure! It actually folds into a particular L-shaped tertiary structure, where one end of it has that anti-codon, and the other end is where an amino acid will get attached with a high-energy covalent bond linking amino acid to the tRNA (meaning that this bond is energetically favorable to hydrolyze. Hydrolyzing it will release enough energy to allow a peptide bond to be made between this amino acid and another amino acid in a coupling like we've seen before, where an energetically favorable reaction is used to drive an energetically unfavorable reaction). So now, it's really important that only the correct amino acid that corresponds with the codon that this tRNA's anti-codon is going to be able to bind to... we've got to make sure that only that amino acid can ever get attached to this tRNA.

G1

· "Gap 1" · First growth stage of interphase Cell grows to nearly its full size, and performs many of its specific biochemical functions that aid the organism

G2

· "Gap 2" · Final growth stage of the cell · Cell finishes growing and prepares to divide. Once the cell has finished duplicating the nucleus, and two centrosomes have appeared in the cytoplasm, mitosis can begin. For a typical eukaryotic cell, this mitotic phase will last about 80 minutes

S

· "Synthesis" Second part of interphase This is an important phase because it is during the S phase that DNA in the nucleus is replicated

What does a linkage ratio tell us, exactly?

· When we're looking at these extensions of Mendelian inheritance - these different patterns - a linkage ratio is basically saying that we have these two traits and they are completely linked. No crossing over is happening with them. So we get more than just the parental types, but we have this complete linkage here, and we're going away from that 9:3:3:1 ratio (1:2:1 for complete linkage)

Discuss the synthesis of each side of a replicon

½ of one side of each replicon was made continuously, because it's leading strands, as that replication bubble was growing. That other strand was made of all these Okazaki fragments

Replication bubble

Segment of a DNA molecule that is unwinding and undergoing replication.

How do we determine the exact sequence?

Sequencing tech

What is the replisome?

complex of multiple proteins involved in replication

Hardy-Weinberg equilibrium (what is it, what does it do, etc)

condition that occurs when the frequency of alleles in a particular gene pool remain constant over time Looks at a population, and says what's going on with the frequency of the alleles in this population? Predicts genotype frequencies So what HW does: basically we can look at the different alleles, and use those different alleles (the frequency, or how common an allele is in a population), to predict the genotypes, and then use the genotypes to predict the phenotypes, of a particular gene If we are assuming that we are looking at a population that isn't changing, we would say that that population is in HW equilibrium

Telomere

Repetitive DNA sequence at the end of a eukaryotic chromosome

Finish this sentence: Within the active site, catalysis occurs by...

...Encouraging formation of the transition state!

Restriction site

A specific sequence on a DNA strand that is recognized as a cut site by a restriction enzyme

What type of knockouts can tell you about function?

Conditional or tissue specific knock-outs can give more specific info about function

How does PCR work?

Dna is heated to separate the strands. Primers are added, polymerase builds new strands. TWO COPIES OF DNA.

How do chromosomes exist in diploid organisms?

Homologous pairs One member of each pair of chromosomes came from dad, one came from mom

How does transcription by eukaryotic RNAP II compare to prokaryotic RNAP? (Initiation and termination)

- Different mechanism of promoter recognition (no sigma factor!) - Mechanism of transcription termination by RNAP II is not well understood (but is not like either of the two mechanisms used by prokaryotic RNAP)

What is common to all cells?

- Plasma membrane that defines the difference between interior of cell and external environment - Cytoplasm (the cytosol is the aqueous liquid within the cytoplasm) - DNA - Ribosomes (might be considered organelles, but are not membrane bound)

Contractile ring

A thin band of actin and myosin filaments that wraps around the midsection of an animal cell undergoing cytoplasmic division. It contracts and pinches the cytoplasm in two.

Who were the first scientists to study enzyme-catalyzed reactions in a specific way?

Leonor Michaelis and Maud Menten

Sister chromatids

Replicated forms of a chromosome joined together by the centromere and eventually separated during mitosis

Cistron is another word for...

"Cistron" is another name for "gene."

3 ways in which photosynthesis is the reverse of respiration

(1) Starts by oxidizing H2O to O2 while reducing NADP+ to NADPH. Remember that ETC in mitochondria oxidizes NADH to NAD+ while reducing O2 to H2O Yet, both processes manage to drive ATP synthesis (2) The ATP and NADPH made in chloroplasts are used right in the chloroplast - ATP as an energy source and NADPH as reducing power - in pathways that reduce CO2 to make glucose molecules and polymerize them to make starch (endergonic, building up processes) Contrast to respiration, which oxidizes glucose completely to CO2 (exergonic) (3) In photosynthesis, CO2 is an input and O2 is a waste product Vs respiration, where O2 is an input and CO2 is a waste product

Why is the fidelity of RNAP lower than that of DNAP? Why is this okay in RNAP and not DNAP?

"Fidelity" is lower than that of DNA replication due to decreased proofreading ability compared to DNA polymerase This is okay because... RNA polymerase does not really have this ability (has slight proofreading capabilities, but its nowhere near as good as DNA polymerase). So, RNA polymerase occasionally does make mistakes (more often than DNA polymerase does) If RNA polymerase makes a mistake, yeah it might make a change in a codon that's a silent or missense mistake... but genes are transcribed many times by RNA polymerase. So there are many transcripts of that gene floating around, and probably only one of them has that mistake, and the others are all fine. And there are quality control systems in cells that can get rid of proteins that are grossly misfolded, etc, because they had a wrong amino acid. And proteins themselves don't hang around forever! So having an occasional mistake, and making an occasional bad protein, when there are lots of good copies of RNA floating around, is not as much of an issue as if DNA polymerase that's replicating the DNA to pass on to daughter cells is making a change that will become a permanent change in the genetic code for the next generation. So that's probably why DNA polymerase evolved to have that proofreading ability! Probably gave selective advantage to organisms who were not making as many mistakes in passing on their genetic code from one generation to the next. RNA polymerase would not have that same selective pressure to it

Why are G1 and G2 called 'gap' phases?

"G1" and "G2" stand for "gaps" between "M" and "S" phases — not much activity is visible in the microscope during these times, but much is happening!

Nuclear pores

"Gatekeeper": only lets proteins earmarked for the nucleus in Quality control: checks mRNA and makes sure they folded correctly

What's the punchline (main purpose) of next-gen sequencing?

"Next-Gen" Sequencing eliminates electrophoresis; allows us to detect each nucleotide as it's added

What type of nucleotides does Sanger sequencing use?

"Sanger" sequencing uses dideoxynucleotide chain terminators

How could we figure out % crossover tetrads?

% crossover tetrads is twice the percentage of recombinant gametes produced

Eukaryotes

- Membrane bound nucleus and organelles -Vesicles (cargo transport) -DNA stored in nucleus (packaged into linear structure called "chromatin" (vs. prokaryotes - stored in one big circular chromosome, no free ends)

Is electrophoresis necessary with next-gen sequencing? Why or why not?

- No electrophoresis is necessary - nucleotide addition is detected by flashes of light detected by a computer

Are reactions automated or monitored by humans with next-gen sequencing?

- Reactions are fully automated and milliions of reactions are done simultaneously

Why aren't primers needed with next-gen sequencing?

- Specific primers are not needed, because "linkers" are ligated to the ends of genomic DNA fragments; then the only primer needed is one that is complementary to the common linker that has been attached to all the fragments!

Why is it important that microtubules act as rails or tracks through the cytoplasm?

-Highway for transport of what the cell needs -Microtubule motor: a dyenin motor protein, powered by ATP, moving along a microtubule track (binding and hydrolysis of ATP each cause conformation changes in the motor protein, allowing it to "walk" along the MT track) -dyenin - towards minus end (nucleus) -kinesins - movement towards plus end -ex neurotransmitter movement

4 steps: Screen a library for specific inserts of interest by hybridization

1. Colonies of plasmid-containing bacteria, each from a clone from the clone library, are grown on agar. 2. A replica of the plate is made by pressing a filter against the colonies. Some cells from each colony adhere to the filter. 3. The filter is washed with a solution that denatures the DNA and contains the radioactively labeled probe. The probe contains nucleotide sequences complementary to the gene of interest and binds to cells containing the gene. 4. Only those colonies containing the gene will retain the probe and emit radioactivity on film placed over the filter. 5. A comparison with the original plate identifies the colony containing the gene. Voiceover: You can use a similar technique... similar to the southern blot, because it uses nitrocellulose filter paper, but now you just stick the piece of paper right onto the Petri dish, and it has bacteria on it in the same pattern as what was on the plate, and then you treat that paper in ways that break open the bacteria and denature the DNA in them, while keeping them in the place where they originally were on the filter, so you can probe it, and find a radioactive spot, so you then you know which of the original clones had the DNA that you're interested in

How will peptide bonds be formed? 3 steps (brief!!/describe this graphic)

1. Condensation reaction to form peptide bond is unfavorable 2. Hydrolysis of high energy bond is favorable 3. Next peptide bond will be made between new C terminus and next aa-tRNA *** Here's a messenger RNA (there's no ribosome, because that makes the image too busy, but envision that). On the left, there's an AUG start codon, that's positioned in the P site of a fully assembled ribosome. It's got that initiator tRNA that recognizes the AUG start codon, and it's got a methionine amino acid covalently attached to it by its carboxyl group in a high energy bond. Even though there's a lot of space drawn between the two nucleotide groups, the group to the right is the next 3 nucleotides (had to separate them to fit the structure, but use your imagination!). The one on the right is a triptophanyl tRNA recognizing a UGG codon, like we just talked about a couple of minutes ago. It's sitting in the A site of the ribosome. If these amino acids were just floating around in solution, the condensation reaction between this amino group and this carboxyl group would be unfavorable, but the bond between the amino acid and the tRNA sitting in the p site is going to get hydrolyzed, and concurrently, the amino acid carboxyl group (rather than being covalently attached to the tRNA), is going to get transferred onto the amino group in a covalent peptide bond. So peptidyl transferase comes in, and the result of that is that the dipeptide is now attached to the second tRNA (the one on the right), which is now called a peptidyl tRNA. Now we have a dipeptide, attached to the tRNA by the carboxyl group of the second amino acid. The ribosome is now going to move three nucleotides along the messenger RNA, so that this tRNA carrying its dipeptide will be sitting in the P site, and another aminoacyl-tRNA will be able to come into the A site where the next codon is shown. The former first tRNA, which no longer has an amino acid attached to it, will be in the E site, and will be ejected from the ribosome. The next peptide bond will be made between the carbon of the carboxyl group attached to the tRNA and the next aa-tRNA

What are the 3 main (and one intermediate) stages in cellular respiration, aka the aerobic catabolism of glucose?

1. Glycolysis pathway 2. [linking reaction] - this is because the input for the citric acid cycle is not exactly the end product of glycolysis 3. Citric acid cycle 4. Oxidative phosphorylation

Cellular respiration (major stages)

1. Glycolysis. Turns glucose into pyruvate, takes place in the cytoplasm; all subsequent stages in mitochondrion 2. [linking reaction]. Single reaction turning pyruvate into acetate in the form of acetyl-CoA 3. Citric acid cycle. Finishes oxidizing acetate to CO2 4. Oxidative phosphorylation. Electron transport chain, ATP synthase

4 categories of membrane proteins How are they bound to the membrane?

1. Linkers 2. Transporters 3. Receptors 4. Enzymes

Name 2 technological offshoots of understanding DNA polymerase

1. PCR 2. DNA sequencing

What is the secretory pathway of the Golgi?

1. Proteins deposited into the ER lumen 2. Certain modifications can happen in ER lumen 3. Transport vesicle moves proteins to golgi cis face 4. Certain modifications can happen in golgi as vesicle moves from cis towards trans face 5. Golgi trans face sorts proteins into vesicles destined for a) plasma membrane (fusion with membrane can be regulated or constitutive) or b) lysosome (to function there as membrane transporters, H+ pumps, or acid hydrolysis)

What are the steps of factor-independent transcription termination? Describe

1. RNAP "pauses" at the termination signal. RNA within termination region contains 2 segments of complementary sequence followed by a run of U's (on the newly-synthesized RNA) 2. RNA folds into base paired hairpin structure (something about the pause gives it the opportunity for this to happen) 3. Disassembly (RNA released, DNA duplex reforms, and RNAP released) (formation of hairpin structure has an allosteric effect on RNAP) In factor independent termination, there is a hairpin structure in the RNA involved, and that its interaction with RNA polymerase leads to disassembly of the transcription complex

Brief overview of the process of DNA replication

1. Strand separation: DNA strands separate when hydrogen bonds between complementary base pairs are broken 2. Base pairing: Each strand of DNA can serve as a template for the formation of a new strand. Free nucleotides attach to 3n ends according to complementary base pairing 3. Polymerization: When the new strands polymerize to form a sugar-phosphate backbone, secondary structure is restored Conclusion: the original molecule has been copied. Each copy has one strand from the original DNA molecule and one new strand

How do we know that mitochondria is descended from prokaryotes?

1. circular DNA inside 2. Own ribosomes inside 3. Divides by fission

How many protofilaments do microtubules have? How are they arranged?

13 protofilaments 9 + 0 arrangment: 9 groups of 3 microtubules, nothing in the middle The 13 protofilaments make up the individual microtubule, the microtubles are then paired to create the outer doublet microtublues, then the 9 doublet microtubules will be arranged in a circle to create the centromere. One way to think about it is using the example of a bundle of bamboo, where each bamboo stalk is one microtubule (made of the 13 protofilaments), and the entire bundle together is what would make up the centromere or the Cilia.

Avery-MacLeod-McCarty experiment

1944 Main point: DNA is the "transforming factor" described in the Griffith experiment Summary: Oswald Avery and colleagues expanded upon the findings of Frederick Griffith to demonstrate that DNA is the genetic material. They prepared cultures containing the heat-killed S strain and then removed lipids and carbohydrates from the solution. Next, they treated the solutions with different digestive enzymes (DNase, RNase or protease) to destroy the targeted compound. Finally, they introduced living R strain cells to the culture to see which cultures would develop transformed S strain bacteria. Only in the culture treated with DNase did the S strain bacteria fail to grow (i.e. no DNA = no transformation). This indicated that DNA was the genetic component that was being transferred between cells. Despite this finding, the scientific community was reluctant to accept the role of DNA as a genetic material. It was only 8 years later, when Hershey and Chase conducted their experiment, that the concept gained traction.

Hershey & Chase experiment

1952 Main point: Confirmed that DNA is the genetic material. Summary: Hershey and Chase used T2 phage, a bacteriophage. The phage infects a bacterium by attaching to it and injecting its genetic material into it. They labeled the phage DNA with radioactive Phosphorus-32. They then followed the phages while they infected E. coli. They found that the radioactive element left on the phage's DNA was only in the bacterium, and not in the phage, meaning that the DNA had entered the bacterium. In a second experiment, Hershey and Chase put labels on the phage protein with radioactive Sulfur-35. After the phage was attached to the bacterium, the radioactive element was found in the phage, but not in the bacterium. This means that the phage's proteins stayed on the outside of the bacterium. These results showed that the genetic material that infects the bacteria is DNA. S radioactivity found only in the medium, P radioactivity found only inside the bacterial cells This experiment is often referred to as the "Waring Blender" experiment! This established that yeah, we knew this was true for bacteria (from Griffith's experiments, and Avery et al), and now we also know that bacteriophages use DNA as their genetic material

Messelson-Stahl Experiment

1957 Main point: supported the semi-conservative model Summary: Meselson and Stahl tested the hypothesis of DNA replication. They cultured bacteria in a 15N medium (heavier isotope) and then shifted the bacteria to a 14N medium (lighter isotope). After one replication cycle, the DNA was all of intermediate density, which ruled out the conservative replication model. After two replication cycles, two bands of DNA were seen, one of intermediate density and one of light density. This result rules out the dispersive replication model, which predicted that after replication cycle 1, the DNA density of all DNA molecules will gradually become lower, so no intermediate density DNA should remain at replication cycle 2. One the other hand, this result is exactly what the semiconservative model predicted: half should be 15N-14N intermediate density DNA and half should be 14N-14N light density DNA. Therefore, the experiment supported the semiconservative model is correct. Result: two different densities observed after two generations, so they knew that semiconservative model was correct

How many ATPs are expended for the attachment of an amino acid to tRNA? How many GTPs expended during elongation for each amino acid added to the chain?

2 ATPs expended for attachment of an amino acid to a tRNA, 2 GTPs expended during elongation for each amino acid added to chain

Describe the structure of mitochondria

2 membranes (phospholipid bilayer, called the inner and outer membranes) Intermembrane space Inner membrane has large surface area, to allow it to hold more proteins to aid respiratory processes Outer membrane has large pores, inner membrane has proteins that carry out final stage of aerobic metabolism and act as a small molecule filter Folds in the inner membrane (crista/cristae) Space inside of inner membrane = mitochondrial matrix

What are the 4 special codons? Why are they special?

4 particular codons that serve very important purposes in terms of the process of actually reading the message Start codon: AUG, codes for methionine · Ribosome needs to recognize this as its place to start reading codons · When first synthesized, every single protein has methionine at the beginning of its chain Proteins are synthesized starting at their N terminus, working towards their C terminus. So the N terminal amino acid is always encoded by AUG and is therefore methionine 3 stop codons: UAA, UAG, UGA · Do not code for any amino acid, but signal for the ribosome that this is the end of the info for this protein and you should stop synthesizing the polypeptide chain

How many possible codons are there? How do you arrive at this number?

64 4^3 This table is showing us that the order of nucleotides in a messenger RNA is read in three letter words, going along the messenger RNA from somewhere near its 5' end and moving towards its 3' end, reading 3 nucleotides at a time, and considering that there are 4 possible nucleotides, and thinking of how many different 3 letter words you can make... 4^3 gives you 64 different codons Table shows all 64 possible codons! Tables like this are always organized in a particular kind of way, where reading down a column the first letter of each of the codons goes through all U's, all C's, all A's, all G's, and then going across a row, all of the second letters are all the U's, all the C's, all the A's, all the G's... there are other patterns too!

Southern blot (steps)

A DNA sample is electrophoresed on a gel and then transferred to a filter. The filter is then soaked in a denaturant and subsequently exposed to a labeled DNA probe that recognizes and anneals to its complementary strand. The resulting ds labeled piece of DNA is visualized when the filter is exposed to film. Voiceover: You take your gel (in which DNA has already been denatured), and put it on a filter paper, and a sponge to soak up the water. The DNA moves with the water, and then the DNA gets stuck on the filter paper. Then you can bake it onto the filter paper, so now you have this filter paper with the same pattern of DNA, except now it's been immobilized. You immerse the paper into a solution that has your radioactive denatured probe in it, and you let it sit for awhile, and this is when that hybridization occurs! That probe base pairs onto the paper wherever there's an immobilized piece of DNA complimentary to it. Then, you use an X-RAY film or a phosphoimager to view the results

What is the TATA box?

A DNA sequence in eukaryotic promoters crucial in forming the transcription initiation complex (where RNAP II will bind)

T/F: a new GTP is hyrolyzed every time a new aa-tRNA enters the A site, and every time a translocation occurs

A GTP is hydrolyzed every time a new aa-tRNA enters the A site, and every time translocation occurs; these steps require additional proteins called elongation factors (EF-Tu and EF-G).

Replication fork

A Y-shaped region on a replicating DNA molecule where new strands are growing

Restriction endonuclease

A bacterial enzyme that recognizes a specific DNA nucleotide sequence and that cuts the double helix at a specific site within the sequence

Endergonic

A chemical reaction that requires the input of energy in order to proceed (positive delta G)

What is the end replication problem?

A problem completing synthesis of the lagging strand at the end of a linear chromosome Here, we're assuming that we've reached the ends of these linear chromosomes. Remember that the dark grey is parental DNA, and the lighter grey is newly made DNA. One strand was made continuously in the 5' to 3' direction, on a leading strand, and it was synthesized right up to the end of the chromosome, but the other one was made via Okazaki fragments, each one starting with an RNA primer. The enzyme that degrades the RNA primer comes along, and removes that primer, but there is no way for DNA to get filled in there! Because DNA polymerase always needs a primer! So we end up with a little area of single stranded DNA, that we can refer to as the 3' overhang, at the end. And if we imagine forward to the next generation... after mitosis, one set of DNA goes to one daughter cell, the other DNA goes to another daughter cell. But then, when each of these chromosomes gets to be replicated the next time around, both of these strands are parental DNA at the next generation, and one of the two parental strands is now lacking something at one of its strands, because of the overhang! In fact, the top strand (bottom set in graphic) will be a leading strand template in the next generation, and it will only copy up to the overhang. So that chromosome is now going to be automatically shorter, and at each subsequent generation, the chromosomes will get shorter at their ends, unless there is a way to get over the end replication problem at linear chromosomes

Chemiosmosis

A process for synthesizing ATP using the energy of an electrochemical gradient and the ATP synthase enzyme

Chemiosmosis aka chemiosmotic synthesis of ATP

A process for synthesizing ATP using the energy of an electrochemical gradient and the ATP synthase enzyme Occurs in the mitochondrion. Electron transport chain (the thing that takes the reducing power of FADH2 and NADH and puts the hydrogens onto oxygens to make water) releases a lot of energy, captures a lot of energy in the form of a proton gradient, across the inner membrane of a mitochondrion Basically, ATP synthase sits in the inner membrane of the mitochondria, and allows protons to flow through it, which is an energetically favorable process because they're traveling towards their area of lower concentration In this process, causes another part of ATP synthase molecule (which has the active sites for making ATP) to drive the synthesis of ATP. Also an enzymatic reaction process, but the energy here will come from a proton concentration gradient

What is G0? Can you give an example of a cell type that spends a lot of time here?

A process where cells are unlikely to divide, but still carry out their normal functions Ex: neurons

Motor protein

A protein that causes sliding along two actin strands, involved in muscle contraction

What is a primer? Why do we care?

