Exam 2

Only <2% of human genomic DNA is transcribed into mRNA and then used to make protein however, most of the human genome is transcribed into other kinds of RNA that is crucial for normal development (scientists don't yet fully understand why this global transcription is necessary). This is why histones must be shuffled around constantly> for RNA polymerase to access these regions.

% of total genome that does not code for a protein, pretty good predictor of complexity, other vertebrates are fairly high but not as high as human, other organisms have very efficient gnome Total number of coding genes between humans/salamanders (more genes, larger genome), but humans have more non-coding information which likely results in more, histones have to look after entire genome because still large amount unknown Epigenetics - the study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself

DNA replication in Eukaryotes is more complex than that in Prokaryotes. Can you give three ways this is so?

- 1. More DNA than prokaryotic cells 2. Linear chromosomes (must deal with ends, no space to put a primer to work last little bit, telomerase is used to deal with linear ends) 3. DNA complexed with nucleosomes 4. Eukaryotic chromosomes contain multiple ORIs 5. Many more genes and thus proteins required DNA, linear, nucleosomes, ORIs, genes DOGLN

What is chromatin? What do I mean by "chromatin is dynamic in Eukaryotes"?

- Chromatin - complex of DNA, RNA, histones, and nonhistone proteins that make up uncoiled chromosomes, characteristic of eukaryotic interphase nucleus

Know how chromatin state dictates gene expression: RNA polymerase requires access to genomic DNA to generate RNA messages. DNA that is packed up (in chromosomes or heterochromatin like a centromere or telomere) or that has been chemically modified (e.g., methylation) is functionally silent, despite the presence of genetic information that is stored within.

- Chromosomes cannot be easily accessed, chromatin can be more easily accessed than chromosomes

In general terms, what kind activity do Prokaryotic DNA Polymerases I, II, IV, and V account for?

- DNA Pol I - elongate existing DNA strand, remove RNA primer and replace with DNA, demonstrates 5' to 3' exonuclease activity DNA Pol II - elongate existing DNA strand, DNA repair that has been damaged by external forces, encoded by a gene activated by disruption of DNA synthesis at replication fork DNA Pol IV - DNA repair that has been damaged by external forces DNA Pol V - DNA repair that has been damaged by external forces

Know what Okazaki fragments are, and why there needs to be a "lagging strand."

- Discontinuous DNA synthesis of the lagging strand, some of newly formed DNA hydrogen bonded to template strand is present as small fragments containing 1000 to 2000 nucleotides, RNA primers are part of such fragments, these pieces are called Okazaki fragments are converted into longer and longer DNA strands of higher molecular weight as synthesis proceeds Lagging strand requires enzymes able to remove RNA primers and unite Okazaki fragment into lagging strand must work from 5' to 3'

Know why DNA Ligase is needed (as opposed to DNA Polymerase I) to seal single-stranded "nicks" left when nucleotides are added to replace an RNA primer.

- Discontinuous DNA synthesis of the lagging strand, some of newly formed DNA hydrogen bonded to template strand is present as small fragments containing 1000 to 2000 nucleotides, RNA primers are part of such fragments, these pieces are called Okazaki fragments are converted into longer and longer DNA strands of higher molecular weight as synthesis proceeds Lagging strand requires enzymes able to remove RNA primers and unite Okazaki fragment into lagging strand, DNA pol I removes primers and replaces missing nucleotides, DNA ligase is capable of catalyzing the formation of the phosphodiester bond that seals the nick between the discontinuously synthesized strands Discontinuous DNA synthesis of the lagging strand, some of newly formed DNA hydrogen bonded to template strand is present as small fragments containing 1000 to 2000 nucleotides, RNA primers are part of such fragments, these pieces are called Okazaki fragments are converted into longer and longer DNA strands of higher molecular weight as synthesis proceeds Lagging strand requires enzymes able to remove RNA primers and unite Okazaki fragment into lagging strand, DNA pol I removes primers and replaces missing nucleotides, DNA ligase is capable of catalyzing the formation of the phosphodiester bond that seals the nick between the discontinuously synthesized strands DNA pol I requires the 3' OH

Why were we targeting genes like cytochrome C (as opposed to a species specific gene) in the various insect species?

- Genes such as cytochrome C, 12s rRNA, 16s rRNA, and NAD were targeted because they are found within all species regardless of genus/species due to their essential-ness for life, additionally they are largely conserved among species and consist of smaller changes that can be effectively mapped for creating phylogenetic trees at interphase eukaryotic chromosomes uncoil and decondense into a form called chromatin, during interphase chromatin is dispersed throughout nucleus, during cell division chromatin coils and condenses back into visible chromosomes "Chromatin is dynamic in eukaryotes" able to change/likely to change, in instance of DNA the structure is changing, chromatins and histones go back and forth between closed and opened, chemical modifications to histone tails, net charges are changed Euchromatin - usually uncoiled and active, appears unstained during interphase Heterochromatin - usually always condensed areas are mostly inactive, appears stained during interphase, genetically inactive (lacks genes or contains repressed genes), replicates later in S phase than euchromatin, telomere maintains chromosome integrity, centromere involved in chromosome movement

Understand how chemical modifications (know the 3 types) to histones and the DNA itself account for the dynamic nature of euchromatin.