A short segment of DNA that acts as the starting point for a new strand A primer is a short nucleic acid sequence that provides a starting point for DNA synthesis. In living organisms, primers are short strands of RNA. A primer must be synthesized by an enzyme called primase, which is a type of RNA polymerase, before DNA replication can occur. The synthesis of a primer is necessary because the enzymes that synthesize DNA, which are called DNA polymerases, can only attach new DNA nucleotides to an existing strand of nucleotides. The primer therefore serves to prime and lay a foundation for DNA synthesis. The primers are removed before DNA replication is complete, and the gaps in the sequence are filled in with DNA by DNA polymerases. Free 3' end on a growing strand This is really important! DNA polymerase can't start DNA strand de novo. Aka, can't start one from scratch. So DNA polymerase can't take two individual nucleotides and join them together, and make a dinucleotide, even if it has a template telling it what to join together. It needs to be adding to the 3' end of an existing primer. So it can add nucleotides to a growing chain, but it can't start from scratch

Aster fibers

A star-shaped structure formed in the cytoplasm of a cell and having ray like fibers that surround the centrioles during mitosis

Glycocalyx

A sugar halo that acts as a distinctive covering of the cell and is composed of oligosaccharides linked to membrane components. Creates an additional barrier outside of the membrane itself. Also provides defense to protect membrane, so cells do not accidentally merge with other cells. Finally, can also serve as identifying features for the cell.

Citric acid cycle (general description)

ALSO CALLED THE KREBS CYCLE. Third step in cellular respiration, aka the aerobic catabolism of glucose. Finishes oxidizing acetate to CO2. Occurs in mitochondrial matrix. Makes citrate, does a series of changes to that citrate that eventually removes some carbons from it in the form of CO2, so it ends up as a 4 carbon molecule that through a few more steps gets turned back into that four carbon molecule called oxaloacetate that is able to take on another acetyl group from acetyl CoA Goes in cycles - in each cycle, a couple of carbon dioxide molecules come off, a few NADHs are made, an FADH2 is made, and there's even one step that is a substrate-level phosphorylation that makes GTP/ATP (energetically equivalent)

What process is ATP made by ATP synthase produced by?

ATP made by ATP synthase is made by chemiosmosis

Substrate-level phosphorylation

ATP made via an enzyme reaction using the high energy phosphate bond a substrate to drive the formation of ATP (i.e. the delta G for the hydrolysis of the phosphorylated substrate is more negative than the positive delta G of adding a third phosphate onto ADP) Enzyme catalyzes a reaction that transfers a phosphate group from an intermediate along a pathway that has a phosphate group attached to it in a high-energy bond (so, favorable to hydrolyze —> delta G of clipping phosphate off in this hydrolysis process is more negative than the positive delta g of putting phosphate onto ADP, so the overall process is delta G negative) Literally, phosphate that gets put onto ADP to make ATP comes off of a substrate molecule that is acted upon in an enzyme catalyzed reaction

When is ATP used in translation? How about GTP?

ATP used to attach amino acid to tRNA GTP used during elongation, for each amino acid added to chain

How is signal transduction different than receptor mediated endocytosis?

Adrenaline hormone binding to a receptor on a cell, and triggering the transduction of a signal into that cell, that results in a change in gene expression. This is different than receptor mediated endocytosis. Here, a receptor on the cell's surface gets a signal - a ligand - bound to it, and then it doesn't get endocytosed into the cell, but rather it interacts with other proteins, either that are present in the membrane, or other proteins inside the cell, and causes changes in them, and like a domino effect, each change can lead to a subsequent change, and ultimately cause a change in gene expression

How many ATP are produced per molecule of glucose (aerobic catabolism)?

Aerobic catabolism of glucose (cellular respiration): leads to its complete oxidation to CO2 (as in combustion) and releases lots of energy - enough o store away ~30 molecules of ATP per molecule of glucose!

Give an overview of the steps of glycolysis in aerobic vs anaerobic conditions.

Aerobic conditions (respiration): glucose —> pyruvate —> acetyl CoA —> CO2 Anaerobic conditions (fermentation): glucose —> pyruvate —> lactate Instead of being brought into the mitochondrion to undergo linking reaction and TCA cycle, pyruvate (in human bodies) is turned into lactate

Broadly speaking, variables that impact an enzyme's native structure impact its activity by influencing what?

Affinity, turnover number, or both

When are helicase recruited? What do they do?

After replication initiation factors recognize origins of replication in the DNA. Helicase use energy from ATP hydrolysis to peel apart the two strands of DNA at these origins of replication. These origins of replication are usually pretty AT rich. The helicase enzymes create a replication bubble, by pulling apart the DNA at these origins of replication.

What happens after splicing?

After the introns have been removed by splicing, there are also a couple of additional processing events that happen, one at the 5' end and one at the 3' end Only after all those processing events occur can the messenger RNA be recognized at the nuclear pore as something that should be allowed out of the nucleus in order for a ribosome to have access to it and translate it (so at this point, is it still pre-mRNA or is it now mRNA? At this point it's mRNA)

Why is 'big data' relevant to next-gen sequencing?

All of this next gen sequencing, and all of the associated computer algorithms and power, have led to "big data" Huge amounts of sequenced information, and annotations of that information There's a project called Encode, for example, that's taking all of the sequence information from sequenced genomes and searching for ORFs, consensus sequences that are known to indicate junctions of exons and introns, core promoter sequences like the TATA boxes, known recognition sites for certain regulatory proteins... going through and annotating all of these things in these genomes, and then whatever information is known about the protein that's encoded by that particular region of DNA sequence, and then whatever information is known about mutations in that sequence, etc

What can you do to study genes that you have sequenced?

All of this sequenced information gives you the tools where you can create organisms where you have changed their genes You can input a sequence that will recombine with an existing gene and essentially knock out that gene (you do this in embryonic stem cells), and you can insert those stem cells into an early embryo and put it into a surrogate mouse, let that embryo develop, and then you get mice where some of their cells have this gene knocked out, and then you can mate those mice with each other, and ultimately end up with some mice in which all of their cells throughout their body have this gene knocked out, and you can study the effects of knocking out that gene You can also knock in! Wherein instead of knocking out the gene, you replicate it with a different version of the gene (mutation). And you get mice with that mutation, and observe the impact Another way to silence genes: RNAi. Gene knockdown or gene silencing

What do all organisms use energy for?

All organisms can use chemical energy to drive their energy-requiring processes, and in doing so will convert energy to other forms (other chemical forms, or even mechanical, electrical, etc)

Gap junction

Allows the passage of small water-soluble ions and molecules in the cytosol. Tubes that connect one cytoplasm to another cytoplasm. Allows materials to flow between cells. Allows cells to communicate with adjacent cells (that it's attached to). Protein is connexin. Example: cardiac muscles. Heart muscles all need to contract in unison, and need to contract in a particular order. All of the muscle cells in your heart have gap junctions, so that if one cell activates and contracts, it causes the next cell to activate and contract, because they're connected directly. Without gap junctions, your heart can't contract in the manner that it does. Common in cardiac muscle intercalated discs, smooth muscle and other places - allows ion and small signal molecule exchange. Example: Defect in connexin gene in cells of the inner ear's cochlea — the most common cause of autosomal deafness

ATP synthase

Also known as "the lollipop" ATPase. Large protein that uses energy from H+ ions to bind ADP and a phosphate group together to produce ATP. Uses energy harvested by electron transport chain redox reactions to drive ATP synthesis. More specifically, uses energy from a proton gradient to drive the condensation of ADP + Pi to form ATP. This is called chemiosmotic coupling (or chemiosmosis for short) Located in the inner membrane. Two parts: F0(in membrane) F1(sticking off of membrane)

What is an allosteric effector? What type of bonding takes place with these?

An allosteric effector is a ligand that binds at an "effector site" distinct from the active site. Causes a conformational change that affects the function of the active site. Can be activating or inhibitory

Why is real-time PCR important?

Although RT-PCR is good at detecting RNA molecules (and regular PCR is good at detecting DNA molecules), they are not great at precisely quantifying them. An important development was the introduction of "real-time PCR" (sometimes also referred to as RT-PCR, but safer to refer to it as qPCR so as not to confuse with the method that uses reverse transcriptase). In this method, accumulation of amplified product is detected in real time through the increase in fluorescence emitted by tags released from oligonucleotide probes during the amplification process. This allows quick, sensitive, and accurate quantification of relative or absolute amounts of a particular DNA of interest in an unknown sample. (The following link has a video that explains the use of fluorescent tags in qPCR nicely: https://www.youtube.com/watch?v=kvQWKcMdyS4. But be careful because he misleading refers to qPCR as RT-PCR. He later goes on to decide how qPCR can be combined with the use of reverse transcriptase in order to quantify RNA molecules in a sample. In this case the correct way of describing the method is qRT-PCR.) The following link has a nice explanation of how qPCR data is analyzed to determine absolute or relative amounts of the molecule of interest in samples: https://www.youtube.com/watch?v=y8tHiH0BzGY.

Where can DNA polymerase add nucleotides? In what way does it move along the template strand?

Always and only at the 3' end of a primer. It is only able to grow chains in the 5' to the 3' direction as it moves along a template strand in the 3' to the 5' direction

When do amino acids attach to tRNAs?

Amino acids get attached to tRNAs before entering the ribosome A-site

What is PCR about, in 2 words?

Amplifying segments

What do we mean by 'addition of a poly A tail' at the 3' end?

An enzyme comes along and recognizes the 3' end of the mRNA and without the help of any template, just adds a whole bunch of adenine residues to the 3' end of that mRNA Specialized type of polymerase called poly A polymerase that does not need a promoter and does not need a template, but actually participates in the transcription termination process, and is sitting there to extend the 3' end of the mRNA by adding that poly A tail Poly A tail also plays a role in the translation process and also helps stabilize the messenger rNA against degradation enzymes that might chew it up before it ought to be

Primase

An enzyme that joins RNA nucleotides to make the primer using the parental DNA strand as a template Specialized RNA polymerase that can synthesize primers using ribonucleotides that are complementary to the DNA template. Can do this anywhere as long as a single-stranded DNA template is available Primase sitting down on exposed single-stranded region of DNA, joining nucleotides together to make a primer. Then, DNA polymerase recognizes the RNA primer available, and then starts adding DNA nucleotides to the 3' end of it. So the nucleic acid chain that is being made now, part of it is RNA! The 5' several nucleotides are RNA. And then there's a phosphodiester bond connecting an RNA nucleotide to a DNA nucleotide, and then from then on, all the nucleotides are DNA

Protein phosphatase

An enzyme that removes phosphate groups from (dephosphorylates) proteins, often functioning to reverse the effect of a protein kinase.

Protein kinase

An enzyme that transfers phosphate groups from ATP to a protein, thus phosphorylating the protein.

Intermediate filaments

An intermediate filament is a strong fiber composed of intermediate filament protein subunits. Rigid proteins with little flexibility. Tough, fibrous molecules twined together in an overlapping arrangement (kind of like a rope). They contribute to cell shape and flexibility, like bones. Examples: keratin, neurofilaments (neurons, nerve cells), nuclear lamins (lives just inside the nuclear membrane of neurons) Have characteristics that allow them to stick together, which is how they assemble themselves

What two pathways make up metabolism?

Anabolism + catabolism

Fermentation

Anaerobic catabolism of glucose The pyruvate is reduced, and the NADH is oxidized (to NAD+)

What happens after a mature mRNA is transported from the nucleus to the cytoplasm?

As soon as a messenger RNA molecule is transported from the nucleus to the cytoplasm, ribosomes begin to translate the sequence into amino acids. Typically, many ribosomes translate the mRNA simultaneously. Each ribosome begins at the 5' end of the mRNA, and progresses steadily towards the 3' end. New ribosomes attach to the 5' end at the same rate as the previous ones move out of the way. These multiple initiations allow the cell to make much more protein from a single message, than if one ribosome had to complete the task before another could begin. When a ribosome reaches a stop codon, the ribosome and the new protein dissociate from each other, and from the mRNA.

Terminator (detail)

As the RNA polymerase moves along with its transcription bubble, it's going to recognize some sort of signal that we'll refer to as a "terminator" as its signal to stop transcription, release the RNA that its made, let go of the DNA, and let the two strands of the transcription bubble go back together. So the DNA itself is unchanged after an RNA polymerase has done this transcription process! Which is quite different than DNA replication

Primase

Associated with helicase, because it is going to be laying down a new RNA primer periodically at the fork on the lagging strand template

What are the assumptions of Hardy-Weinberg equilibrium?

Assumptions (proportions of genotypes do not change in a population as long as): 1. No mutations take place 2. No genes or alleles are transferred to or from other sources (no immigration or emigration) 3. No coming into the population, or leaving the population, due to migration/movement 4. Random mating is occurring. Assuming that every individual has equal probability/opportunity of mating with any other individual 5. The population size is very large 6. No selection occurs. No natural selection going on! In a population, every single organism is as likely to survive as every other organism.

Where are ribosomes found in prokaryotes? In eukaryotes?

At any given moment, some ribosomes in a eukaryotic cell are floating free in the cytoplasm and others are attached to membrane of the rough endoplasmic reticulum (RER). Similarly, in prokaryotes, some ribosomes are floating free in the cytoplasm and others are attached to the inner side of the plasma membrane. In both cell types, all protein synthesis starts on a free ribosome. Synthesis of certain proteins continues after the free ribosome has attached to a membrane.

What are you looking at, if you are looking at inidividual chromosomes? What point is this in the cell cycle?

At the beginning of mitosis, all of the DNA compacts into its most compacted form, and you can see these sister chromatid pairs. This is the only time you can see individual chromosomes. If you are looking at pictures of individual chromosomes, know that what you are looking at is sister chromatid pairs that are identical to each other and strongly connected to each other by proteins that are holding them to each other. This is called a mitotic chromosome

How does RNAP begin if it doesn't use a primer?

At the start position that was defined by the promoter, it started by joining together two individual nucleotides to make a dinucleotide and then continued to add to the 3' end

After Griffith, Avery et al, Hershey & Chase, and Hammerling, what did the scientific community know? What didn't it know?

At this point, after these experiments, the scientific community was convinced that DNA is what carries the genetic information. It's been shown in bacteria, bacterial viruses, and some examples of eukaryotic cells There were for sure other experiments in addition to these, that probably demonstrated that this was true in multiple types of bacteria, and multiple types of eukaryotes, etc But! They still didn't know the structure of DNA, and people were pretty amazed at this finding that DNA carries genetic information And of course, that was what led to the very intense search for understanding the structure of DNA

Transmembrane linker proteins

Attach to actin microfilaments of the cytoskeleton and bind adjacent cells Some anchor things to the inside, some anchor things to the outside, some anchor things on the outside the the inside

Why is ATP used for movement and GTP used for growth in microtubules?

Because otherwise, how would the cell know what to do? (See: Nexin example) Would grow and move at the same time! So ATP for movement, GTP for growth.

Because there are only 20 amino acids, what do we know about the way that they are coded by codons?

Because there are only 20 amino acids, we know that there are many amino acids that are encoded by multiple codons Ex: All 4 in one box are proline, all 6 in another box are leucine, etc

Cytokinesis in plant cells

Because they have these rigid cell walls, there's no use for a cleavage furrow. You can't literally pinch a plant cell in half! So vesicles containing cell wall material emerge from the Golgi apparatus, which itself has doubled itself and moved towards both ends of the cell. These get sent along polar microtubules towards the former metaphase plate, and then all of those vesicles fuse with each other and with the plasma membrane on both sides. Finally, that creates a continuous cell membranes around each of the daughter cells, with an area of cell wall in between them

Nucleating proteins

Bind to actin and create filaments to push the cell in one direction

What happens to a substrate when it binds to an active site?

Binding into an active site will also change a shift in the overall shape (conformation) of the substrate. (See image: hydrogen bonding, hydrophilic interactions, etc between backbone and R groups)

Capping protein

Blocks the end of actin to prevent growth

Severing proteins

Break actin filaments all along the length of the filament (ex: in retreat from something toxic)

Single-strand binding protein (SSB)

Brown circles on graphic Coats the exposed single-strand of DNA on the lagging strand template in order to keep it from accidentally making base pairing that might fold it upon itself (recall: RNA can base pair upon itself and make hairpin structure). We also want to make sure that the energy that that helicase expended to peel apart the two strands isn't wasted by this strand of DNA happening to re-pair with the leading strand template, and blocking the ability of the leading strand to be synthesized. So SSB holds that DNA in an extended conformation until the DNA polymerase for the lagging strand is ready to copy it

How do you use real-time PCR?

But basically, the way it allows you to quantitate things is, the more of the relative DNA that you're interested in, relative to others, is there... the quicker enough of its amplified product starts to accumulate to a level that where it crosses the threshold of detectability You use these graphs to figure out, what was the time at which this particular amplified fragment became detectable? The quicker it was detected, the more of the original sample was there. You do this to figure out exactly how much was there

Why are changes in nucleotide sequence important?

Changes ("mutations") in DNA nucleotide sequence are what drive evolution (and can also cause diseases) As organisms reproduce from generation to generation, there are occasionally mistakes made in DNA replication, or chemical or environmental insults that cause damage to DNA that change bases, and then don't get repaired before DNA is replicated, so over time, genome sequences change, and this is what drives evolution Natural selection will favor any changes that are beneficial, or create increased fitness of the organism for survival in its particular environment Some changes, or mutations, in DNA cause defects that may be selected against, and are responsible for many of the human diseases that we know of, and are, in many cases, able to cope with medically, so those people are not selected against

Can you provide an example of a way in which change in amino acid sequence can affect protein function?

Changes in amino acid sequence can affect protein function Ex: sickle cell anemia One of the amino acids in a chain of hemoglobin is changed because of a point mutation in a single codon (glutamate to valine, missense) Mutation happens to be on surface Valine is hydrophobic, so the presence of that sticky, oily residue on the surface of the hemoglobin causes the hemoglobin to clump together, and those aggregates of hemoglobin cause a deformation of the hemoglobin, which causes them to get clogged in capillaries Substitution of a valine (hydrophobid) for a glutamic acid (negatively charged) creates a "sticky" patch on the surface of the hemoglobin molecule that makes the hemoglobins clump together and results in distortion of the shape of the rbc.

Do changes in protein-coding DNA sequences always lead to changes in amino acid sequence?

Changes in protein-coding DNA sequences sometimes, but not always, lead to changes in amino acid sequence

What is chromatin remodeling? How does it happen?

Chromatin remodeling makes a region of the genome accessible the dynamic modification of chromatin architecture to allow access of condensed genomic DNA to the regulatory transcription machinery proteins, and thereby control gene expression The tightness of packaging (euchromatin vs. heterochromatin) is itself regulated through various covalent modifications of the histone proteins in specific regions of the genome, and this affects the accessibility of the DNA to the basal transcription factors as well as to the regulatory transcription factors. Graphic: Shows example where this particular gene and its regulatory sequences... although, sitting right near a region of tightly-packed chromatin, has been opened up by the presence of a chromatin-remodeling complex that bound somewhere in its regulatory region, and helped loosen the packaging, and then ultimately allows other regulatory proteins to come in and bind to enhancer or silencer regions

Can chromosomes have sister chromatids in cytokinesis?

Chromosomes cannot have sister chromatids in cytokinesis, because this after telophase, when they separate!

Telophase (detailed)

Chromosomes de-condense Gene activity resumes because they are recondensing. Nucleolus reappears because gene activity resumes Nuclear envelope reforms around collection of chromosomes that has been pulled near to that spindle pole. 2 new nuclei start to form

Cilia vs flagella

Cilia: rowing like pattern Flagella: undulating motion Both have core of microtubles sheathed in an extension of plasma membrane Cilia- occur in large number, differ in beating pattern (oars) Flagella- one or few. Way more common in bacteria. Flagella - sperm (in humans) Cilia - fallopian tubes, upper respiratory tract (move mucus from lungs up into your throat)

Differentiate between the 4 protein complexes of the ETC. Which one(s) is/are Q, succinate dehydrogenase, NADH, FADH2 associated with? Why does this matter?

Complexes 1, 3, and 4 pump protons. Complex 2 does not. Succinate dehydrogenase of CAC is a peripheral membrane protein associated with complex II. Goes into its active site. Next intermediate in citric acid cycle floats away. FAD is reduced to FADH2, and it can pass its electrons onto a carrier in complex II. Q is an electron carrier called ubiquinone. Very similar to NAD and FAD in that it's able to interconvert between an oxidized state and a reduced state (in reduced state, has extra hydrogen atoms attached), but also has hydrophobic tail that makes it soluble in the membrane. Picks up hydrogens from complex I and complex II and delivers them to complex III. Path taken by electrons through ETC is either from complex 1 —> 3 —-> 4, because coenzyme Q shuttles them (this is the path of NADH), or complex 2 —> 3 —> 4, shuttled by coenzyme Q (this is the path of FADH2).

How is the S phase in the cell cycle similar to the process for replicating a prokaryotic chromosome?

Conceptually, what happens in S phase is identical to the process for replicating a prokaryotic chromosome. There is an origin of replication where two strands of DNA get pried apart, and that creates a replication bubble, which grows in both directions. Each end is referred to as as 'replication fork,' and that's where the current active polymerization of DNA nucleotides is happening

Describe the coupling of ATP hydrolysis to a condensation reaction. Is this an endergonic or exergonic process?

Condensation reactions are typically endergonic (will not happen spontaneously without the input of energy). Consider the condensation reaction of molecule A and B. This often happens in two steps. Step 1: Phosphate is taken off of ATP, and is attached to one of the molecules. Then, attached to molecule B, the phosphate group retains some of its high energy bond (molecule B is phosphorylated). Step 2: Then, molecule A comes in, and attacks the phosphorylated B, in such a way that the inorganic phosphate gets released, and molecule A ends up covalently attached to molecule B. The thing that drove this process (making it favorable) was breaking the high energy bond of phosphate to molecule B

What is albinism and how does it appear in pedigrees?

Condition in which the pigment melanin is not produced Form of albinism due to a nonfunctional allele of the enzyme tyrosinase Males and females affected equally Most affected individuals have unaffected parents

Can you give an example of a signal transduction pathway that impacts transcription?

Cortisol - upregulates expression of anti-inflammatory proteins in many cell types; in a liver cell it will upregulate genes needed for gluconeogenesis. Estrogen - upregulates expression of ovalbumin for chick eggs. Progesterone - upregulates yolk protein expression for chick eggs. Adrenaline - in a skeletal muscle cell upregulates genes involved in cellular respiration as well as those that will cause breakdown of glycogen (in both cases to promote ATP production)

Three types of active transport? (and what differentiates between them?)

Coupled transporter, ATP-driven pump, light-driven pump (differentiation between the three is based on type of energy source)

Can crossing over happen between the p and q arms?

Crossing over can happen between the p and q arms (across the centromere), but it's way less likely, just because of the way they associate in meiosis When the centromere is in the middle, the p and q arm distinction kinda goes away So yes, if you have a really big q arm, you have more crossing over happening there... crossing over is a random event Is complicated by interference, which is the more crossing over events you have, the less likely you are to have another crossing over event

Can you tell if there's crossing over just by looking at the gametes? If so, how can you tell?

Crossing over exchange alleles Basically, just remember that if every gamete looks parental, there's no crossing over. If you have gametes that don't look parental, then you have crossing over occurring

When is crossing over more likely to happen (with respect to linked genes)?

Crossing over is more likely to interrupt a linkage the further apart they are

Where does translation begin?

Cytoplasm

What is STR profiling? Why do we care?

DNA "fingerprinting" (STR profiling) The principle: Different individuals have different numbers of repeats at the same STR locus, so PCR gives different length amplified products There are lots of different STR loci in the human genome, each with different flanking sequences Voiceover: PCR products give different lengths in different individuals. So this is an example of DNA fingerprinting in a bunch of different individuals, using primers from a bunch of different loci that are different STRs. So the primers for the different loci are all labeled differently... represent sizes of their amplified fragments from their particular copy of their STR. Different people have different sizes) If you include enough loci, you can end up with these fingerprints that are highly individual · 0.0001% likelihood of two individuals having a matching fingerprints at random. So you can use that to identify suspects from the DNA at crime scenes

What is the basic flow of a protein from DNA onward? (include forks)

DNA (transcriptional control) RNA transcript (RNA processing control) mRNA (mRNA transport and localization control) mRNA [(mRNA degradation and control) —> degraded mRNA OR] (translational control) protein [(protein degradation control) —> degraded protein OR] (protein activity control) inactive protein/active protein

What do we mean when we say that DNA polymerase cannot start a DNA chain 'de novo'? How do we circumvent this problem?

DNA Polymerase cannot start a DNA chain "de novo" (i.e. from two single nucleotides); this has to do with its ability to proofread its work It can only add nucleotides to the 3' end of an already existing nucleic acid chain (say, at least ~10 nucleotides long). To get around this problem, a short "primer" made of RNA is synthesized by an enzyme called "primase", which works by an identical mechanism to DNA polymerase except that it uses RNA nucleotide substrates (ATP, GTP, CTP, UTP; "NTP's") and can start chains de novo (it is essentially a specialized RNA polymerase). The primase then moves out of the way and lets DNA polymerase add DNA nucleotides onto the 3' end of the RNA primer. The newly made DNA strand grows 5'®3' as DNA polymerase moves in the 3'®5' direction along the template strand (this is because the two strands are antiparallel).

DNA Gyrase

DNA gyrase - this is an enzyme that sits on the parental DNA ahead of the replication fork and fixes the tangles that accumulate as the replication bubble grows.

What does DNA polymerase require?

DNA polymerase requires... - Template strand to decide what nucleotides to add - Substrates (dNTP's) (dATP, dGTP, dCTP, dTTP) - Mg2+: Most enzymes that use nucleotides as substrates also require magnesium ions in their active sites, because those magnesium ions interact with the phosphate groups of the nucleotides and hold them in a proper orientation for the enzyme active site to catalyze the reaction - Primer: Free 3' end on a growing strand This is really important! DNA polymerase can't start DNA strand de novo. Aka, can't start one from scratch. So DNA polymerase can't take two individual nucleotides and join them together, and make a dinucleotide, even if it has a template telling it what to join together. It needs to be adding to the 3' end of an existing primer. So it can add nucleotides to a growing chain, but it can't start from scratch

What is an NTP?

DNA synthesis uses dNTPs as substrates, while RNA synthesis uses NTPs as substrates. NTPs cannot be converted directly to dNTPs. Nucleic acid synthesis is catalyzed by either DNA polymerase or RNA polymerase for DNA and RNA synthesis respectively.

Recombinant DNA molecule

DNA that has been formed artificially by combining constituents from different organisms

Describe the interphase nucleus. What assumption do these observations rest upon?

DNA wrapped up with histone proteins to make chromatin Packaging of DNA into chromatin is related to whether the genes in that part of the chromatin are being expressed or not All of this information rests on the assumption that we a priori understand that DNA is the genetic material that directs cells and gives organisms their specific characteristics

Given point mutation, provide definition and potential consequence: Frameshift

Definition: Addition or deletion of a nucleotide (one, two, or maybe even three nucleotides) Consequence: Reading frame is shifted, altering the meaning of all subsequent codons; almost always deleterious One thing to think about - if you add or delete three, you retain the reading frame - you just add or delete one full codon. So adding or removing a single amino acid along a polypeptide chain might not be so bad... but only mentioning this because this might show up on the MCAT. "What would be worse, adding 1 2 or 3 extra base pairs?" Or which one might not have a chance of creating such an issue?

Given point mutation, provide definition and potential consequence: Missense

Definition: Change in nucleotide sequence that changes the amino acid specified by the codon Consequence: Change in primary structure of protein; may be beneficial, neutral, or deleterious (depending on circumstances of amino acid in the protein, e.g. acidity, basicity, charge)

Given point mutation, provide definition and potential consequence: Silent

Definition: Change in nucleotide sequence that does not change the amino acid specified by a codon Consequence: No change in phenotype; neutral with respect to fitness

Given point mutation, provide definition and potential consequence: Nonsense

Definition: Change in nucleotide sequence that results in an early stop codon Consequence: Leads to mRNA breakdown or a shortened polypeptide; usually deleterious (especially if it occurs early in the ORF. If it's very close to the end of the ORF, close to the C terminus, it might not have much effect... but in general, assume that a nonsense mutation will destroy the function of the protein)

Denaturing gel electrophoresis

Denaturing: urea or formamide to keep strands separated (by competing for H-bonds); can resolve single-nucleotide differences in length Heat DNA sample before loading onto gel... not only do you heat it, but your sample buffer has a chemical in it (urea or formamide are common), which compete for the hydrogen bonding that the two strands use to complementarily pair with each other, thus keeping the strands as single strands. You also have the formamide (or whatever chemical) at saturating concentrations within the gel itself, so that as the DNA moves through the gel, it stays single stranded! Important: you can separate single strands of DNA or RNA from each other with very high resolution (can separate 100 vs 101). This ability to get very high resolution is what enabled the first methods of actually determining the nucleotide sequence of DNA molecules

Can you provide some examples of applications of CRISPR?

Designed to target a particular place in a genome and cut it The cell typically responds to breaks like that by inducing repair processes that try to put the ends back together, but typically end up deleting a couple nucleotides or adding a nucleotide, so that the function of that gene has been destroyed... but if you provide along with your CRISPR cas9 system a donor DNA with an alternative sequence, then recombination mechanisms in that host cell will let your donor DNA be used to replace the sequence that you've targeted You can also use a cas9 protein that has been deliberately mutated so that it won't cut the DNA, but it will still land on the DNA at the site you're interested in, and you can attached other cas9 proteins... look up recording here

Autoradiography

Detecting radioactive molecules using photographic film is called autoradiography

How does the ribosome small subunit locate the start codon? Is it the same or different in proks vs euks? Why?

Different! Because of the difference in the organization of genes in prokaryotes and eukaryotes Euks: Bind at 5' cap and scan for 1st AUG. In eukaryotes, the messenger RNAs are always monocistronic, so there's always only one start codon that needs to be located. So, a very simple approach is used! Where initiation factors that are bound to the small subunit of the ribosome (and also along with a tRNA that has a methionine amino acid attached to it) - this complex binds to the 5' cap of a messenger RNA, and simply scans along, and basically just goes to the first AUG that it finds and starts translating. In reality, there is some role of the sequence context of the AUG, so there are of course exceptions to the rule, but for our purposes, the rule is: bind at the 5' cap, and scan along until you find the 1st AUG Proks: Remember that prokaryotic mRNAs are often "polycistronic": more than one ORF, and each ORF has its own ribosome binding site.

Describe the movement of RNAP along the template strand

Direction of RNAP movement = moving 3' to 5' along template strand to grow RNA chain 5' to 3'

When are distinct chromosomes visible? What do they look like?

Distinct chromosomes are only visible during a short phase when the cell is actually undergoing mitosis (M phase - Mitosis is part of M phase, and M phase also includes cytokinesis) Mitosis means the chromosomes appear thread-like

Sliding clamp

Donut shaped red things in the graphic, next to DNA polymerase Tethers DNA polymerase to the DNA, so that it doesn't fall off of the DNA when it's not supposed to. The DNA polymerase on the lagging strand does need to hop off of the DNA and reassociate closer to the fork at the 3' end of a new RNA primer, and so its sliding clamp will be associated with some machinery at the fork... this lagging strand is actually looped back towards the fork, so that the sliding clamp and this DNA polymerase can be associated with the fork, and when that DNA polymerase bumps into the next Okazaki fragment, that's going to trigger that sliding clamp to open up, let the DNA polymerase jump to the new RNA primer, and then the sliding clamp will go back onto it at that point

Nuclear envelope

Double membrane perforated with pores that control the flow of materials in and out of the nucleus Composed of 2 phospholipid bilayers with aqueous area inbetween (perinuclear space)

Describe the structure of the double helix. How is it held together?

Double-helix, two sugar-phosphate backbones that are anti-parallel and are wound around each other in helical shape with base pairs in the middle and hydrogen bonding occurring The double helix is stabilized (held in its shape) by: H-bonding between bases Base "stacking" (hydrophobic & van der Waals interactions between basepairs)

DNA duplex

Duplex DNA is another name for double-stranded DNA. This means that the nucleotides of two DNA sequences have bonded together and then coiled to form a double helix. This double-stranded structure facilitates the stable duplication of genetic material, a requirement for cell division.

What is the cell up to during interphase?

During interphase, cells are doing the specialized function that they're meant to be doing in your body. Might be secreting enzymes, or hormones, or importing glucose and making glycogen, or breaking down glycogen and exporting glucose Doing all normal stuff during interphase, and growing in preparation for next cell division

What does RNA polymerase do during transcription?

During transcription, RNAP (not shown here) only unwinds the DNA locally (only ~20 basepairs) to form a "transcription bubble" and the bubble moves with it as it makes RNA After making its first phosphodiester bond between two nucleotides, RNAP elongates that RNA chain by adding nucleotides one at a time to the 3' end of the growing chain.

During translation, how many sites does tRNA move through within a ribosome? Explain.

During translation, each tRNA will progress through 3 locations (A, P & E) within a ribosome During translation, each tRNA will progress through 3 locations (A, P & E) within a ribosome Aa-tRNA enters the A site, escorted by an elongation factor. It gets moved into the P site after the peptidyl transferase has moved the previous tRNA's attached peptide to it (making it the new peptidyl-tRNA). The ribosome trans locates to the next codon (toward the RNA 3' end, with help from an elongation factor) as this happens When its peptide gets transferred to the new aa-tRNA (leaving it as an uncharged tRNA) and the ribosome moves gain, it gets pushed into the E-site and will be ejected from the ribosome

During translation, how many sites on the ribosome are occupied at any given time? Why?

During translation, only TWO sites are occupied at any given time though! (A new aa-tRNA is not allowed to enter the A-site until the uncharged tRNA has been ejected from the E-site.)

Because enzyme/substrate interactions are reversible, we know that...

E + S (free enzyme and substrate) exists in equilibrium with ES (enzyme-substrate complex)

How many hydrogen bonds hold each type of base pairs together?

Each AT base pair is held together by two hydrogen bonds, and each GC pair is held together by 3 hydrogen bonds.

The DNA sequence at an origin of replication is typically pretty rich in AT base pairs - can you think of why that might be?

Each AT base pair is held together by two hydrogen bonds, and each GC pair is held together by 3 hydrogen bonds. So regions of DNA that are enriched in AT base pairs relative to GC base pairs are a little bit easier to pull apart.

What happens after helicase create a replication bubble?

Each end of the replication bubble is going to become a replication fork, because it's the fork at the end of the bubble where the double stranded parental DNA is forking open at the edges of the bubble, and new DNA is going to be made at the ends of that bubble, and the bubble is going to grow.

How does pH impact enzyme affinity?

Each enzyme has an optimum pH range. Changing the pH outside of this range will slow enzyme activity. Extreme pH values can cause enzymes to denature. Consider also - the change of pH can lead to the ionization of amino acids and other molecules, which with necessarily change the shape and structure of proteins (further damaging their function)

What does the central dogma have to do with macromolecules?

Each of these processes uses information from the previously existing molecule (either DNA or RNA) to grow a macromolecule In each case, this growth of a macromolecule is going to have 3 stages that we'll describe: initiation (how does the process get started? How does the enzyme that does it know where to start its process on the piece of information that it has available to it?), elongation (how does it then elongate that macromolecule chain?), termination (how does it end the process? What kind of signal does it recognize that tells it 'okay, now you've made enough of this polymer, and you should stop this process)

Briefly describe the relationship between proteins, cells, and genes. Does every gene code a protein? If not, what do those other guys do?

Each protein in a cell is encoded by a gene in its DNA (reminder)... but not every gene encodes a protein! There are genes or segments of chromosomes that are transcribed by RNA polymerase, but are not RNA molecules that encode protein. Rather, their RNA molecules that are going to carry out some other function typically involved in helping ribosomes synthesize proteins

How many centromeres does a sister chromatid pair have?

Each sister chromatid has a centromere sequence region within it, but the entire complex of the sister chromatid pair, where kinetochore proteins are bound to these centromere sequence regions, is called A CENTROMERE. A single sister-chromatid pair has a single centromere

Does glycolysis take place under aerobic or anaerobic conditions?

Either! Both!

Why can't ethidium bromide detect single strands?

Ethidium bromide isn't good at this because single strands aren't base paired, so it's harder for it to bind

How can we detect DNA on gels (traditionally)? How does this work? What is the issue with this?

Ethidium bromide! Classically, and still most commonly used, for detecting DNA (at least on native gels) Intercalates into helix. Flat, aromatic molecule, so it likes to insert itself between adjacent base pairs on double helical DNA (remember - base pairs are flat ring structures, and the surfaces are hydrophobic, so ethidium bromide likes to stick itself in there) When it intercalates, it fluoresces, so you can see it under UV light Mutagenic, so not very safe! Have to wear gloves! Basically, this stain lets you see DNA under visible light

What are the main differences between prokaryotic and eukaryotic RNAP? What are the similarities?

Eukaryotes: 3 types of RNAP for 3 types of RNA (our main focus: RNAP II, which makes mRNA) Prokaryotes: 1 type of RNAP for all 3 types of RNA -Their mechanisms for promoter recognition (transcription initiation) are different - Their mechanisms for termination transcription are different - In between starting and stopping, mechanisms are pretty much identical

What's happening at the point on the Michaelis-Menten curve when you're close to Vmax?

Even if an enzyme has relatively low affinity for substrate, if enough concentration of substrate is present, essentially all of the enzyme will be occupied with substrate. At very high [S], every active site is constantly occupied (the enzyme is "saturated" with substrate). The reaction cannot go any faster unless more enzyme is added (remember that Vmax depends on turnover # and enzyme concentration) This is how you get that condition where when the product floats away, the enzyme is immediately occupied by another substrate molecule. The equilibrium is dictating that enzymes and substrates spend very little time floating separately, and are always together as a complex At the point in the Michaelis-Menten curve when you're close to the Vmax, there are essentially no enzyme molecules sitting and waiting for a substrate to bind

If the concentration of the substrate is much higher than the concentration of the enzyme, does that mean that every enzyme molecule has a substrate molecule bound to it?

Even if the concentration of the substrate is much higher than the concentration of the enzyme, that doesn't mean that every enzyme molecule has a substrate molecule bound to it. The ratio between enzyme-substrate complex and free enzyme/free substrate is defined by the equilibrium, which in turn depends on the affinity of the enzyme for its substrate

Why do cells separate DNA into heterochromatin and euchromatin?

Every cell contains all genetic material, but not every type of cell needs to use all of that material (ex: liver cell needs to express different proteins than a brain cell). Heterochromatin vs euchromatin allows cells to separate DNA that they want to express from DNA that they do not want to express.

Why do we need to regulate genes?

Every cell in your body has the same genome, but clearly every cell has different jobs, need to be expressing different proteins, etc The same cell, over the course of its lifetime, may even have differences in gene expression

What is evolution the study of? What does evolution result in?

Evolution results in a change in the genetic composition of a population Evolution is the study of how genetic frequencies change over time

What is FAD?

FAD is a coenzyme that is typically permanently held within an enzyme as a prosthetic group, and when it needs to pass its hydrogens onto some other molecule, the protein that it's in is going to be part of a larger multi-protein complex, and it will pass its hydrogens to another place in the same protein

What is an Immunoblot? What is an ELISA?

Example of using antibodies to detect specific proteins Immunoblot (aka "Western blot") We are looking at an image of a protein gel (separates proteins by sizes!). The proteins were blotted onto nitrocellulose paper, and that paper was blotted with antibodies against a certain protein. So the image shows the places on the protein gel where the antibodies were detecting the presence of certain proteins This is used a lot in research You can use this technique to detect a protein in a sample that you got from a patient. You can have many wells, and each well has antibodies covalently attached to the bottom of the well. You put different samples in different wells, and if the protein is detected by an antibody, it'll stick to the antibody, and you can wash away anything that didn't bind, and then you can add more antibodies, that will also stick to the protein, that has something attached to the protein that will allow you to detect the presence of that protein (enzyme, add to substrate, change color, etc) ^This is called an enzyme-linked immunosorbent assay (ELISA)

What is FISH? Why do we care?

Example of what you can do with sequenced information! FISH · Fluorescent in situ hybridization · Can detect gene amplifications right in cells! Example: HER-2 (see: notes - related to breast cancer) Takeaway: this kind of technique is actually used to detect whether tumors is using amplified HER-2, because there are drugs that can target that protein. But you only want to use that drug on tumors that are dependent on that kind of protein!

Can you give an example of a famous case where STR profiling was called as evidence?

Example: OJ Simpson case

Why does Hess's Law apply to enzymatic reactions? Can you give an example?

Example: conversion of glutamic acid to glutamine by the enzyme glutamine synthase Enzyme that synthesizes amino acid glutamine, and does this by synthesizing an amino group from ammonia and attaching it to glutamic acid (glutamine is just glutamic acid with an NH2 group instead of an OH) If this enzyme were going to try to catalyze this reaction directly, it would be using glutamate and ammonia as its reactants, and it would be a condensation reaction that produced glutamine and released a molecule of water. But that is endergonic, so it's done in two steps. First, a phosphate group gets attached to the glutamic acid (high energy linkage to glutamic acid). In a second step, the ammonia comes in and is able to displace that phosphate and create the glutamine molecule. Basically, this is Hess's law. The sum of reactions of Glu + NH3 —> Gln + H2O plus the ATP —> ADP + Pi is negative, so this becomes a spontaneous reaction (negative enough to make the whole thing negative)

Can you give a general example of allosteric regulation in bacteria?

Example: regulation of operons in bacteria Genes that are involved in a given process are often grouped together under the control of a single promoter in prokaryotes. Such a group of genes is called an operon (and the mRNA made from the operon is 'polycistronic'). Therefore, all the genes for that process that can be coregulated by controlling the ability of RNAP holoenzyme to recognize the single promoter of the operon This specific example shows an operon that contains several genes, each encoding an enzyme that catalyzes a step in the anabolic pathway that synthesizes the amino acid tryptophan. This group of genes, along with the promoter that controls its expression, is called the "tryptophan operon", or "trp operon" for short.

So what can you do with this sequenced information, to increase your ability to annotate the sequenced information? To associate important functions or characteristics with certain sequences? Provide several examples

FISH, antibodies to detect specific proteins (diagnostic, e.g.), chromatin-immunoprecipitation, CRISPR

Can you give a specific example of allosteric regulation in bacteria?

Example: the trip operon contains genes encoding all the enzymes needed to synthesize the amino acid tryptophan The pathway in bacteria that synthesizes the amino acid tryptophan has 5 steps, each catalyzed by a different enzyme Each of these enzymes is encoded by a gene. These genes only need to be expressed when there is not sufficient dietary tryptophan (so cell needs to make its own) There is a metabolic pathway, composed of several steps, whose final product is tryptophan! Which the cell might need, if there is no tryptophan available in the environment · This operon only needs to be expressed when here isn't sufficient tryptophan available This operon is regulated by a repressor protein that is controlled allosterically by tryptophan The level of expression of this operon will depend on whether or not there is tryptophan present to bind to this repressor protein If tryptophan present, it will allosterically affect the repressor protein to bind to the operon sequence and block RNAP from transcribing the operon If the bacteria runs out of tryptophan, then the repressor will come off of the DNA and let the RNAP transcribe the operon

Exonuclease

Exonucleases also hydrolyze p-diester bonds, but can only attack the phosphate connecting the endmost nucleotide to the chain. (So, exonucleases "chew" the DNA progressively one nucleotide at a time from the end; some start at a 5' end, some at a 3' end.

Why is expression of protein-coding genes in eukaryotes more complicated than in prokaryotes?

Expression of protein-coding genes in eukaryotes is more complicated than in prokaryotes, because of the differences in where they store the bulk of their genetic information Prokaryotes: In a prokaryote, translation of mRNA can begin even before transcription has finished! The two stages of the central dogma can basically occur simultaneously, because there's just the one large compartment within the cell The DNA is being transcribed to RNA, and in many cases, even before the RNA is finished being made, a ribosome has latched onto it and started to translate its information into a protein Eukaryotes: Transcription process is going on inside the nucleus, and ribosomes are in the cytoplasm, and do their translation of messenger RNA out there. In addition, the initial product of transcription of a protein coding gene in eukaryotes needs to go through some processing steps before it's considered a mature mRNA

Is F0F1 ATP synthase part of the ETC?

F0F1 ATP synthase is totally separate from this chain. Makes use of proton gradient produced by ETC, but functions independently of ETC (isn't part of it, doesn't interact directly with it —-> this is the basic point of Racker and Stoeckenius experiment)

What's necessary for three point mapping (in terms of parent genotype)?

F1 parent must be heterozygous for all three genes under consideration

Why is allosteric regulation relevant to transcription termination in prokaryotes?

Factor-independent: Step 2: RNA folds into base paired hairpin structure (something about the pause gives it the opportunity for this to happen) Rho: When Rho catches up to the RNAP, it has a similar effect as the RNA hairpin did in the factor independent version of termination (allosteric effector causing conformational change that triggers termination)

Using key features, differentiate between aerobic and anerobic catabolism of glucose

Fermentation and cellular respiration begin in the same way, with glycolysis. In fermentation, however, the pyruvate made in glycolysis does not continue through oxidation and the citric acid cycle, and the electron transport chain does not run. Because the electron transport chain isn't functional, the NADH made in glycolysis cannot drop its electrons off there to turn back into NAD+. The purpose of the extra reactions in fermentation, then, is to regenerate the electron carrier NAD+ from the NADH produced in glycolysis. The extra reactions accomplish this by letting NADH drop its electrons off with an organic molecule (such as pyruvate, the end product of glycolysis). This drop-off allows glycolysis to keep running by ensuring a steady supply of NAD+.

F-actin

Filamentous actin. A fibrous protein made of a long chain of G actin molecules twisted into a helix. This is what gives us the filament

Glycolysis (general description)

First step in cellular respiration, aka the aerobic catabolism of glucose. Takes place under aerobic and anaerobic conditions. Turns glucose into pyruvate while reducing NAD+ to NADH. Some energy is also stored as ATP through substrate-level phosphorylation. Takes place in cytoplasm of all cells (including prokaryotes and eukaryotes); all subsequent stages in mitochondrion Creates 2 ATPs per glucose, requiring ~7kcal per each (uses ATP). So in total, gain about ~15 kcal (for roughly 686 kcal you could get for complete oxidation of glucose... ~2%, not awesome). But! Some energy has been stored as reducing power in NADH, and not all of that has been realized

Name three factors that cause gene expression to vary

Gene expression varies... 1. During development 2. In different cell types within an organism 3. Within single cells according to changes in their environment

Gene flow

Gene flow A very potent agent of change. Individuals or gametes move from one population to another Where you have changes within larger populations, and localized changes that lead to populations and sub-populations changing their allele frequencies

How does Michaelis-Menten Kinetics vary from the general rules governing reactant concentration and rate of reaction?

General rule: the relationship between reactant concentration and rate of reaction is linear. The more reactant concentration, the faster the rate of reaction But when Michaelis and Menten did this, it showed something different! Michaelis and Menten observed that enzyme-catalyzed reactions display "saturation kinetics" (see: "showed saturation behavior"). Observed 'Vmax': maximum rate of reaction Also observed that the substrate concentration need to approach Vmax varies considerably between different enzymes

What is a basal transcription factor?

General transcription factors, also known as basal transcriptional factors, are a class of protein transcription factors that bind to specific sites on DNA to activate transcription of genetic information from DNA to messenger RNA

What do arrows/other diagram markers mean when talkin gene regulation?

Generally, an arrow indicates that something is activating something else, and a line with a bar indicates that something is inhibiting something else

Are genes that are close to each other likely to have a chiasmata form between them?

Genes that are relatively close to each other are less likely to have a chiasma form between them (less likely that crossing over will occur) The closer you are, the less likely you are to have this crossing over occurring

Genetic drift

Genetic drift Statistical accidents. The random fluctuation in allele frequencies increases as population size decreases Really complex. When you have a population, and for some reason, something happens, and you get a sub-part of that population that starts interbreeding with itself. Because of this sampling error, you don't get a good picture of what that population really looks like, and that gets exacerbated over populations Ex: founder effect · Imagine a population with equal number black cats and white cats (2/3 black). But then say 3 white cats get stuck on an island. Eventually you'll have a whole population of white cats. This is genetic drift. This is called the founder effect: the founders of this population had reduced genetic variation Similar: genetic bottleneck · You have a few phenotypes that are focused on

What is required for evolution to occur?

Genetic variation is required for evolution to occur

Why are germ cells 'immortal'?

Germ cells (eggs, sperm) are "immortal" due to the presence of an enzyme (telomerase) that can replenish telomeres!

What does glycolysis require to happen? In this same vein, what can we define fermentation as?

Glycolysis requires a continuous supply of NAD+ NAD is needed as an input for glycolysis. Under normal (aerobic) conditions, NAD, when turned into NADH, will be used in oxidative phosphorylation - will donate its reducing power to oxygen to make water - and that will regenerate the NAD, in order to allow glycolysis to continue. But under anaerobic conditions, that oxidative phosphorylation is not happening, so very soon, if you don't have another way of recycling that NADH, you're going to run out of NAD input that would be needed for glycolysis to continue. So, fermentation is simply a way of regenerating NAD+ to keep glycolysis going in anaerobic conditions

In one short summary, relate Griffith, Avery et al, and Hershey & Chase to the idea that DNA carries genetic information

Griffith's "transforming principle" Avery et al's characterization of the "transforming principle" Hershey & Chase definitively prove DNA as the genetic material

Cilia

Hairlike projections that extend from the plasma membrane and are used for locomotion. Lots of them in one area, short, used to help move fluid. "Power stroke" - bend and then snaps back, pushing liquid in the direction of the bend

What's the role of helicase enzymes once the replication bubble has been created?

Helicase enzymes expand the replication bubble, revealing additional template for continued DNA synthesis

Mitotic chromosome

Highly condensed duplicated chromosome in which the two new chromosomes (also called sister chromatids) are still held together at the centromere. The structure chromosomes adopt during mitosis.

Which experiment is often referred to as the "Waring blender experiment" and why?

Hershey and Chase, because... T2 bacteriophages are labeled with appropriate radioactive isotopes —> bacteriophages infect bacterial cells —> bacterial cells are violently agitated with a kitchen blender to remove the phage protein coats (before bacteria explode on their own) —> S radioactivity found only in the medium, P radioactivity found only inside the bacterial cells Why put them in a blender before they explode?Because they want to catch this while the bacteria are still early in this stage of infection, to find out which part of the virus went in The physical shearing force of the blender knocked off any part of the viral particle that was still sitting on the outside (so whatever didn't go in), leaving behind whatever had gotten in to the cells The blender blades are not fine enough to cause the bacterial cells themselves to rupture, so the cells are still intact, but anything that was loosely attached to their surfaces has been ripped off

Heterochromatin vs euchromatin

HeteroChromatin = Highly Condensed (transcriptionally inactive) euchromatin = less condensed, transcriptionally active ("truly transcribed")

How does fatty acid saturation impact membrane fluidity?

High ratio of saturated fats --> not a lot of mobility, can become brittle High ratio of unsaturated fats --> creates space in between for movement, but too much fluidity

How many homologous pairs/chromosomes do humans have? What are these pairs called?

Humans have 23 homologous pairs, 46 chromosomes altogether One pair is sex chromosomes (X or Y), other 22 pairs are called autosomes

Is the hydrolysis of ATP exergonic or endergonic? Why?

Hydrolysis of ATP to ADP (breaking off third phosphate) Breaks into ADP + Pi. This reaction has a delta G of -7.3 kcal/mole, so quite a favorable reaction. The bonds between phosphates on this nucleotide are "high energy bonds," so breaking them lowers the overall energy of the molecule, making this a favorable reaction

Transmembrane domain

Hydrophobic region of the phospholipid bilayer of the membrane

Describe the hypothesis, prediction, and test of the Creighton-McClintock experiment

Hypothesis: crossing over involves a physical exchange of genetic material Prediction: recombination of visible differences in a chromosome should correlate with genetic recombination of alleles Test Two visible chromosome markers · Yellow extension marker · Green knob marker Combined with two genetic markers · Kernel color Texture

Monozygotic

Identical twins - sex must be the same

If all 5 assumptions for HW equilibrium are true, what can we know about allele and genotype frequencies?

If all 5 assumptions for Hardy-Weinberg equilibrium are true, allele and genotype frequencies do not change from one generation to the next In reality, most populations will not meet all 5 assumptions

What do we mean by the word 'degenerate' in this context?

If an amino acid can be coded by multiple codons, we say that that code is "degenerate"

If... Parents: AABB, aabb. Heterozygous for A, heterozygous for B, both of dominant alleles on one chromosome, both of recessive alleles on the other chromosome If completely linked, what should we see (in terms of gametes)? If crossing over were to happen, what should we see (in terms of gametes)?

If completely linked... We'd expect to see the two dominant together, and the two recessive together (in the gametes) If crossing over were to happen... we'll get some new chromatids (gametes) that we haven't seen before! Noncrossover (recombinant) gamete: AB Crossover gamete: Ab Crossover gamete: aB Noncrossover (recombinant) gamete: ab

In a SCO: What will you see if genes are close together? What about if they're far apart?

If genes are close together... (you won't see the results). Segments of two nonsister chromatids are exchanged... but the linkage between the A and B alleles and between the a and b alleles is unchanged If genes are far apart... (you will see the results). Segments of two nonsister chromatids are exchanged... and the alleles have recombined in two of the four gametes

What do the 3 different models predict about the density of DNA after 1 generation of cell division? After 2 generations?

If semi-conservative is correct: all of the DNA after one generation of cell-division should have 1 strand heavy, 1 strand light. So all DNA has same density, intermediate labelled parental DNA and unlabeled control If conservative model is correct: two different DNA densities, one identical to labeled parental DNA and one identical to unlabeled control If dispersive model is correct: all DNA has same density, intermediate labelled parental DNA and unlabeled control Basically ^ you can distinguish conservative model from the other two, but dispersive and semi-conservative look similar to each other

What ratio would you expect with a dihybrid cross? What would you expect with linked genes?

If you were doing a dihybrid cross, you'd expect to see a 9:3:3:1 ratio. But if linkage is going on, you're going to see something else!

How do we derive the calculations for recombination frequency? Give a general explanation using an example wherein we have 50 meiotic divisions, wherein 49 divisions have no crossing over occurring.

If you were to imagine crossing over occurring at some frequency - won't happen 100% of the time, if it does happen 100% of the time, what does that mean in terms of what we see, etc — see: example from slide · Ex: 50 meiotic divisions happen. In 49 of those, no crossing over happens. But in that 1, meiosis with crossover occurs. So in one tetrad, we have 4 final parts, with no crossing over. In the other tetrad, we have 2 recombinants. So out of our total 200 gametes, we have 2/200 that are recombinant gametes. So that gives us 1 % = 1 map unit = 1 cM

What happens if you have two genes really close together on a chromosome? How is this different than if you have two genes really far apart? Why do these differences occur?

Imagine two genes really close together vs really far apart on a chromosome. You will see a lot more crossing over happening between the genes that are really far apart. This is because crossing over happens randomly along a chromosome, and because there's more space, just from a probability perspective, more crossovers are going to happen. Please understand: Even with the close together genes, crossing over is still happening, it's just not interrupting them If a crossover happens not between the two genes we're looking at, we can't see evidence of it. When we have two genes farther apart, and that crossover is happening anywhere along the chromosome, there's just more distance between them

What are the differences that we see in a population generally due to?

In a population, the differences that we see are usually due to genetics

What are replication initiation factors?

In any given chromosome, there will be one or more places of DNA sequence that are going to be recognized by proteins called replication initiation factors. These factors are going to recognize origins of replication in the DNA.

How is transcription terminated in prokaryotes?

In bacteria (prokaryotes), the terminator causes RNA polymerase to stop transcription in 2 steps: 1) the DNA sequence causes the RNAP to "pause" (like sitting at a stop light, but in theory could still keep going if the light turned green) 2) the RNA within the transcription bubble gets peeled away from the DNA template strand (releasing the completed RNA molecule) and the two DNA strands repair with each other, and this results in the RNAP falling off of the DNA. There are two types of terminator sequences in bacteria that differ in the way they achieve the 2nd step above. One is called "factor-independent" because it does not involve any other macromolecules besides the DNA, RNA and the RNAP. The other makes use of a protein called "Rho factor" and is therefore called "Rho-dependent".

Compare/contrast chloroplast vs mitochondria electron transport energy diagrams

In both cases, favorable redox reactions are being used to pump protons. Then, that proton gradient is used to drive ATP synthase in the the thylakoid membrane via an enzyme complex that is exactly analogous to the F0F1 in mitochondria (Image is of chloroplasts)

In eukaryotes: how is it possible for the same gene to encode multiple proteins?

In eukaryotes, alternative splicing can allow the same gene to encode multiple proteins! Many, if not most, eukaryotic genes have multiple exons and introns, and it's only beginning to be understood how the snRPS and spliceosomes are regulated, and are able to be sure to remove all of the introns, and not skip over any exons... that is all still being learned What is known is that in fact in many cases, there is a deliberate variation in how splicing goes, in any particular gene. Consider (example on slide) this gene, that has four exons and some introns with it. Here is its pre-mRNA. Due to differences in how this pre-mRNA gets spliced, there can be a mature mRNA that has exons 1, 2, and 3 joined together, or a different mRNA, that has exons 1 and 2 and has skipped over exon 3 and has joined exon 4 to exon 2. And obviously, these methods of alternative splicing are going to encode different proteins. The N terminal of them will be the same, because they were both encoded by exons 1 and 2, but once we get into exon 3 and exon 4 on these 2 different mRNAs, we're going to be making different protein chains

Why do we care about nuclear pores in this context?

In eukaryotes, the "nuclear pore complex" provides quality control to ensure that only fully processed, "mature" mRNAs exit the nucleus

Describe the gene arrangement of eukaryotes

In eukaryotes, there are no operons. Each gene has its own promoter and the resulting mRNAs are monocistronic (alternative splicing can lead to production of multiple different mRNAs from the same gene, but still each mRNA is monocistronic) Of course, keep in mind that eukaryotic genes typically have introns in them, so this diagram is oversimplified for depicting a typical eukaryotic gene. It would have introns and it would be made into pre-mRNA, and then there could be potentially alternative splicing that would result in the production of multiple different, mature messenger RNAs, but each messenger RNA would be monocistronic (single open reading frame in it that needs to be translated by a ribosome)

Why are telomeres related to the aging process?

In germ cells, there is an enzyme that replenishes the repeating telomere sequence, so that the DNA in those germ cells doesn't shorten with repeated generations Germ cells (eggs, sperm) are "immortal" due to the presence of an enzyme (telomerase) that can replenish telomeres! Somatic cells do not have this enzyme, so over generations, chromosomes lose information from their ends - this contributes to the aging process!

Describe the gene arrangement of prokaryotes

In prokaryotes, groups of genes related to a common process (e.g. synthesis of a particular amino acid, or uptake and catabolism of a particular sugar) are often clustered together in the genome as an operon that is transcribed from a single promoter, generating a polycistronic mRNA (multiple ORFs) RNA polymerase will bind to that promoter, and that group of genes that's sitting together downstream of that one promoter are referred to as an 'operon.' The mRNA that the RNA polymerase makes as it transcribes, starting at that promoter, and going all the way through the group of genes until it finds a terminator, has multiple ORFs: one for each protein that's encoded in the operon. So we say that this mRNA is polycistronic (cistron = another term for ORF)

Shine-Dalgaro sequence

In prokaryotes, rather than this initiation complex that has the initiator tRNA bound to it - rather than its binding to the 5' end and just scanning for the first AUG, it floats around and just bumps into the messenger RNA at random places. What it's looking for is particular sequences just on the upstream side of an AUG start codon. Bind at any ribosome binding site, "Shine-dalgarno sequence"

Differentiate between transcription and translation in prokaryotes vs eukaryotes

In prokaryotes, the mRNA synthesized by RNAp polymerase does not have to be processed before it can be translated by ribosomes. Also, because there is no nuclear membrane, mRNA translation can begin while transcription continues, resulting in a coupling of transcription and translation. In eukaryotes, the primary RNA transcript is a precursor mRNA (pre-mRNA) molecule, which is processed in the nucleus by the addition of a 5' cap and a 3' poly A tail and the removal of introns. Only when that fully mature mRNA is transported to the cytoplasm can translation occur.

What's the mitotic spindle composed of? How does it come together?

In prophase: a lot (or even most) of the microtubules in the cytoskeleton collapse, and begin to be renucleated from these two centrosomes.

How does the data from real time PCR differ from regular PCR?

In regular PCR, you get a gel In real-time PCR, you get a graph

How does DNA replication differ between prokaryotes and eukaryotes? (initiation process)

In terms of the initiation process, the main process between prokaryotes and eukaryotes is that prokaryotic chromosomes are circular, and they typically have one origin of replication, and that one replication bubble will grow and grow and grow until the two forks meet at the opposite side of that circular chromosome (180 degrees away). Eukaryotic chromosomes are linear, and they are way way larger than a prokaryotic chromosome. If a eukaryotic chromosome only had a single origin of replication, it would take a very very long time for that single replication bubble to grow and result in replication of the whole chromosome. Because of that eukaryotic chromosomes typically have multiple origins of replication, and each of them starts a replication bubble, and as those bubbles grow, they end up fusing with each other, and eventually the whole chromosome is replicated

Sealing junctions

Type of tight junction. 100% seal space between cells, so that nothing can squeeze in between the two, not even water

What do we mean by 'chance deviation'? Can you give an example?

Independent assortment & random fertilization are influenced by chance The larger the sample size, the less effect chance has Ex: coin flip. If you flip a coin 5 times, you should get heads 2.5 time (1/2 probability). But you can't have a half flip, so you'll get heads or tails more times than the other. But that doesn't mean you have an unbalanced coin! If we were to flip the coin 5000 times, we'd have closer to 50/50 Chance deviation from an expected outcome is diminished by larger sample size Chi squared and us: We are going to compare our experimental results to what we expect based on a null hypothesis

Summarize stage 1 of replication

Initiation! - Origins of replication (ori): Specific sites on DNA where replication begins; typically AT-rich - Recognized by replication initiation factors (proteins); "melted" open by helicase enzymes (using energy from ATP hydrolysis) to form a "replication bubble" - Each end of a replication bubble will be a "replication fork" where DNA synthesis will occur

Voiceover: translation process. Compare/contrast with transcription?

Just like the process of transcription, and even the process of DNA replication, we have initiation, elongation, and termination phases. As ever, initiation is going to involve locating a start codon that defines the reading frame for the ORF, and that elongation will continue as the ribosome reads along, and reads the nucleotides 3 bases at a time, as codons, to put in amino acids, until it reaches a stop codon that tells it that it's time to terminate the process In any given sequence of nucleotides in a messenger RNA there are three different possible reading frames, each of which would specify a different order of amino acids. So on this slide, you'll see that each of these is showing the same order of nucleotides, but using different reading frames, we come out with three different orders of amino acids. So it's very important that the ribosome be able to accurately locate the intended AUG start codon in order to put it in the right reading frame in order to get the right order of amino acids

Northern blot

Just use of RNA in the gel of a Southern Blot, not a scientist named Northern

Which symbiotic relationship is most often emphasized in this biotechnology unit?

Keep in mind the symbiotic relationship between technology and science. Science helps the development of technology, then new technologies aid the furthering of scientific discovery

Adhesion junctions (function, types)

Keep things together, but things can pass through. Keeps cells close together, but also allows fluid etc to pass through the space between cells. Types: Aherens, desmosomes, hemidesmosomes

Where do microtubules attach to centromeres? Why does this attachment matter?

Kinetochore structures (made of proteins) are the attachment sites for microtubules at the centromere regions This is what ultimately pulls the two sister chromatids apart from each other during mitosis

In bacteria with lac operons - is glucose the only alternative source of fuel? Explain why or why not

Lactose isn't the only alternative fuel source for these bacteria! So there are other operons whose genes are needed for metabolizing other alternative fuel sources, and all of them also need to be regulated in a way such that they are only turned on when that particular fuel source is available, and glucose is not available, since glucose is the preferred source. So there are several operons, dispersed throughout the E. coli genome, that all have binding sites for this CAP protein, so that... if glucose is available, simultaneously, does not allow/activate the expression of all these other alternative fuel source operons. SO, a group of operons like that, that is under the control of a single regulatory protein, like CAP protein, in this example, is called a regulon

Ribosome

Large RNA-protein complexes where protein synthesis occurs, "universal organelle." Either found free in cytoplasm or associated with internal membranes. 2 subunits: ribosomal RNA (rRNA) and proteins Travel into the nucleus and aggregate in the area of the chromatin that contains RNA that will be part of the ribosome. Ribosomes start being assembled in the nucleolus.

Can you do Sanger reactions all in one tube? If so, how?

Later, they changed it so you could do all 4 reactions in the same tube Each of the dideoxies themselves was labelled with a different colored fluorescent molecule, and just get different colored bands for them, and then you could even do it in a way that collected data digitally and showed different colored peaks for each of the DNA molecules

How can you use the complementarity of nucleic acid sequences as a tool? Can you provide examples?

Leading up to this, I've been emphasizing the importance of using complementarity of nucleic acid sequences as a tool, to detect the presence of its complementary strand Ex: microarrays, Southern blotting (using radioactive probe to detect fragments that have been separated from each other on a gel), Northern blotting (RNA fragments)...

Ligase

Ligases join a 3'OH to a 5'PO4 at the ends of two DNA chains that are abutted to each other

Summarize the "light reactions" and "dark reactions" of photosynthesis

Light-dependent reactions that absorb light within the photosystems, and pass electrons to the electron transport system. This produces a proton gradient and allows ATP synthase to make ATP. The products of the redox reactions are oxygen and NADPH. The ATP and the NADPH actually never leave the chloroplast, but are used by a pathway called the Calvin Cycle (this cycle uses the energy from ATP and the reducing power from NADPH to take carbon dioxide gas that has diffused into the chloroplast from the outside environment and turn it into sugar —> these reactions are sometimes called "dark reactions" because they are independent of light. If NADPH and NADP are supplied, these processes can happen in the dark. So during the day, plants are doing the light reactions, and they're building up their NADP and NADPH, and at night, in the dark, they're continuing to do these reactions to make sugar. Light dependent and light independent are both anabolic. Both energy requiring processes that are overall energy unfavorable (products are higher energy than starting reactants) Anabolic/catabolic classification depends on end energy of products as compared to energy of reactants

What's the limit of recombination and at this point how many tetrads are involved? What does that mean?

Limit of recombination is 50% (50cM) - occurs in 100% of tetrads - act like unlinked

How would we sequence a large restriction fragment using Sanger?

Let's say we want to sequence a large restriction fragment that we isolated from a clone that we made. The fragment has 5' and 3' ends. So what we're going to do is use a kinase enzyme to put a radioactive label at the 5' ends of this DNA. Then, I'm going to take some of this DNA, and cut it with a restriction enzyme (a different one than I had used to create the fragment). Once it's been cut - so we have 2 strands - I'm going to denature the strands, and purify the labelled strand (the upper strand, in this example, that has the radioactive label on it). So now we have 4 fragments. Then, I'm going to take some of my original fragment, and denature it, and add my labeled primer (which is the radioactively labeled fragment from above.... This radioactive bit becomes the primer). Put a big excess of the labelled primer, so that chances are, each of the bottom strands that got denatured from the big fragment get a labelled primer base paired to them Finally, we lower the temperature, and that allows complimentary strands to reanneal We end up with something that looks like (see slide). There are 2 denatured strands, and only one of those strands has a primer bound to it (because the primer is complimentary to ONE of the strands), and the primer has a 3' OH on it DNAP sit down at the 3' OH end of the primer, and use dNTP as substrates. When the DNAP comes to a c in the template (what is the c???) they have a choice between adding a regular dNTP or a ddNTP, and you set it up so that there's a way higher concentration of the regular nucleotide than of the ddNTP, so that when the DNAP reaches each position, they have a higher probability of putting in the regular nucleotide than of putting in the ddNTP So with your many, many copies of this template, and many many DNAP simultaneously synthesizing and randomly happening to incorporate a ddNTP, you end up with a whole bunch of sequences

Is it energetically taxing to make proteins? If so, what implications does that have for organisms?

Making proteins is energetically expensive!! 2 ATPs expended for attachment of an amino acid to a tRNA, 2 GTPs expended during elongation for each amino acid added to chain A typical polypeptide has hundreds of amino acids, so it will be important to regulate gene expression to make only those proteins that are needed at any given time Transcription (to make the messenger RNA) also uses energy —> might be best to not even transcribe genes whose products are not needed at the moment But in some cases it can be beneficial to regulate at the translational level

Where are microtubules found?

Many different places! 1. Interphase - scattered throughout cytoplasm when cell is not dividing. Emanate out from centrosome (near nucleus) - "microtubule organizing center, MTOC." Act as rails or tracks through the cytoplasm 2. In mitosis - two cell centers, microtubules emanate out of the poles (replicated centrosome). Microtubules are attached to chromosomes of which they are about to help move to opposite poles, guides/helps with scaffolding for division. 3. In ciliated and flagellated cells (ex: sperm cell)

Why is lacZ relevant here?

Many plasmids that are used for this have this gene called LacZ. LacZ is one of the genes in the Lac operon. This is an enzyme that lets bacteria metabolize lactose, and there's an artificial substrate for this enzyme that when this enzyme is present in this bacteria and you include this substrate in the media, and it gets acted on by the lac Z enzyme (lac Z is the gene, the enzyme is called beta glactosidase) - the substrate turns blue. And so if a bacterium took up just the vector DNA that didn't have insert, has this LacZ gene, and in the presence of that substrate the colony turns blue. But if the DNA was inserted into the polylinker site which is engineered into the middle of the lac Z gene, then that inserted DNA interrupts the open reading from for lac Z, and that means that these bacteria that have inserts don't turn the substrate blue. So any white colony here, is one that has insert. So now I know what has inserts, and what doesn't! All the white ones are part of my "library" of different fragments of mosquito DNA (see above) If I wanted to know which one of these has a particular gene that I'm interested in, then I can use my 'hybridization technology' and use a radioactive probe with a DNA sequence that I know to be identical to what I'm looking for, or similar to what I'm looking for, and I can use that as a probe to find which one of these colonies, if any, has that gene in it TL;DR: can use as a probe to determine which colony has DNA inserted

Which two units are important to chromosomal mapping?

Map units (mu) and centimorgans (cM) These are the same

What is the mediator in transcription?

Mediator can integrate input from multiple regulatory transcription factors (some of which may actually inhibit transcription) Activators bind to enhancers and increase transcription. Repressors bind to silencers to decrease transcription. Allows very fine control of expression in response to multiple signals In many cases "chromatin remodeling" modulates gene expression by altering tightness of DNA packaging

When we talk about recombination, are we talking about meiosis or mitosis?

Meiosis!

Linkers

Membrane bound, link other proteins together. Attach to things like actin to keep them near the membrane. Ex: cross membrane, linked to actin on the inside. Keeps actin along the inside lining of the membrane, which enables the creation of the actin cortex.

How are membrane lipids distributed throughout the bilayer? Why is this important?

Membrane lipids are asymmetrically distributed within the bilayer. Different phospholipids make the outside and inside of the cell. The variation in R groups on phospholipids makes it possible to orient proteins in the membrane in a certain direction. The types of lipids present also affect membrane fluidity, especially the length of fatty acid chains and their degree of saturation

Cell cortex

Mesh of actin proteins that sit just inside the membrane of the cell to give its shape

Metaphase (detailed)

Metaphase Chromosomes align at the equator (metaphase plate) There are regulatory processes in place to ensure that each sister chromatid pair is attached to a microtubule from one centrosomes, and the other gets attached to a microtubule from the other At this point, the centrosomes are called the 'poles' of the mitotic spindle In metaphase, because each each sister-chromatid pair is attached both to one pole, and to the other, and the microtubules are exerting some force on those sister-chromatid pairs, it's like a tug of war, and the chromosomes start jiggling around as they're being pulled apart by these two poles Eventually, because there are relatively equal forces being applied from both poles, all of these chromosomes end up being kind of lined up with each other at the equator of the mitotic spindle Think of it like a plane that's cutting what was formerly the nucleus (no longer exists bc nuclear envelope disintegrated) in half

Metaphase (basic)

Metaphase · Chromosomes align · Sister chromatids align along the center of the cell, so that both chromatids face towards opposite poles of the cell. Now, the sister chromatids are ready to be separated

Do all enzymes show Michaelis-Menten kinetics?

No! Allosteric enzymes have a sigmoidal curve rather than a hyperbolic one

Interphase

Most cells spend most of their time here 3 stages: G1, S, G2

What is a plasmid and why do we care?

Most common type of cloning vector! Small circular DNA molecules that are able to replicate inside of bacteria because they have an origin of replication in them, and also carry a gene to confer antibiotic resistance to the bacterium, so that when you put this plasmid into the bacterium, you will be able to select for bacteria that received the plasmid into them... and these vectors typically have what we call a polylinker region, which is a region that has been engineered into it Note: this lacZ gene must have this polylinker in phase and with neutral or silent changes in the DNA sequence to still allow functional beta-galactosidase to be made

Why do enzymes that use nucleotides as substrates also need Mg2+

Most enzymes that use nucleotides as substrates also require magnesium ions in their active sites, because those magnesium ions interact with the phosphate groups of the nucleotides and hold them in a proper orientation for the enzyme active site to catalyze the reaction

Are most eukaryotic core promoters weak or strong? What does this mean for the basal transcription factors?

Most eukaryotic core promoters are "weak" - basal transcription factors have trouble finding them without help!

Where was knowledge about DNA and proteins at in the early 20th century?

Most people in the early twentieth century expected that it would be protein that carried genetic information They didn't know anything yet about the structure of DNA. They only knew that it was composed of nucleotides, and that there were four different kinds of nucleotides They knew that proteins, on the other hand, were composed of 20 different types of amino acids, and that proteins are the 'workhorse' of the cell, and there are many varieties achieved by the variety of amino acids, and somewhere, that information for creating that huge diversity of proteins must be stored Was hard to imagine that a molecule that contains only 4 different components could somehow contain information to direct the assembly of this huge variety of proteins that need 20 different amino acids put together in so many variations Most people thought that DNA was serving some kind of structural function! They imagined that these 4 nucleotides were just present in a constantly and monotonous repeating sequence that couldn't contain any information This is where people were at the beginning of this set of experiments...

What is the most well-studied basal transcription factor? What does it do?

Most well-studied basal factor is called TATA binding protein (TBP) Binds to TATA box first, and then all the other factors assemble with it, and together that complex recruits the core RNAP

Myosin motors

Motor proteins that interact with actin filaments in cytokinesis, causing them to slide relative to each other so that they can pinch the cell

How do actin contribute to movement? What else do they do?

Movement: Ex: actin in cilia. Microfilament make the shift, but the actin allow the rigidity that enables powerful movement. Also, actin assists in the expansion/reduction of cell membrane (in conjunction with microtubules). Actin also creates the shape of the cell. Cell fusion (ex: fusing of sperm and egg). Keeps cells together so they can exchange material

Can the ratio of heterochromatin to euchromatin in a cell change?

Much heterochromatin converts to euchromatin in an "activated" lymphocyte Depending on what an interphase cell happens to be doing, the ratio of heterochromatin to euchromatin can change, depending on which genes need to be activated, and which do not. Different cell types differ from each other in the amount of heterochromatin, and which parts of their genome are in heterochromatin vs euchromatin form, and those can change according to the conditions that the cell finds itself in

Is glucose the only molecule that feeds into the cellular respiration pathway?

NOPE. Catabolic pathways for molecules other than glucose feed into the same cellular respiration pathway Even though we've been talking specifically about how glucose is catabolized to CO2, the metabolic pathways in our bodies also deal with breaking down macromolecules into their monomer components. Other sugars can also feed into the glycolysis pathway at various points, and the carbons from breakdown of fatty acids end up as acetyl groups on coenzyme A, and feed into the TCA cycle. The glycerol can feed into glycolysis. Various amino acids feed into this pathway at different points - some have pathways that convert them to pyruvate, some have pathways that convert them to intermediates of TCA cycle, etc. We've just been looking at one central process, but this is just a way of showing how other things are linked in.

What does RNAP require? What doesn't it require? What can it do to the parental DNA?

NTPs are substrates No primer required DNA template required Has ability to unwind and rewind parental DNA (trxn bubble moves w/RNAP toward terminator)

Native gel electrophoresis

Native: mild conditions to keep DNA double-stranded (and in some cases even remaining complexed with proteins)

How does natural selection impact allele frequencies?

Natural selection can lead to change in allele frequencies - frequencies of alleles of a gene from generation to generation Mechanism by which evolution works.. Recall that alleles are different versions of genes. Changes in how often we see either allele in a population is evolution (in the simplest sense)

What is cloning in this context?

Using plasmids to clone and manipulate DNA

What's a negative control? How about a positive control?

Negative control: a regulatory protein that's interfering with transcription by RNA polymerase Very often, there is a protein called a repressor protein that can bind to a sequence that will happen to be present overlapping, or just downstream of, the place where the RNAP holoenzyme would bind to the promoter. That sequence where the repressor protein would recognize and bind is called its operator sequence. And if the repressor is bound at that operator, that physically blocks - it either blocks the RNAP from binding to the promoter, if that operator sequence is actually overlapping with the promoter (literally, if the repressor is bound there, the RNAP can't even see the promoter), or if it's slightly downstream, it's just going to block the RNAP from doing anything once it's bound to the promoter Positive control This is most often true at promoters that are relatively weak, aka their "basal level of transcription" is quite low, because they're weak promoters. Often, promoters like that have a sequence adjacent to the promoter (but upstream!, so the protein binding doesn't block the RNAP), and that activator protein binds to its recognition sequence, and the presence of that activator protein increases the ability of RNAP to bind to the promoter

Does negative regulation exist in eukaryotic transcription?

Negative regulation also does exist (though less common); eukaryotic repressor proteins bind to DNA sequences called "silencers" within the regulatory region

Introns

Noncoding segments of nucleic acid that lie between coding sequences.

Nonrandom mating

Nonrandom mating Inbreeding is the most common form. It does not alter allele frequency but reduces the proportion of heterozygotes Ex: sexual selection · How do organisms choose mates? Most organisms do not mate randomly. Almost every organism that does sexual reproduction... there's some way that they choose their mates. So when we talk about non-random mating, this can be in reference to artificial breeding (farming), or inbreeding (this links into genetic drift)

Does PCR tell you exact sequence?

Nope!

Are all membrane proteins able to move freely about the membrane?

Not all membrane proteins are able to move freely within the membrane (despite the fact that the membrane is a "lipid sea"). It's important for the cell to be able to control where proteins are located.

What do we mean by "strong" and "weak" promoters?

Not all promoters have exactly the same nucleotide sequence Some have higher affinity for RNAP holoenzyme than others. ("Strong" vs. "weak" promoters)

Do all promoters have the same nucleotide sequence? Why or why not?

Not all promoters have exactly the same nucleotide sequence, so some are better at attracting holoenzyme than others

What's the net result of the Krebs cycle?

One turn of the cycle produces three NADH, one GTP (or ATP), and one FADH2, and releases two molecules of CO2

Why do cells undergo fermentation? What does this process entail? Where does it take place? What does it yield?

Not enough oxygen to complete the full aerobic catabolism of glucose, so cells undergo fermentation. In this process, glucose is only partially oxidized (to the smaller carbon-containing molecule pyruvate) rather than completely to CO2 (Pyruvate has 3 carbons, vs 6 carbons in glucose). Takes place completely in the cytoplasm (no involvement of mitochondria). Harvests much less energy than aerobic catabolism —> much less ATP per glucose. Only goes partway through aerobic catabolism process! So this makes sense. Cellular respiration: ~30 ATP per glucose, fermentation: ~2 ATP per glucose. You should know - under anaerobic conditions, you only get 2 ATP per glucose for fermentation. Way less efficient

Why are anticodons important?

Not only does the tRNA fold into this secondary structure! It actually folds into a particular L-shaped tertiary structure, where one end of it has that anti-codon, and the other end is where an amino acid will get attached with a high-energy covalent bond linking amino acid to the tRNA (meaning that this bond is energetically favorable to hydrolyze. Hydrolyzing it will release enough energy to allow a peptide bond to be made between this amino acid and another amino acid in a coupling like we've seen before, where an energetically favorable reaction is used to drive an energetically unfavorable reaction). So now, it's really important that only the correct amino acid that corresponds with the codon that this tRNA's anti-codon is going to be able to bind to... we've got to make sure that only that amino acid can ever get attached to this tRNA.

Why are mediators necessary?

Obviously, if multiple factors are going to regulate a promoter, they can't all bind immediately adjacent to the promoter. Eukaryotic regulatory transcription factors often bind at sequences quite distant from the promoter. Most have a positive impact on transcription, but some act negatively. The transcription factors themselves are often regulated by phosphorylation or by allosteric interactions. Remember also that eukaryotic chromosomes have DNA packaged with nucleosomes. The tightness of packaging (euchromatin vs. heterochromatin) is itself regulated through various covalent modifications of the histone proteins in specific regions of the genome, and this affects the accessibility of the DNA to the basal transcription factors as well as to the regulatory transcription factors.

Nucleosomes (structure, function, distribution pattern throughout the cell)

Octamers of histone "core" proteins wrap DNA around themselves (gets ~140 base pairs of DNA spooled around it), giving it the appearance of "beads on a string" (8 histone proteins) Along these very long chromosomes, roughly every 200 base pairs, there's another one of these nucleosomes In between adjacent nucleosomes, there's ~60 base pairs of 'linker DNA.' Overall, this structure looks like beads on a string

Can anything be an allosteric effector?

Often nucleotides, but can also be amino acids, ions, can be other small molecules. In some cases, another whole protein can come along and bind to an enzyme and cause a conformational change ("allosteric interaction between two proteins")

What is Okazaki's discovery? Why do we care?

Okazaki discovered how the replication of the DNA on that other strand of the replication fork is one On one parental strand at each replication fork, new DNA is laid down discontinuously in small fragments, each synthesized 5' to 3' (pointing away from the replication fork! Figured out that that strand of new DNA, rather than being made continuously with growth towards the replication fork, as we know DNA polymerase can do, he discovered that this other strand is down discontinuously. It's made in little pieces, and each little piece is made in the 5' to 3' direction, and is done in a kind of 'back stitching' kind of way

Now visualize DNA synthesis on both parental strands at a replication fork. If a DNA chain can only be grown in the 5' to 3' direction, how can both parent strands at a replication fork be copied?

Okazaki's discovery BABY

Relate PCR and Southern blots

Once enough sequencing had been done by these "old-fashioned" approaches, it became possible to do PCR. This meant you could quickly amplify a fragment of interest (rather than growing up huge quantities of bacteria and doing plasmid preps). You could label an amplified fragment from one organism and use it as a probe to detect a clone of a related gene in another organism (through Southern blotting). Then you could grow up lots of that clone to isolate enough DNA to sequence that gene using the old-fashioned approach.

How/why was PCR a natural evolution from these 'old ways' of sequencing?

Once enough sequencing had been done by these "old-fashioned" approaches, it became possible to do PCR. This meant you could quickly amplify a fragment of interest (rather than growing up huge quantities of bacteria and doing plasmid preps). You could label an amplified fragment from one organism and use it as a probe to detect a clone of a related gene in another organism (through Southern blotting). Then you could grow up lots of that clone to isolate enough DNA to sequence that gene using the old-fashioned approach. PCR can also be used to detect specific mRNA molecules in samples, by preceding the amplification step by a reaction that uses reverse transcriptase to make a complementary DNA strand to the mRNA (hence the name "RT-PCR" for the technique.)

How do you analyze results of a Sanger?

Once you finish DNA synthesis, you denature it, and you run it out on your gel, and you only see the radioactive bits, so you see where the DNAP had to stop because that particular dNTP was needed Loaded in gel: remember, smallest ones move farthest. So the smallest bits are at the bottom (if the gel was loaded at the top) and they represent the places where DNAP had to stop the earliest because it added a ddNTP In the gel, recall that the smallest fragments are at the 5' end of whatever we're looking at (assuming that none of the ladder ran off of the gel)... "the first nucleotide beyond the 3' end of that primer" Reading 5' to 3' going up the gel (towards the top), and this is complimentary to the template strand. So the template strand itself, running 3' to 5' (for this example) is TACG... etc In order to do this kind of sequencing, you need to have large amounts of this DNA! You need to isolate it, clone it, cut it, denature (check steps), run another gel to label fragment that will be a primer, etc... very labor intensive and time consuming!

Generally, when can DNA hybridization be applied?

Once you had isolated a gene of one species that was of interest to you, you could use the DNA of that species as a probe to look for a similar gene in another organism!

How do we know if something is 'dominant' in pedigree analysis? Can you give an example? If we know that a member of a pedigree has a dominant trait, what do we know about their parents?

One copy, trait expressed (AA, Aa). Ex: Huntington's disease Does not skip generations For offspring to have trait at least one parent must have trait

What 3 types of processing does pre-mRNA need to undergo before it can be considered 'mature' mRNA (and released through the nuclear pore?

Only after a pre-mRNA has undergone all 3 types of processing (5'-capping, splicing, and 3' polyadenylation) is it considered a "mature" mRNA and allowed to leave the nucleus!

Can anything attach to tRNA?

Only the particular amino acid corresponding to the codon/anticodon pair can get attached to the tRNA

Lysosomes

Organelles containing digestive enzymes. Aid in the digestion of macromolecules arriving from outside the cell (ex: food vesicles). Recycling of organelles from within the cell. Can also attack bacteria Endycytosis

Gap junctions

Points that provide cytoplasmic channels from one cell to another with special membrane proteins. Type: communication junctions.

How can you use PCR to detect specific mRNA molecules in samples?

PCR can also be used to detect specific mRNA molecules in samples, by preceding the amplification step by a reaction that uses reverse transcriptase to make a complementary DNA strand to the mRNA (hence the name "RT-PCR" for the technique.)

Why is PCR useful in forensics?

PCR lets you amplify particular regions of DNA and get large amounts of them... and because of that, it's useful in forensics, where you might be collecting really tiny bits of a suspect's DNA and need to amplify it in order to analyze it

What is PCR good for? What is it not good for?

PCR lets you amplify particular regions of DNA and get large amounts of them... and because of that, it's useful in forensics, where you might be collecting really tiny bits of a suspect's DNA and need to amplify it in order to analyze it... ... but it's not that useful for quantifying/comparing how many different pieces of DNA/RNA there might be relative to each other in your original sample. Just amplifies everything and lets you see what's there

At what step in the 'flow' of a protein can you regulate amount of active protein?

PSYCH - you can technically do it at any of the steps, but some are preferable to others...

Electron transport chain

Part of oxidative phosphorylation. A sequence of electron carrier molecules (membrane proteins) that shuttle electrons during the redox reactions that release energy used to make ATP. ETC are a series of proteins in inner mitochondrial membrane. Accepts electrons from NADH and FADH2, and ultimately passes those electrons on to oxygen to make water NADH enters at the beginning of the ETC. From there, redox reactions move horizontally. At the end, oxygen is used and hydrogen is added to it to make water. Simultaneously, protons move vertically, from the matrix to the inter membrane space Reduction of oxygen by electrons (in form of H atoms) from NADH or FADH2 via the ETC results in the pumping of protons (H+) to form a gradient of protons across the membrane

What is the lacZ gene?

Part of the lac operon

What are the limitations of pedigrees? What can we use them for?

Pedigrees don't allow us to know everything about individuals that we're going to analyze, but we we are able to do is say "what do we know?" And what does that tell us, and what can we figure out from that

Walk me through the process of using recombinant DNA technology to synthesize insulin

People with diabetes need to inject insulin in order to be able to control the level of glucose in their bloodstream. Before the days of recombinant DNA technology, the only way to get insulin would be to get it from animals. Mostly from pigs, sometimes from cows... from bloodstream or pancreas. They would have to purify insulin from these animals, and it takes a lot of animals! And a lot of expensive, time consuming purification technology, to get enough of this insulin to supply to patients. But once recombinant DNA technology was available, we could use that to make recombinant insulin, and we could make human insulin, and have the bacteria mass produce it for us. So to do that, the gene for insulin (like any other eukaryotic gene) has introns in it, so you have get the mature messenger RNA, gets translated, and then there's some complicated modification/processing events that the protein undergoes... so the mature insulin looks like [bottom of graphic]: two separate polypeptide chains, even though it was made as one polypeptide chain, and has these disulfide bonds To produce this in bacteria, they had to use some previous cloning technologies to make a gene for just the A chain and a gene for just the B chain, that were each cDNAs (see: microarrays), aka reverse transcriptase, so there's no introns... put a C-DNA copy of the a-chain You can grow huge vats of this stuff! Very powerful recombinant DNA technology

Peptide bonds are energetically unfavorable, so how do they get made?

Peptide bond formation is energetically unfavorable The way that we depicted the synthesis of polypeptides at the beginning of a course, just simply by condensation reactions between two amino acids floating around in aqueous solution, this is an unfavorable reaction. But the high energy bond, between an amino acid and a tRNA - that getting hydro lined is going to supply the energy to allow the peptide bond to get made

Phagocytosis

Phagocytosis is the process by which a cell uses its plasma membrane to engulf a large particle, giving rise to an internal compartment called the phagosome. It is one type of endocytosis. In a multicellular organism's immune system, phagocytosis is a major mechanism used to remove pathogens and cell debris. (cell eating) Large particle moved into cell. Predominant way in which a lot of single called organisms are able to consume food. Membrane envelopes food particle into food vacuole, and once it's in there, the vacuole breaks it down. Also enables white blood cells to break down bacteria or fungi. If there are bacteria moving about your body, white blood cells arrive, and take in the bacteria via phagocytosis. Then, they break it down

What is a phosphoimager? Why do we care?

Phosphoimager lets us get away from all that XRAY imaging! Advantages over film - Enhanced sensitivity - Shorter exposure times - Wider linear dynamic range - Reusable - No chemicals/no darkroom - Digital image - Basically, we no longer use X-RAY films or dark rooms. Today's tech is faster, reusable, etc

What kind of lipids are in membranes?

Phospholipids, cholesterol, liposugars

Relationship between phosphorylation and mitosis?

Phosphorylation events trigger progression through mitosis Phosphorylation events can trigger progression from one stage to the next by causing conformational changes in proteins

Do plants have mitotic spindles?

Plants have mitotic spindles, but no centrioles, and they don't have aster fibers

Main differences between plant and animal cells

Plants: rigid cell walls, chloroplasts (photosynthesis), large central vacuoles (storage, waste disposal)

What is a plasmid?

Plasmid: a small (1000-10000 by) circular dDNA that is distinct from the normal chromosomal DNA

Prometaphase (detailed)

Prometaphase (this is the very end of prophase, and indications that things are starting to begin in metaphase) Begins when the nuclear membrane (nuclear envelope) is broken down. At the same time, microtubule strands (spindle fibers) are growing from the centrosomes. These strands attach to a protein structure called the kinetochore. One kinetochore is attached to the centromere of each sister chromatid. Microtubules attach to the chromosomes. Microtubules that are radiating out from two centrosomes are able to grab onto kinetochore complexes that are on sister chromatin pairs. These new microtubules that are extending from the duplicated centrosomes are called the mitotic spindle Chromatin resolves into chromosomes, each consisting of two chromatids joined at the centromere

Promoter vs primer

Promoter is a sequence in DNA that RNA polymerase needs to recognize in order to use that DNA as its template to make an RNA. Making the RNA does not need a primer because RNA polymerase can start its chain de novo. A primer is a molecule that is needed in addition to a signal for where to start in DNA replication. In DNA replication, you have an ORF that initiator proteins recognize as the place where they should recruit helicase enzymes to to open replication bubble, but then DNA polymerase... even that isn't enough for DNA polymerase to make DNA. It needs a primer that is actually going to be covalently incorporated into the DNA chain that it's making. Uses that short RNA primer and adds DNA nucleotides onto the end of it DNA polymerase does not need a promoter. DNA polymerase needs a replication bubble to have been established. And the DNA polymerase gets recruited to that bubble by the protein components of the replisome that have already assembled there, and they recruit the DNA polymerase there

Prophase (detailed)

Prophase · The DNA condenses, organizes, and the classic chromosome structure appears (cell division starts to occur here, because the chromosomes condense) Gene activity stops, because the chromatin condenses so much (packaged even more tightly than heterochromatin, and we already know that heterochromatin is pretty inactive in terms of gene expression). Nucleolus (region where ribosomal RNA is made, and where ribosomes are getting assembled with that RNA to make proteins, and that is only visible because of gene expression activity) disappears This is when we first see the classic mitotic chromosome structure. This occurs through a condensation process. At the same time, microtubules in the cytoskeleton have some dramatic action happening: mitotic spindle starts to form. The nucleolus disappears, and the two centrosomes start to move apart from each other (still outside of the nucleus, just moving to opposite ends of the nucleus). At the end of prophase, the nuclear envelope itself breaks down, and that's the event that is the beginning of prometaphase

Give a full list of the proteins involved in replication PIPSGLSH

Proteins involved in replication: initiator proteins - unwind DNA at replication origin to form replication bubble primase - synthesizes RNA primers "de novo" using NTP substrates DNA polymerase - synthesizes DNA continuously on leading strand template and discontinuously on lagging strand template; requires primer (in addition to template and dNTP substrates) to get started ["sliding clamp" - actually a subunit of DNAP that tethers the enzyme to its template so it doesn't fall off while synthesizing] Helicase - unwinds DNA at fork using energy from ATP hydrolysis to expand the replication bubble [Single-strand binding protein (SSB) - binds to newly revealed template strands to keep them from re-pairing or folding back on themselves before DNAP synthesizes the 2nd strand] DNA ligase - seals together Okazaki fragments after RNA primer has been replaced with DNA. (Note that a special form of DNA polymerase - not the same one that's making the Okazaki fragments - has the special ability to remove the RNA primer and replace it with DNA; this has to happen before the ligase can seal together the Okazaki fragments.) DNA gyrase - this is an enzyme that sits on the parental DNA ahead of the replication fork and fixes the tangles that accumulate as the replication bubble grows.

Active site

Proteins tend to have, as part of their native structure, basically a pocket or crevice where small molecules can come in and interact non-covalently. In an enzyme, this crevice is called the active site

Bundling proteins

Proteins which cross-link unbranched actin filaments into parallel arrays Found in cilia, flagella

Why do you put a generous amount of primer into the test tube for Sanger?

Put a big excess of the labelled primer, so that chances are, each of the bottom strands that got denatured from the big fragment get a labelled primer base paired to them Finally, we lower the temperature, and that allows complimentary strands to reanneal

What is the current state of the art tech for measuring quantities of target sequences that you're interested in quantifying?

QPCR is currently a state of the art technology for measuring quantities of target sequences that you're interested in quantifying

Why is 'real time PCR' dubbed 'real time'?

Quantitative measurement of genes or mRNA (gPCR; also known as 'real time PCR") Called 'real time' because the detection and measurement of DNA/RNA in your sample occurs in real time as the amplification is going on. In regular PCR, after you've done your amplification, you run your sample out on a gel, and you see what pieces of DNA are there. In QPCR, you don't run a gel, but the primers that you use have fluorescent tags on them, whose ability to be detected changes when they become part of an amplified DNA molecule. So the primers floating around are either invisible to begin with, and then become not invisible when they become part of the DNA, or vice versa... so you can tell how many primers have become incorporated into a DNA molecule as you go along, and you can watch that amount increase with time. So that data you get from this is a graph, rather than a gel

How do we know that sequencing tech was developed before PCR?

RECALL - PCR is about amplifying segments. But there's sequencing tech that can tell us the exact sequence! You have to know the sequence to do PCR! Because you have to know sequence for primers, to know what primers to use for PCR (so sequencing was developed before PCR)

What is RNAi?

RNA interference; dsRNA complementary to mRNA sequence of interest is synthesized and transfected into human cells, where it separates and promotes degradation of target mRNA, creating a knock-out

Does RNAP need to unwind DNA to recognize and bind to a promoter?

RNA polymerase can recognize and bind to a promoter without unwinding the DNA Can initially attach to DNA anywhere along the molecule (can bind "no specifically"), but only loosely bound to the sugar phosphate backbone. Can slide along DNA. While its doing that, it can sample what the chemical features are of those major and minor groups, which are displaying the chemical groups sticking out on the edges of the base pairs. The chemical landscapes change as its moving along the DNA, and there are amino acid residues in the RNA polymerase that once they get to the region of the promoter, have R groups that are positioned exactly right to make some fairly strong non covalent interactions with the major groove. So then, RNA polymerase stops sliding, because it knows it's at a promoter, and there's a conformational change that basically triggers the ability of the RNA polymerase to peel open the DNA and create open the transcription bubble (the conformational change happens in the RNA polymerase)

Which is more error prone, RNAP or DNAP? Why?

RNA polymerase is more error prone than DNA polymerase, and it's thought that that is at least partly because it doesn't require a primer, and is just starting from scratch and because of the mechanism of doing that, is not as good at recognizing that it might be using the wrong bases DNA polymerase actually has built-in proofreading capability

In the case of lac operon: Why does it matter that the lac promoter is relatively weak?

Regulated by a repressor protein ("lac repressor") that binds to a sequence called the "lac operator" and is controlled allosterically by lactose. If lac repressor binds to to lac operator, it will block the ability of RNAP to transcribe the operon Also regulated by an activator protein ("CAP" protein) that is controlled allosterically by glucose (the lac promoter itself is relatively weak). Because weak, the promoter depends on the presence of the activator protein, in order to actually transcribe... in fact, it depends on the presence of the activator protein and the absence of the repressor protein! Because even if the activator protein is bound, the RNAP is not going to be able to bind, or at least not able to transcribe, if the repressor is bound to the operator

After the TATA box, how large is the remaining regulatory region? What sort of things are involved in this region, and why?

Remaining regulatory region is typically quite large (can be several thousands of basepairs and can even include sequences downstream of the gene's transcribed region!) Even though in this diagram, they're only showing upstream... So there can be a variety of regulatory proteins that combine in this very large regulatory region and influence the ability of the basal transcription machinery and RNAP to bind at the core promoter, and start transcription

Receptor-mediated endocytosis

Requires receptors sitting on surface of cell. When they respond to a molecule, a signal, whatever, then they will create a vesicle from the cell membrane that captures whatever the cell receptor responded to. This is how cells gather cholesterol for their membranes. Ex: LDL, HDL. LDL is the vesicle! There are LDL receptors that sit on the surface. When the lipoprotein (LDL) attaches to the LDL receptor protein, that signals the cell to go through endocytosis and bring in the LDL vesicle into the cell. There, they break it down, and the cell uses the cholesterol inside the protein to do whatever it needs. This also explains why having a large amount of LDL in your blood is an issue. Your cell is only going to endocytose cholesterol if it needs cholesterol. If it doesn't need it, it's going to send out a signal to stop producing LDL receptor proteins for the membrane. If it stops producing those proteins, then it's no longer taking in LDL. This causes LDL to go unused, and to start building up in your circulatory system. If you have that buildup, eventually it's going to have to deposit it somewhere, and since it's already in your circulatory system, that's where it's going to end up. And so begins the cascade of causing damage to the lining of your vessels, formation of clots, etc. TL;DR: it comes down to can the cell absorb enough LDL for normal function without much leftover? This is why excess LDL is a bad sign, because it means that you're either consuming too much, or your cells aren't using enough

What's the main issue with native gel electrophoresis? How do we get around this?

Resolution isn't very high with native - can't distinguish between difference of a single base pair (can do 500 from 550, but can't do 100 from 101). Need to use denaturing for this You can separate single strands of DNA or RNA from each other with very high resolution (can separate 100 vs 101). This ability to get very high resolution is what enabled the first methods of actually determining the nucleotide sequence of DNA molecules

Peroxisome

Responsible for oxidation of lipids. Process produces hydrogen peroxide, but peroxisomes have enzymes that break down hydrogen peroxide into water and oxygen.

Provide an example of next-gen sequencing. Explain: Should realize how, conceptually, this approach allows sequencing of many more DNA molecules simultaneously, and more quickly, than what came before

Reversible terminator ("Illumina") - Specific primers are not needed, because "linkers" are ligated to the ends of genomic DNA fragments; then the only primer needed is one that is complementary to the common linker that has been attached to all the fragments! - The linkers get covalently attached to the "flow chip" where the sequencing reactions will actually be done. Primers complementary to the linkers are used to amplify all of the fragments right on the chip prior to the sequencing reactions - Reactions are fully automated and milliions of reactions are done simultaneously - No electrophoresis is necessary - nucleotide addition is detected by flashes of light detected by a computer - Random fragmentation of the genome generates overlapping sequences that need to be assembled together to get the entire genome sequence - Complicated computer algorithms are used to do this. "Reads" in both directions (both strands) of any given region, as well as statistical criteria related to the number of reads in any given region are used to gain confidence in the deduced sequence's accuracy. (One of the biggest contributions to the genomics revolution was the development of such software as well as database software for organizing and analyzing all the information, and the availability of computers powerful enough to do all of this!) - Similar approaches can be used to sequence all the RNAs in a cell sample (RNA-seq)

What are the steps of Rho-dependent transcription termination? Describe

Rho factor will bind to the RNA that is hanging out of the RNAP at the recognition site, and it will use energy from ATP hydrolysis to chug along the messenger RNA in the 5' to 3' direction, so it's chasing along behind the RNAP, and eventually, it catches up to the RNAP, which is made more likely by signals in the DNA that cause the RNAP to pause temporarily, so when it catches up to the RNAP, it has a similar effect as the RNA hairpin did in the factor independent version of termination (allosteric effector causing conformational change that triggers termination) Rho unwinds DNA-RNA hybrid at the transcription bubble. Transcription is terminated. RNA polymerase, Rho factor, and the RNA strand are released Rho factor has helicase activity - this is what allows it to unwind the DNA-RNA hybrid (to peel the RNA product away from the template strand)

How does Rho unwind DNA-RNA hybrid? Where does it do this?

Rho unwinds DNA-RNA hybrid at the transcription bubble. Transcription is terminated. RNA polymerase, Rho factor, and the RNA strand are released Rho factor has helicase activity - this is what allows it to unwind the DNA-RNA hybrid (to peel the RNA product away from the template strand)

In one sentence, summarize the difference between Rho and factor independent transcription termination

Rho uses ATPase activity to chug along RNA and disassemble transcription complex at termination site. Factor-independent termination is mediated solely by interactions between the RNAP, RNA and DNA

How was Sanger sequencing discovered?

Sanger realized that you could take a piece of DNA... it was known that DNA polymerase needs a primer. Sanger realized that you could create a reaction in a test tube that had a template strand of DNA whose sequence you were interested in determining, and a single stranded fragment of DNA that was hybridized to it as a primer, and that if you put in DNA polymerase and nucleotides, the DNAP would extend it But he also very cleverly figured out that in order to extend the DNA chain - recall: DNA polymerase is always adding on to the 3' end of a growing chain. It uses the 3' end OH group at the sugar at the end of the chain to create a phosphodiester bond between that and the next nucleotide. Well, if you include in your mixture chemically created nucleotides that are missing the hydroxyl at the 3' position (these are called dideoxynucleotides, dDNTPs)....

Nexin

Scaffolding protein, connecting all microtubules in 9+2 arrangement (cilia, flagella). With the addition of ATP, nexin cause microtubules to slide against one another (one goes up, the other stays still). Linking proteins along the path ensure that when this shift occurs, the microtubules bend

Tight junction

Seals neighboring cells together in an epithelial sheet to prevent leakage of molecules between them. Bind outer membrane of each cell. Surround and line the entire cell itself (apical side) and attach to every neighboring cell. Forms a seal all the way around the cell walls, so nothing from the lumen can pass between the cells and get into the tissue or blood on the base-lateral side. Restricts movement within the plane of the membrane such that proteins (including channels and transporters) and phospholipids in the apical membrane domain cannot freely diffuse into the base-lateral domain and vice-versa. This is especially important in the lumen, because in the lumen, you don't want things to just flow into your body. For example, there's a lot of stuff that you eat that you don't want to let inside your body. So tight junctions prevent that type of thing.

[linking reaction] (general description)

Second step in cellular respiration, aka the aerobic catabolism of gluocse. Happens because the input for the citric acid cycle is not exactly the end product of glycolysis. Chemical reaction that turns the pyruvate into acetate (acetyl group), which is carried by coenzyme A into the citric acid cycle Oxidation of pyruvate by pyruvate dehydrogenase (a large multi-subunit enzyme complex). Kind of makes sense that this would be an important regulatory step, because it's at that decision point between "are we going to use this pyruvate to complete the aerobic catabolism, or are we going to stop here and use the fermentation process to get whatever energy we can out of the glucose, because we don't have oxygen available?"

What do we mean by "screen for plasmids with insert"?

So now I've got a bunch of different colonies on my plate, each represented by one of these spots. Each was formed from a single bacterium that landed on that plate, and had a single plasmid in it. I know that all of these have plasmid because I selected for the antibiotic resistance, but other than that, I have no way of knowing which one of them actually have insert in them, and which ones might be ones where the ligase just happened to seal the vector closed without getting an insert. SO, to determine that, we use color! Here, the blue and white is what we use to screen for whether the plasmids have inserts

Why do you need to denature the DNA with Sanger?

So that DNAP can get to work making complimentary strands!

What is the initial amino acid (the one that came in with the initiation complex) in a polypeptide chain? Where is it located?

So the initial amino acid that came in with the initiation complex, with the AUG recognizing tRNA, is a methionine - it's the amino terminus, and it's always at the very end of the chain that's being extruded from the ribosome

How does DNA polymerase add dNTP monomers to the growing chain?

So the parental DNA has a g, so we will need a C - incoming substrate is DCTP. As this gets added to that strand by the activities of the active site of the DNA polymerase enzyme, 2 of the 3 phosphates are going to get removed, and they'll get clipped off as a molecule of inorganic phosphate. As that happens, the remaining phosphate gets joined to the 3' hydroxyl of the existing DNA strand, to create the phosphodiester bond

Pre-mRNA splicing (voiceover)

Splicing is done by assemblies of RNA and protein called 'spliceosomes'. Spliceosomes are assembled from a number of smaller RNA and protein assemblies called snRNPs SnRNPs ("snurps") are small, nuclear, riboucleoprotein complexes that have the enzymatic activity to do the splicing. Like a ribosome, they are complexes of RNA and protein, and like the ribosome, the enzymatic activity that they have for doing the splicing is in the RNA part Multiple snRNPs recognize splicing signals in each intron in the pre-mRNA and interact with each other to form a 'spliceosome'. Multiple snRNPs have different jobs of recognizing different parts of a pre-mRNA. Some are specialized for recognizing the sequences at the junctions between introns and flanking exons, others are designed to recognize a particular sequence within the intron sequence... once they've all recognized the thing they're supposed to recognize, they all get together and assemble together to form the spliceosome, which then clips out the intron and splices the flanking exons together covalently with a phosphodiester bond connecting them, so it's continuous RNA The spliceosome cuts out the intron and splices together the ends of the flanking exons. The enzymatic activity is actually contained in the RNA part of the snRNPs, not the protein!

Replication: Initiation

Stage 1 of DNA replication Origins of replication (ORI): specific sites on DNA where replication begins; typically AT-rich Recognized by replication initiation factors (proteins); "melted" open by helicase enzymes (using energy from ATP hydrolysis) to form a "replication bubble" Each end of a replication bubble will be a "replication fork" where DNA synthesis will occur

What does population genetics study?

Study of properties of genes in a population

Why is the lacZ gene used in cloning?

Successful clones form white colonies

What is succinate dehydrogenase and why do we care?

Succinate dehydrogenase engages in a redox reaction that makes FADH2. It is the only enzyme of this cycle that is not floating free in the mitochondrial matrix (recall - NAD can float in and out, but FADH is a prosthetic group that stays in its protein), but rather is a peripheral membrane protein (enzyme) that uses FAD and is going to pass the hydrogens (electrons) from its FADH2 to a membrane protein that it is physically interacting with, rather than letting the FADH float away Only enzyme of the citric acid cycle that is not floating free in mitochondrial matrix

Where do all living things derive energy from?

Sunlight

Transcription Template, product, enzyme, raw material, direction of growth

Template: DNA Product: RNA Enzyme: RNA polymerase Raw material ("substrates"): nucleotides Direction of growth: 5' —> 3'

Differentiate between/related the following terms: mitotic chromosome, sister chromatids, chromosome, centromere region

Terminology note: a mitotic chromosome has two sister chromatids. As soon as they separate from each other, now each is known as a chromosome. And each one has its own centromere region

If you see multiple ribosomes on an RNA that's in the same image as DNA being transcribed by RNA polymerase, what do you know?

That it's a bacterial cell! Or a prokaryote of some sort

When is regulation at the translational step preferable?

That said, there certainly is regulation that happens at all of these other steps. In fact, regulating at the level of translation can be beneficial when a very quick response is needed, so you can already have mRNAs sitting in the cytoplasm, ready to be translated, if you need a quick response So regulation is not just happening at the transcriptional level! But that's what we're going to o focus on today, because that is a major level of regulation

How many phases does the cell cycle have? What do these four phases represent? Where do they occur?

The "cell cycle" has four phases These phases describe the phases of life of any cell type that undergoes continual rounds of being in interphase, and going through the process of cell division. Occur in any tissue where cells are actually actively reproducing.

How are Okazaki fragments converted into continuous DNA?

The "erasure" of the RNA primers and their replacement by DNA is done by different enzymes in prokaryotes and eukaryotes. You do not need to know the names of these enzymes; just know that the RNA primers are degraded and replaced with DNA. After this is done, adjacent Okazaki fragments (now completely DNA) are joined together by an enzyme called DNA ligase, using energy from ATP. (Ligase exists in both prokaryotes and eukaryotes; the word ligase derives from the Latin root "lig" meaning "tie" or "bind". Here, we're seeing a primer that was made by primase, and then we're seeing DNA polymerase having extended that primer, and bumped into the beginning of the next RNA primer. This causes the DNA polymerase to fall off, but it did not make any phosphodiester bond between that last nucleotide of DNA and the first nucleotide of RNA. Another enzyme comes along and degrades that RNA primer at that junction, between the Okazaki fragments, and another DNA polymerase comes in and removes the RNA and extends what was the 3' of the Okazaki fragment (fills it in with DNA). Then, yet another enzyme called DNA ligase comes in, and it's the enzyme that's able to make a phosphodiester bond connecting two adjacent nucleotides that are sitting base-paired to a template strand but haven't yet been covalently attached to each other. Uses energy from ATP hydrolysis to do that.

Describe the -10 box composition

The -10 box is all A's and T's When we talked about DNA replication, we talked about the origin of replication tending to be AT rich, because there are only 2 hydrogen bonds between A and T (makes it easier to open up). So similarly, the place where the DNA is going to get the nucleation/opening of the DNA to form the transcription bubble, is going to be at this AT rich -10 box

Describe what's happening with the ends of the RNA as the RNAP moves. What if we're talking about a prokaryote?

The 3' end of the RNA is displaced from the transcription bubble as the RNAP moves and grows the RNA at its 3' end. When the RNAP reaches a terminator sequence in the DNA, it will come off the DNA, letting the two DNA strands go back together, and releasing the RNA to float away As the RNA polymerase moves down the template strand, it peels the RNA that it has made off of the template strand, and allows the parental DNA to close behind itself, and it is wedging its way into the parental DNA in front of itself and opening it up, so the transcription bubble stays a relatively constant size until it reaches a terminator sequence If this is a prokaryote - now, the 5' end of the RNA is emerging from that transcription bubble, and is floating in the cytoplasm, which means a ribosome could grab onto it and recognize an AUG start codon and start making a protein and actually be chasing along behind that RNA polymerase that's transcribing the DNA When it reaches the terminator, it will release its RNA, it will release itself from the DNA, and let the DNA go back together

Where is the C terminus of a protein during the elongation phase (what is it attached to)? How is the next peptide bond fomred?

The C terminus is always during the elongation phase covalently attached to a tRNA within the ribosome, and the next peptide bond gets made by hydrolyzing that attachment to the tRNA, and attaching to the amino group of the next amino acid, which in turn is attached to a tRNA by its carboxyl group.

Give an example of operon regulation involving E. coli, glucose, and lactose

The E. coli Trp Operon is regulated by a repressor protein that is controlled allosterically by tryptophan Lactose is a disaccharide that can be catabolized to generate ATP, provided the appropriate enzymes are present However, if lactose and glucose are both present, glucose is the preferred energy source So only want to express lactose-metabolizing enzymes if lactose is present but glucose is absent The group of genes required for catabolism of lactose are grouped together in an operon called the lac operon Voiceover: We're looking at a period of time over the course of a few hours. At time zero, some E. Coli cells were in a growth medium that included both glucose and lactose. Three things are being monitored: The red is the amount of glucose in the medium, the blue is the expression of the genes in the lac operon (group of genes required for metabolizing lactose), and the yellow is showing the growth of the bacteria. What you see here is that these genes for metabolizing lactose don't start to be expressed really at all until the cell has depleted the availability of glucose... and at that point, the cells have been growing steadily, and then there' as lag while the cells gear up to express the genes to let them metabolize the lactose, and then the bacteria start to grow faster again... although, at a slightly lower rate than at what they were growing when they were metabolizing glucose, which goes along with the idea that glucose is the preferable energy source Sometimes, E. Coli might find itself in an environment where it doesn't have glucose in its environment, but it has lactose! There is an operon in E. Coli that will let it break down lactose, and get energy from it. But, if lactose and glucose are both present, we want to regulate this operon so that it only gets expressed if glucose is absent, and lactose is present

Where does catalysis occur?

The active site of an enzyme

What catalyzes the reaction that reads the bond between the amino acid and the tRNA? Why? How is this different than GTP hydrolysis?

The RNA molecules within the large subunit have peptidal transferase activity that catalyzes that reaction that reads the bond between the amino acid and the tRNA. It has particular chemical groups that are arranged in a particular way... it catalyzes the concurrent hydrolysis of this bond, and making the peptide bond between this and the next amino acid. The GTP hydrolysis is something totally separate - that's done by an elongation factor that brings the aminoacyl-tRNA in, and after a short period of time during which that aminoacyl tRNA and the elongation factor can float away if its not making the proper base pairing with the anti-codon, if it's sticking around because it's base paired there, then the elongation factor hydrolyzes its GTP and floats away, and that's the signal for the peptidal transferase to act (but the peptidal transferase is not using the GTP). And then that other elongation factor comes in and uses energy from GTP hydrolysis to push the ribosome to the next position (we don't need to know the specific names of elongation factors)

How is the ability of a repressor/activator to bind to DNA controlled?

The ability of a repressor or activator to bind to its site on DNA can be controlled allosterically Regulated by some small molecule that might be serving as what the cell wants to be detecting in order to control whether the gene should be expressed or not Remember - some allosteric effectors increase the ability of a protein to carry out its function, while others decrease that ability!

Where does RNAP II bind?

The core promoter (often containing an AT-rich region called the TATA box) is where RNAP II will bind

Interpret a Michaelis-Menten curve

The curve towards Vmax as substrate concentration is increased represents a gradual increase in the percentage of enzyme molecules that are occupied with substrate at any given moment The gradual curve reflects the affinity of the enzyme for the substrate So, we define the Km (the substrate concentration that allows this particular enzyme to operate at ½ of its maximum possible velocity) because at that substrate concentration, 50% of the molecules at any given moment are occupied with catalyzing the reaction Example: When [S] is very low, the rate (on y-axis) is much less than Vmax. When [S] is very high, the rate approaches Vmax (difference between measured rate and theoretical Vmax gets vanishingly small)

Nucleolus

The darker region inside of the nucleus where ribosomes are produced. Some cells have one, some cells have multiple (not membrane bound)

Interlocus distance (formal definition)

The degree of crossing over between any two loci on a single chromosome is proportional to the distance between them

Gene arrangements in prokaryotes vs eukaryotes? Why do we care?

The differences in genomes between prokaryotes and eukaryotes has important implications for the process of protein synthesis In prokaryotes, groups of genes related to a common process (e.g. synthesis of a particular amino acid, or uptake and catabolism of a particular sugar) are often clustered together in the genome as an operon that is transcribed from a single promoter, generating a polycistronic mRNA (multiple ORFs). In eukaryotes, there are no operons. Each gene has its own promoter and the resulting mRNAs are monocistronic. (Alternative splicing can lead to production of multiple different mRNAs from the same gene, but still each mRNA is monocistronic.)

How is DCO related to SCO?

The double crossover (can be?) the product of both single crossovers

Where does enzymatic activity take place in snRNPs?

The enzymatic activity is actually contained in the RNA part of the snRNPs, not the protein!!

What is the 3' overhang?

The enzyme that degrades the RNA primer comes along, and removes that primer, but there is no way for DNA to get filled in there! Because DNA polymerase always needs a primer! So we end up with a little area of single stranded DNA, that we can refer to as the 3' overhang, at the end.

Which enzyme is responsible for the elongation phase of DNA replication?

The enzyme that does elongation (synthesis of DNA) is called DNA polymerase

Cleavage furrow

The first sign of cleavage in an animal cell; a shallow groove in the cell surface near the old metaphase plate.

What's the first step in gene expression?

The first step in gene expression is transcription DNA is copied to make RNA —> more specifically, there is a particular class of RNA, called mRNA, where the m stands for messenger (this RNA is the messenger that's carrying the information from the DNA gene to the ribosome that will translate the information into a protein)

Describe anabolism

The flip side of metabolism are are metabolic programs/pathways that use energy to build up molecules from smaller ones. These pathways are endergonic. This is anabolism, and these pathways are called anabolic pathways - Other cellular work - Complex bio molecules - Mechanical work - Osmotic work

What tf is the lac operon? Why do we care? (How is it regulated, and by what?)

The group of genes required for catabolism of lactose are grouped together in an operon called the lac operon Has a promoter, and RNAP that we want to regulate whether it can bind or not, and it has this group of genes that are going to be transcribed Regulated by a repressor protein ("lac repressor") that binds to a sequence called the "lac operator" and is controlled allosterically by lactose. If lac repressor binds to to lac operator, it will block the ability of RNAP to transcribe the operon Also regulated by an activator protein ("CAP" protein) that is controlled allosterically by glucose (the lac promoter itself is relatively weak). Because weak, the promoter depends on the presence of the activator protein, in order to actually transcribe... in fact, it depends on the presence of the activator protein and the absence of the repressor protein! Because even if the activator protein is bound, the RNAP is not going to be able to bind, or at least not able to transcribe, if the repressor is bound to the operator

How does an enzyme's affinity for substrate impact the equilibrium between occupied and unoccupied enzyme? Why does this matter?

The higher affinity for the substrate, the lower the concentration of substrate needed to achieve 1/2Vmax (and therefore the lower the Km)

Sister chromatid pair vs homologous chromosome

The key difference between homologous chromosomes and sister chromatids is that homologous chromosomes may not carry identical information all the time whereas sister chromatids carry identical information all the time. Sister chromatids are used in cell division, like in cell replacement, whereas homologous chromosomes are used in reproductive division, like making a new person. Sister chromatids are genetically the same. That is, they are identical copies of one another specifically created for cell division. In fact, the term sister chromatid is only used during the parts of cell division when the structures are in that X shape, or when the two copies are connected by a centromere. On the other hand, a pair of homologous chromosomes consists of two non-identical copies of a chromosome, one from each parent. For example, one of your skin cells has a copy of chromosome number one from your mother and a copy of chromosome number one from your father. These are a homologous pair and present in all of your skin cells all of the time. But if you should happen to cut yourself, and skin cells are preparing to divide to heal the wound, then all 46 of the chromosomes in those cells have been replicated, and sister chromatids are present.

Replicon

The length of DNA replicated from a single origin

What do we mean when we say NTP?

The main point here is that we're using NTPs as the substrates. N just refers to any of the 4 possible nitrogenous bases

Describe what's happening with this graph

The more copies of the original target DNA that were present, the faster the cleaved fluorescent molecules appear, and the quicker the curve rises (goes up)

What's the relationship between amount of recombination and distance between two genes? How do we use this insight in practice?

The more crossing over that happens, the more of these recombinant gametes you will see. The farther apart two genes are, the more crossing over that happens. This is how we utilize this information for mapping

What's the elongation of translation?

The movement of a ribosome along an mRNA in order to add more amino acids to the polypeptide chain

What type of transport occurs on the electron transport chain?

The movement of protons by the ETC is active transport (creates a gradient). The energy for it comes from the favorable redox reactions mentioned above. (Although this particular source of energy was probably not listed among energy sources for active transport that you learned from Dr. G, it can be grouped with the category of ATP-driven pumps in the sense that in both cases favorable chemical reactions - as opposed to light or the energy stored in a gradient - are what drives the movement of a substance against its gradient.)

Leading strand

The new continuous complementary DNA strand synthesized along the template strand in the mandatory 5' to 3' direction

What is the nuclear pore looking for to allow mRNA to exit the nucleus?

The nuclear pore is looking for the 5' cap, the poly A tail, and evidence that splicing has occurred

What defines the number of chromosomes?

The number of chromosomes is defined by the number of centromeres

H1 histone

The only histone that is not in the nucleosome core (DNA is not wrapped around it. Ties the beads together in a string Not part of the core octamer, but each one binds to a nucleosome and causes it to interact with the adjacent nucleosomes so that ultimately that results in a super structured of a coiling of the nucleosomes relative to each other

Define a relationship between a proton gradient and ATP synthesis

The only thing coupling NADH and FADH2 oxidation to ATP synthesis is the proton gradient. AKA, a proton gradient is both sufficient and necessary for ATP synthesis by the F0F1 ATP synthase

In what instance is the lac operon transcribed?

The operon is only transcribed if the activator protein is bound to its recognition site on the DNA and the repressor protein is not bound to the operator

What do we mean when we say: Each protein in a cell is encoded by a gene in its DNA

The order of amino acids in a polypeptide (its primary structure) is encoded by a sequence of nucleotides in DNA. RNA is involved in extracting this information and translating it into an amino acid sequence.

Describe the process of NAD+ regeneration under anaerobic conditions

The oxidation of a variety of small organic compounds is a process that is utilized by many organisms to garner energy for cellular maintenance and growth. The oxidation of glucose via glycolysis is one such pathway. Several key steps in the oxidation of glucose to pyruvate involve the reduction of the electron/energy shuttle NAD+ to NADH. You were already asked to figure out what options the cell might reasonably have to reoxidize the NADH to NAD+ in order to avoid consuming the available pools of NAD+ and to thus avoid stopping glycolysis. Put differently, during glycolysis, cells can generate large amounts of NADH and slowly exhaust their supplies of NAD+. If glycolysis is to continue, the cell must find a way to regenerate NAD+, either by synthesis or by some form of recycling.In the absence of any other process—that is, if we consider glycolysis alone—it is not immediately obvious what the cell might do. One choice is to try putting the electrons that were once stripped off of the glucose derivatives right back onto the downstream product, pyruvate, or one of its derivatives. We can generalize the process by describing it as the returning of electrons to the molecule that they were once removed, usually to restore pools of an oxidizing agent. This, in short, is fermentation. As we will discuss in a different section, the process of respiration can also regenerate the pools of NAD+ from NADH. Cells lacking respiratory chains or in conditions where using the respiratory chain is unfavorable may choose fermentation as an alternative mechanism for garnering energy from small molecules. In general, cells try to maintain a balance or constant ratio between NADH and NAD+; when this ratio becomes unbalanced, the cell compensates by modulating other reactions to compensate. The only requirement for a fermentation reaction is that it uses a small organic compound as an electron acceptor for NADH and regenerates NAD+. Ex: the reactants are pyruvate, NADH, and a proton. The products are lactate and NAD+. The process of fermentation results in the reduction of pyruvate to form lactic acid and the oxidation of NADH to form NAD+. Electrons from NADH and a proton are used to reduce pyruvate into lactate. If we examine a table of standard reduction potential, we see under standard conditions that a transfer of electrons from NADH to pyruvate to form lactate is exergonic and thus thermodynamically spontaneous. The reduction and oxidation steps of the reaction are coupled and catalyzed by the enzyme lactate dehydrogenase.

In what direction does the ribosome move along mRNA?

The peptide chain grows N®C (new amino acids always added at C terminus), as ribosome moves in 5'®3' direction along mRNA Growing chain always attached, via its C terminus, to tRNA

M phase

The phase of the cell cycle that includes mitosis and cytokinesis

Chiasmata (structure, form, function)

The point of crossing over is called chiasma, or chiasmata Chiasmata are points of genetic exchange. Physical exchange of information. You have a molecule that is complexed around these proteins to form this condensed chromosome. When they cross one another, there's actual breakage and rearrangement of the molecule. The closer you are, the less likely you are to have this crossing over occurring

Product law

The probability of two independent events occurring at the same time The probability is the product of both of those events

What does it actually mean for different promoters to have different nucleotide sequences?

The sequences we see on this slide are called consensus sequences... different genes have slightly different promoters. They will resemble these sequences (on the slide), but a given gene might have one or two base pairs in their -10 and -35 boxes that differ from these consensus sequences... and still be able to be recognized by the holoenzyme, albeit maybe not quite as efficiently. So this makes some promoters better at attracting the holoenzyme than others

Can we get more detail about ribosome small subunit locating start codon in proks? Why can't proks use the same approach as euks?

The story in prokaryotes is different, because prokaryotic RNAs are very often polycistronic. That means that if prokaryotic ribosomes used the same approach that eukaryotic ones do, only the first ORF in any prokaryotic messenger RNA would ever get translated! So we can't use that approach In prokaryotes, rather than this initiation complex that has the initiator tRNA bound to it - rather than its binding to the 5' end and just scanning for the first AUG, it floats around and just bumps into the messenger RNA at random places. What it's looking for is particular sequences just on the upstream side of an AUG start codon. Bind at any ribosome binding site, "Shine-dalgarno sequence" So this complex of initiation factors and small ribosomal subunit and initiator tRNA goes bumping and sliding around and there's an equal probability that any one of the ORFs at any given moment will have a ribosome bind to it and start translating This polycistronic RNA could have multiple polysomes attached to it in a prokaryote, and multiple groups of polysomes... wherein each group would each be making a different kind of protein...

Describe the direction of the growth of a new strand of DNA. How is each new nucleotide selected?

The structure of the parental DNA strand shown is 5' to 3', from bottom to top, and growth of new strand is from top to bottom. It's showing us that each new nucleotide that's going to be added to this growing DNA strand is going to be selected based on its ability to base pair with the next nucleotide on the parental DNA strand, and the incoming substrate for this enzyme is a nucleoside triphosphate

Basic overview of citric acid cycle

The two carbons of the acetate group of acetyl CoA get joined to a 4 carbon molecule to make a 6 carbon molecule called citrate, and then a series of reactions happens that one at a time take a carbon off of these so we get back to a 4 carbon molecule. 3 steps that are redox reactions that produce NADH, and 1 that produces FADH2. There is also a substrate-level phosphorylation step that makes GTP

Once a given chromosome has been replicated in S phase... what happens to the two identical chromosomes?

The two chromosomes, that are identical to each other (sister chromatids), are actually tethered to each other by some proteins as soon as they're replicated during S phase. They never separate from each other physically until the appropriate point during mitosis

Oxidative metabolism

The use of oxygen to breakdown carbohydrates, fats, and proteins to produce energy. This is how mitochondria produces ATP. A chemical process in which oxygen is used to make energy from carbohydrates (sugars). Also called aerobic metabolism, aerobic respiration, and cell respiration.

At what point in cellular respiration is the majority of ATP made? Why/how?

The vast majority of ATP production occurs at the oxidative phosphorylation stage, as a result of electrons being transferred from NADH and FADH2 onto O2 to make H2O

What's happening with the chromatin as DNA is replicated?

The way we've learned it implies that you have to unpackage an entire chromosome from its chromatin structure in order to do that, but we already said that an entire chromosome wouldn't fit in a nucleus? So even though schematically, this is what's happening, in real life in the cell, almost all of the DNA is still compacted into chromatin, and only the little bits at a time that need to be replicated are unwound from their nucleosomes. And then nucleosomes are placed on the new DNA very soon after its formed

How many aa-tRNA synthetase enzymes are there? How specific are they? How are they named?

There are 20 different aa-tRNA synthetase enzymes - one for each of the 20 amino acids. Each is extremely specific - will only bind to that one amino acid, and will only attach it to tRNAs whose anticodons will recognize codons for that amino acid. The generic name for tRNAs that have amino acids attached is aminoacyl-tRNA Each of the 20 aa-tRNA synthetase enzymes also has a specific name - e.g. "tryptopanyl-tRNA synthetase" is the enzyme that attaches tryptophan to tRNAtrp to make trp-tRNAtrp.

How do cells choose whether to pursue catabolism or anabolism?

There are catabolic and anabolic pathways, and cellular conditions and the role of the particular tissue or organ in the body will help make those decisions about which pathways are used. Choices made to pursue catabolic pathways include cellular respiration

How many kinds of aa-tRNA are there? How are they named? Can you give an example?

There are enzymes called aminoacyl-tRNA synthetases (aka aa-tRNA). All it means is that these are enzymes that allow the synthesis of aminoacyl-tRNAs, which is the generic name for an amino acid covalently bound to a tRNA. There are 20 different aminoacyl-tRNA synthetases, one for every amino acid. They very specifically bind to that amino acid, and they're able to bind to any and all of the tRNAs whose anti-codons are going to be specific for that amino acid. So, each one has a name that mentions its amino acid. ex: tryptophanyl tRNA synthetase is the enzyme that covalently attaches the amino acid tryptophan to any tRNA whose anti-codon should be able to recognize a codon for tryptophan). The tRNA itself usually is designated with a superscript representing the codon that its anti-codon is going to be specific for, so it's written like tRNA^(superscript abbreviation, e.g. trp). Once the amino acid is attached, the aminoacyl tRNA is now tryptophanyl-tRNA^Trp

What's a recursive thought about ribosomes and protein synthesis that we can reflect on?

There are genes in the nucleus that are being transcribed and translated by existing ribosomes in the cytoplasm, but these proteins are destined to become parts of newly assembled ribosomes. So ribosomes are in the process of actually making parts for new ribosomes

Are there cell types that don't undergo the cell cycle?

There are many cells that actually don't reproduce - they've reached their final, mature, differentiated form. Suffice to say there are a lot of cells that aren't going to be dividing anymore

Describe catabolism

There are many metabolic pathways that break down large molecules to smaller ones, and extract energy from them, because generally the reactions that are breaking them down are exergonic. This energy can be harvested in the form of ATP. This is catabolism, and these pathways are called catabolic pathways -Stored nutrients - Ingested foods - Solar photons

What types of connections are present in the mitotic spindle?

There are other microtubules extending from the poles that don't connect to the chromosomes, but instead connect to each other non-covalently (actually, connections to kinetochores are also non-covalent connections)

Describe the quality control system of the nuclear pore complex

There are proteins that bind to the 5' cap and the poly A tail, and even proteins that bind to the junctions where two exons were spliced together, and proteins at the nuclear pore complex check for the presence of all of those things before they let the messenger RNA out into the cytoplasm Also, factors that are required to help the ribosomes start translation also bind to the 5' cap and the poly A tail and so those modifications, because of that, play a role in the translation process

Use the following terminology to describe the structure of a ribosome: E-site P-site A-site Large subunit Small subunit mRNA binding site Peptidyl transferase

There is kind of a channel through the small subunit, where the mRNA is going to be pulled through like a tape being read. There are also 3 important sites that kind of traverse parts of both the large and small subunits, called the A site, the P site, and the E site. The A-site stands for amino acid site. This is where the next amino acid attached to its tRNA is going to first enter the ribosome. The P-site, which stands for peptide site, is where a tRNA that is carrying the peptide chain that has been made so far sits. The peptidyl transferase active site is at the interface between the P and A sites. Finally, there's an E-site, where a tRNA, after the amino acid that it's carrying gets removed from it, will go to and be ejected, or exit the ribosome

What is translocation?

Thereafter, aa-tRNAs are delivered to the A-site by proteins called elongation factors. The elongation factors use energy from GTP hydrolysis to deliver the incoming aa-tRNA, and also use energy from GTP to push the ribosome over to the next codon (this process is called translocation).

Antiport (+ provide an example)

This is a form of coupled transport. A membrane transport process that carries one substance in one direction and another in the opposite direction. "Co-transported ion" Ex: Na+/K+ ATPase pump Single most important transmembrane protein in the body (according to Dr. G), because this moves Na+ from the inside of the cell out, and K+ from the outside of the cell in. This pump creates the Na+/K+ gradient mentioned previously. This pump is constantly going. Theorized that 50% of all cellular energy, in all cells, is keeping this pump going. Uneven exchange (2 K+ pumped out for 3 Na+ pumped in). Binding sites for Na+, K+, and ATP: energy from ATP hydrolysis drives movement of 3 Na+ out, and 2 K+ in, both against their concentration gradients. Covalent attachment of phosphate to the protein (and subsequent removal) cause conformational changes that drive the process. phosphorylation!!!

Symport (+ provide an example)

This is a form of coupled transport. A membrane transport process that carries two substances in the same direction across the membrane. Active, because you're going against the gradient of one of the molecules · Ex: Na+-glucose symport. Na+/Ka+ gradient allows us to do work. One way to do this is with a symport, specifically the Na+-glucose symport, which moves sodium and glucose in the same direction. Uses the concentration gradient of sodium to move the glucose inside of the cell (moving against the glucose gradient). (Ex: liver cell! Always has a lot of glucose within the cell. Liver cell is one of the main locations of energy storage. Loves to horde sugar.) So the cell uses the concentration gradient of Na+ to pump glucose into the cell, against the glucose gradient, and then the pumps pump the sodium back out (thus maintaining sodium gradient outside the cell, but keeping glucose inside of the cell)

Describe what's happening in this electron micrograph of protein synthesis in a prokaryotic cell. How can you tell which way the RNA polymerase is moving?

This electron micrograph is showing a particular gene in a bacterial chromosome that is in the process of being concurrently transcribed and translated. So every place that you see one of these big black blobs extending from the DNA chain, that's where an RNA polymerase is busy transcribing, and the big black blobs are the ribosomes. In this diagram, the RNA polymerase molecules are moving to left to right, which you can tell because the RNA gets longer as you move to the right. Promoter is therefore closer to the shorter RNA, and the terminator is closer to the longer RNA. Ribosomes have latched onto the 5' end of the RNA, and they're moving along... chasing along behind the RNA polymerase that's making that RNA, and as each ribosome has moved along that RNA, another ribosome can get on and start translating. We call these groups of ribosomes that are translating the same messenger RNA 'polyribosomes' Basically, they re just all latched on to the same strand of messenger RNA emanating out from the template gene. Can also call them polysomes Ribosomes are moving with RNA polymerase. All of the black glom strands that we see here - they're all making the same protein. It's hard to see, but each ribosome as its moving along (and has moved farther along the RNA) has already synthesized a greater amount of the protein (you see more of it sticking out from the top of the ribosome). They are all copies of the same protein in the process of being made We already mentioned that proteins are synthesized from their N terminus to their C terminus. So it's the N terminus sticking out of these ribosomes, and the growing C terminus is within the ribosome, having amino acids added to it. It's the 5' end of the messenger RNA that's sticking out, because RNA is synthesized 5' to 3' so it's the 3' end that's in the active site with RNA polymerase that's being extended by adding additional nucleotides to it Recall: all of the above is in a bacterial cell! Where you can see multiple ribosomes on an RNA and also in that same image see the DNA itself still being transcribed by RNA polymerase

When does crossing over/recombination happen?

This is all happening in meiosis I, when we have the homologous pairs forming tetrads

DNA hybridization (define)

This is another important concept that came out of our basic research into the structure of DNA and how it works! Use renaturation as a tool to fish for the complementary strand of a probe sequence Based on the idea that two strands of DNA are complementary to each other, or a messenger RNA that was transcribed from a DNA molecule is complementary to one of the strands of that DNA molecule, means that you can use a piece of DNA or RNA as a tool to go fishing for its complementary sequence

Why does the substrate concentration need to approach Vmax vary considerably between different enzymes?

This is because some enzymes have much higher affinities for their substrates than others (those that have lower affinities need higher concentrations of substrate before showing any appreciable binding to substrate)

Cell junctions (function, 3 classes)

This is how cells connect to one another. 3 classes: tight junctions, adherens junctions, communication junctions

How do restriction enzymes create recombinant DNA?

This is illustrating the idea that when you cut a piece of DNA with a restriction enzyme, it creates this thing called 'sticky ends,' because the two strands of DNA were cut at staggered- positions, so each end of double stranded DNA that's generated has a bit of an overhang that is still complementary to the other piece that it used to be attached to So then, if you take some other DNA that you're interested in, and cut it with the same restriction enzyme, then you can mix those DNAs together and add DNA ligase, and lo and behold now two different DNA molecules that didn't used to be attached to each other are now covalently attached to each other in a single, continuous double-stranded DNA This is most commonly done with a cloning vector aka plasmid

Regulation by phosphorylation

This is regulation by covalent modification - the addition of a phosphate group from ATP onto the R group of an amino acid. Specific enzymes are needed to do this (protein kinase, protein phosphatase)

Describe each part of this diagram of RNAP moving along a DNA template

This is showing that this RNA polymerase has initiated transcription somewhere upstream of here, at a promoter. It's moving along in this direction to the right, downstream, towards a terminator. It has a transcription bubble that it's peeled open, and as it's moving forward, it's unwinding the DNA in front of it, and it's rewinding the DNA behind itself. It has an active site, where nucleotide substrates (NTPs) are entering through a tunnel to get to the active site, and get added to the 3' end of the growing RNA chain. And the 5' end of the RNA chain is being extruded through a channel as this complex moves along. The shape that they determined through crystallography reminded people of jaws - the enzyme is clamped around the DNA, so that it's holding it... in a way kind of like the sliding clamp that the DNA polymerase had, it's tethered, but this here is actually part of the RNA polymerase. So it's moving downstream

Describe all elements of this image. What phase of mitosis is shown here? What are all components? (lines, black dots, etc)

This is the mitotic spindle at the culmination of metaphase, once the tug of war has succeeded in lining up all of the chromosomes at the equator (also called the 'metaphase plate'). There are the two poles, lined up, with their centrioles inside of centrosomes, and with microtubules emenating from them. Each sister chromatid pair is attached to both poles by microtubules referred to as 'kinetochore microtubules,' but also there are other microtubules extending from the poles that don't connect to the chromosomes, but instead connect to each other non-covalently (actually, connections to kinetochores are also non-covalent connections). The black circles bridging the 'polar microtubules' are motor proteins, and they help the microtubules slide relative to each other (see - Dr. G lecture). In addition, at least in animal cells, there are also microtubules eminating from the spindle poles that are neither kinetochore microtubules nor polar microtubules, and are referred to as 'aster' microtubules, or 'aster fibers.'

How do the mitochondria factor into metabolism?

This is where the important final stages of the aerobic catabolism of glucose occurs. Pathway starts in cytoplasm, but ends up in the mitochondrion

What is happening at a replication fork?

This is where the new DNA is being synthesized! In real life, both new strands (red) are being synthesized at the same time. For our purposes, we'll start at the 3' end This is representing one end of a replication bubble. It's pointing out to you that the growth of the fork is going to be towards the right. And it's showing you polarity of the strands of the parental DNA (one strand has 5n end going in that left direction, 3' to the right, and the other parental strand is anti parallel to that one), so it's also then showing the polarity that the newly-synthesized DNA strands have to have, in order to be anti-parallel to the parental strands, and we're going to start out at the 3' end of the newly synthesized strand, where a new DNA strand is being grown in the 5' to 3' direction, meaning that growth is at the 3' end

Relate relative DCO frequency to probability?

To bring it back to probability... If we have 2 independent events occurring, the probability of them both happening is going to be lower than either of the one happening on its own. If we are trying to use this mapping technique to look at the order of 3 different genes on the chromosome, the frequency of these double crossover gametes should be very low. Mathematically, it should be the product of the two single crossovers, but we're going to see that that doesn't always hold water

Compare/contrast transcription & translation on the following criteria: Template, product, enzyme, raw material, direction of growth

Transcription - Template: DNA - Product: RNA - Enzyme: RNA polymerase - Raw material ("substrates"): NTPs - Direction of growth: 5' —> 3' Translation - Template: RNA (more specifically, mRNA) -Product: protein (polypeptide chain whose amino acid sequence is dictated by the sequence of codons in the messenger RNA) - Enzyme: peptidyl transferase (within the ribosome. The machine that does it is the ribosome, but the particular enzymatic activity that actually creates peptide bonds is called peptidyl transferase) - Raw material ("substrates"): amino acids (not just plain old amino acids, though - amino acids that are covalently attached to tRNAs. They're called amino acyl tRNAs) - Direction of growth: N —> C

In one sentence, what's up with transcription and translation in bacteria?

Transcription and Translation Are Coupled and Occur Simultaneously in Bacteria

tRNA

Transfer RNAs Carries amino acids to the ribosome Many types of tRNA, each specific for a particular amino acid. These are the molecule that are... they're going to basically serve as the genetic code table for the ribosome, because they have a bit of a little sequence of three nucleotides in them that are going to be complementary to a codon on the messenger RNA. And, each tRNA molecule (many different ones) has a little region called an "anti codon," which is complementary to a codon in the messenger RNA, and that tRNA is going to have the amino acid that corresponds to the codon that its anticodon will base pair to covalently attached to it, and it will carry that amino acid into the ribosome, and allow the ribosome to add that amino acid to the protein

Metabolite transporter

Transmembrane component of lysosome. Allows monomers of broken down particles out of the lysosome and into cells where they can be used

How are transmembrane proteins held in place?

Transmembrane proteins have hydrophobic regions (alpha helices). These region allow them to embed in the hydrophobic interior of the bilayer.

So how do large, uncharged molecules get through cell membranes and into the cell?

Transmembrane proteins!

What is the function of endo/exocytosis?

Transport across membranes of bulk material

How many ways are there for transcription to terminate in prokaryotes?

Two!

Adherens junction

Type of adhesion junction. Bundle of actin filaments that connect an actin bundle in one cell to a similar bundle in a neighboring cell.

What happens during glycolysis under anaerobic conditions?

Under anaerobic conditions, pyruvate goes into the mitochondrion, where pyruvate dehydrogenase converts pyruvate (3C) into acetate (2C) + CO2 (acetate and carbon dioxide) Pyruvate processing into acetate and CO2 happens in the mitochondria in eukaryotes and in the cytoplasm of prokaryotes

Types of transport carriers? (3)

Uniport, symport, antiport

Why do we care about pedigrees? What is required when writing them?

Utilizing a pedigree first requires that we use more or less the same patterns, in terms of writing pedigrees. Pedigree analysis is an important part of understanding genetics, and specifically how it applies to humans traits and disease states.

What's the difference between RNA polymerase and primase?

Unlike the RNA polymerase that makes RNA copies of genes for subsequent translation, primase does not need to recognize a specific nucleotide sequence telling it where to start copying. Primase is also designed to only synthesize a short stretch of RNA before falling off the template strand, whereas the RNA polymerase for gene expression will keep going until it recognizes a signal telling it where to terminate transcription.

What does it mean for genes to be 'unlinked' and how does this relate to Mendel?

Unlinked genes on different chromosomes assort independently This is one of the major tenets of Mendelian inheritance

What is chromatin-immunoprecipitation?

Use antibodies to find out what proteins are interacting with different parts of the genome

Can you give an example of a way in which recombinant DNA technology has been majorly helpful?

Use of Recombinant DNA Technology to Overexpress and Purify Large Quantities of a Desired Protein (e.g. Human Insulin!)

How might we use microarrays to examine differences in gene expression? Give an example

Use of this technology to compare what messenger RNAs are present in different amounts in a tadpole vs. the fertilized egg it came from A microarray is a small rectangular chip. Each spot on a DNA microarray has many copies of one gene (covalently attached to the spot) from the frog genome Recall: as with other organisms, all the cells in a frog contain the whole genome ·You take your fertilized egg and your tadpole, and you isolate mRNAs from cells at early stages of development. Each mRNA sample represents all the genes expressed in the cells at that stage Use reverse transcriptase (recall - telomerase is a reverse transcriptase that carries its own RNA template. Also, one of the exceptions to the central dogma... is retroviruses, because they have reverse transcriptase enzyme!)... convert mRNAs to cDNAs by reverse transcriptase, using fluorescently labeled deoxyribonucleotide triphosphates (provide these dNTP substrates to the enzyme) Add the cDNAs to a microarray; fluorescent cDNAs anneal to complementary sequences on the microarray First denature the cDNA to ensure that they're single stranded So any DNA strands in cDNA ample that are complimentary to the chip will base pair and sit on the chip at the places where there are sequences complimentary to it, so you can wash away any of the cDNA that didn't base pair to something on the chip (this is called "removal of unhybridized cDNAs) Results: each fluorescent spot represents a gene expressed in the cells Colors: Unchanged expression: yellow Higher-expression at a single-cell stage: green Higher expression at tadpole stage: red Think of it like a continuum of expression... with red at one end and green at the other

ATP-driven pump (active transport)

Uses ATP to drive something against concentration gradient. ATP-driven pump creates concentration gradient, and coupled transporter takes advantage of this concentration gradient. Basically, these two types work together a lot

What does Vmax depend on?

Vmax (rate of reaction) depends on turnover # and enzyme concentration, and the substrate concentration needed to approach Vmax varies considerably between different enzymes

How is translation terminated? (voiceover)

We're moving along in elongation, and we get to a point where the codon that's sitting in the A site of the ribosome is not one that is recognized by any tRNA. It's one of the three stop codons. And it turns out that there's a protein called a release factor that goes into the A site and recognizes those stop codons (any one of those stop codons) What this release factor does is - it activates the peptidal transferase activity in a way that causes it to only hydrolyze the bond that's attaching the polypeptide chain to the tRNA that's in the P site, so that releases the completed polypeptide chain, and the ribosome itself falls apart, and the small and the large subunits are ready to move along and to start over again

Facilitated diffusion (passive transport) What is it, how does it work, which type of transmembrane proteins are involved?

What is it: Molecules will move without additional energy being put into movement. A specific protein acts as a carrier or channel to facilitate the membrane for a specific solute How does it work: Net movement by diffusion occurs only in the direction favored by the concentration gradient ("passive" - no energy input). Without a concentration gradient, there is no means for these proteins to transport anything. Concentration gradient somethings referred to as "chemical gradient," or if charged ions are involved, the "electrochemical gradient". This is called diffusion: movement from high concentration to low concentration What type of proteins are involved: Carrier protein, channel protein

What is a two-point cross? Why do we care? Can you give an example?

When 2 gene loci are examined When we are looking at any two genes, there is a way that we can utilize the amount, or frequency of crossing over, in order to map these genes - in order to say how far apart they are, relatively, on a chromosome Example: bugs on slide Different body colors, different wing shapes. Starts off with a fruit fly that is heterozygous for both of these traits (showing dominant normal) Fruit fly people use + to indicate dominant allele. If we look at F1 generation (offspring of two true-breeding fruit flies...) F1: b+b vg+vg But if we look at the gametes that are produced... · b+vg+ (parental type) · bvg (parental type) · b+vg (recombinant) · bvg+ (recombinant) Crossing over occurred when F1 individual underwent meiosis

How does a catalyst impact the overall energy of a reaction?

When a catalyst decreases the activation energy, it decreases the energy for both directions of a reaction

Describe how dyenin walks on microtubules

When doublet is isolated, ATP is harnessed and allows dyenin (the motors) to start "walking" towards the minus end of the microtubule (the "walking" motion is actually conformational changes in the motor protein). Doublets are held together by nexin - this causes sliding and bending.

What is incomplete linkage? Why do we care?

When you have two genes on the same chromosome, if they are really tight together, they very well move as if they were completely linked. But if there's any distance or any space between them, you already know that crossing over happens. And when that happens, we start to see recombination here We are going to look at recombination mathematically. We are going to look at the amount of recombination as a measure of how far apart two genes are. Happens as a result of crossing over. Genes far apart on the same chromosome have incomplete linkage Crossing over leads to recombination - Ex: § Parental gametes (AB and AB) § Non-parental gametes (aB and Ab)

What are we assuming re: alleles when discussing HW equilibrium?

When you're thinking about any population, if we're talking about a gene that only has two alleles, and those alleles have a simple dominant-recessive relationship, HW equilibrium works really nicely, and all of these formulas fit. For all of the other modes of inheritance that we talked about, or multiple alleles, or anything like that, this gets much more complicated. It can still be done - we're just not going to do it. So, we are going to assume that we are looking at genes that have just 2 alleles, and that one is dominant over the other. We also have to assume that there's no epistasis going on, no pleiotropy going on, back to the very beginning with Mendelian inheritance

How do X-linked traits appear in pedigrees?

X-linked traits in a pedigree will be predominantly males, but it doesn't have to always be males

Can primase start chains de novo? How about RNA polymerase?

Yes

Do dominant autosomal traits appear equally between the sexes?

Yes

Do recessive autosomal traits appear equally between the sexes?

Yes

Is the promoter included in the operon?

Yes

Do plants have mitochondria too? What do they use theirs for?

Yes! But they make their own sugar (and starch) rather than needing to eat it, like us. They do this by photosynthesis that occurs in their chloroplasts (use energy from light to make the sugar/starch that can be used by their mitochondria for energy)

What do we mean by "creating a "Library" of Cloned Genomic DNA Fragments"?

You can isolate whole genomic DNA, and cut it with a particular restriction enzyme, so that you've got a mixture of all the possible DNA fragments from genome whose ends correspond to the cleaved ends made by that restriction enzyme. And I cut my vector plasmid with that same restriction enzyme, and I put this whole mixture of DNA into a test tube with a bunch of my cut plasmid molecules, and add DNA ligase, and now I've got this whole complicated mixture of plasmids, some of which might have just sealed back together without getting mosquito DNA inserted, but many of whom will have mosquito DNA inserted. And if I do a large enough sample, I will hopefully have a representative of every single genomic fragment of the mosquito DNA in this library (in this collection that I'm going to put into the bacteria to make my library). So then I take these plasmids that I've sealed up with ligase in my test tube, and there are some chemical procedures you can use to make bacterial cells take up DNA. And I put them on a Petri dish that has an antibiotic in it that my plasmid is encoding resistance for, so that only bacteria that successfully took up my plasmid are going to grow on this plate

What type of internal controls are involved with real-time PCR?

You do this to put internal controls - you can be detecting multiple amplified fragments at the same time, and compare what time they start to be detectable. You basically spike your sample with known amounts of certain fragments, and that lets you calibrate! Oh, if there was one of these fragments per microliter at the start of my reaction, that one that I know that I put in one copy of... that appeared at this time, and took an hour to show up. One that I put in ten copies of per microliter, that one showed up at maybe 30 minutes instead of at an hour, and that one showed up in 10 minutes... so that lets you calibrate and then you compare the times of appearance of the unknowns that you're interested in, and then compare to known quantities that you had put in QPCR is currently a state of the art technology for measuring quantities of target sequences that you're interested in quantifying

Are repressors and activators present all the time?

You should assume that these repressor or activator proteins themselves are present all of the time, but their ability to bind to their recognition sites on the DNA is being controlled allosterically by some small molecule

Is DNA gel electrophoresis the only type of electrophoresis that can be used to study DNA?

You should realize that this is only one type of electrophoresis that can be used to study DNA. This here is called 'native' electrophoresis, because the DNA molecules are in their native, double stranded state when you do this type of electrophoresis

What are consensus sequences?

[Awaiting Judy response]

What is a microarray?

a grid of DNA segments of known sequence that is used to test and map DNA fragments, antibodies, or proteins.

Protofilament

a linear chain of protein subunits joined end to end, which associates laterally with other such chains to form cytoskeletal components (microtubules)

What is a point mutation?

a mutation affecting only one or very few nucleotides in a gene sequence

What does the A site do? What does the A stand for?

amino acid aminoacyl-tRNA binding site

What is a sticky end?

an end of a DNA double helix at which a few unpaired nucleotides of one strand extend beyond the other (result of restriction enzyme cut)

Founder effect

change in allele frequencies as a result of the migration of a small subgroup of a population

Exon

expressed sequence of DNA; codes for a protein

mRNA

messenger RNA specifies amino acid sequence of a polypeptide Used by ribosomes to provide the information for synthesizing a protein

RNAP III

tRNA. Recognizes promoters tRNA

Genome

the complete instructions for making an organism, consisting of all the genetic material in that organism's chromosomes

Proteome

the entire complement of proteins that is or can be expressed by a cell, tissue, or organism

Genome annotation

the process of identifying the location of genes and functional sequences within the genome sequence Genome annotation is the process of identifying functional elements along the sequence of a genome, thus giving meaning to it. It is necessary because the sequencing of DNA produces sequences of unknown function

Name the 7 enzymes that can be used for manipulating DNA

•Endonucleases •Exonucleases •Kinases, phosphatases •Ligase •Topoisomerases •Polymerases