- Histones - positively charged proteins associated with chromosomal DNA in eukaryotes, contain large amounts of lysine and arginine, makes electrostatic bonding to negatively charged phosphate possible (five main types H1, H2A, H2B, H3, H4) Chromatin remodeling - to accommodate DNA-protein interactions chromatin structure must change, to allow replication and gene expression chromatin must relax compact structure/expose regions of DNA to regulatory proteins/have a reversal mechanism for inactivity Chemical modifications are important for genetic function, histone tails provide potential targets along chromatin fiber for chemical modifications: 1. acetylation - addition of an acetyl group to the positively charge amino acid lysine effectively changes the net charge of the protein by neutralizing the positive charge, linked to gene activation enzyme: histone acetyltrasferase (HAT), addition of acetyl group neutralizes (+) charge 2. methylation methyl groups can be added to both arginine and lysine rediues in histones and this change has been correlated with gene activity

Know the 2 types of chemical bonds holding double stranded DNA together. Know how each type contributes to the usefulness of DNA as the genetic ingredient to every cell on the planet.

- Hydrogen bonds between base pairs (very weak electrostatic attraction between a covalently bonded hydrogen atom and an atom with an unshared electron pair, hydrogen atom assumes a partial positive charge while unshared electron pair characteristic of covalently bonded oxygen and nitrogen atoms assume a partial negative charge, opposite charges result in hydrogen bond), greater number provide stability - Phosphodiester bonds - nucleotides are linked by these between phosphate group at C-5' position and OH group on C-3' position (forms polynucleotide chains), each structure has C-5' end and C-3' end, two joined nucleotides form a dinucleotide, three form a tri-

Know the primary structure of a nucleotide: sugar, phosphate and nitrogenous base. Know what each piece contributes to overall function. Be able to sketch a double-stranded DNA chain (not the full chemical structure, but include important attachment point(s) and know what 5' to 3' means).

- Nucleotides consist of: nitrogenous base (2 flavors, 4 in total), pentose sugar (positioning of oxygen is important), phosphate group Two types of nitrogenous bases: - Purines (nine-member double ring): Adenine, Guanine - Pyrimidine (six-member ring): Cytosine, Thymine, for RNA Uracil replaces Thymine Nucleoside: contains nitrogenous base and pentose sugar, molecule is composed of purine or pyrimidine base and ribose or deoxyribose sugar Nucleotide: nucleoside with 1-3 phosphate groups added Pentose sugars give name and overall structures, RNA contains ribose, DNA contains deoxyribose (hydrogen atom rather than hydroxyl group at C-2' position), phosphate acts like glue for the backbone, nitrogenous base do not contribute to support Watson and Crick model: two long polynucleotide chains are coiled around a central axis forming right handed double helix, antiparallel chains (C-5' to C-3' run in opposite directions), nitrogenous bases of opposite chains are paired as result of hydrogen bonds (A-T, G-C) COMPLEMENTARITY, major grooves alternate with minor groove winding along length of molecule, double helix A and T have two bonds G and C have three bonds

How is DNA packaged up to fit inside the nucleus? (Fig 12-9) Histone proteins are the building blocks of this packaging. DNA is wrapped around histone complexes (nucleosomes) and there are multiple layers of folding that lead to dense chromosomal arrangements. Euchromatin transitions between loose and packed, heterochromatin does not very often (there are fewer genes in heterochromatic regions). Prokaryotes don't display such organizational hierarchy because they lack the need for histones. Why do Eukaryotes need so many extra gene expression control mechanisms? >>> the answer lies in their multicellularity

- REVIEW General model of the association of histones and DNA to form nucleosomes, illustrating the way in which each thickness of fiber may be coiled into a more condensed structure, ultimately producing a metaphase chromosome enzyme: methyltransferase, adds methyl groups to arginine and lysine residues in histones 3. phosphorylation phosphate groups can be added to the hydroxyl group of the amino acids serine and histidine introducing a negative charge on the protein enzyme: kinase, adds phosphates groups to hydroxyl groups of amino acids serine and histidine Methylation and phosphorylation result from the action of enzymes called methyltransferases and kinases RWE: Readers, Writers, Erasers Euchromatin usually uncoiled and active, appears unstained during interphase Heterochromatin - usually always condensed areas are mostly inactive, appears stained during interphase, genetically inactive (lacks genes or contains repressed genes), replicates later in S phase than euchromatin, telomere maintains chromosome integrity, centromere involved in chromosome movement

Due to 5' to 3' DNA polymerase activity, there is discontinuous synthesis of the lagging strand. What biochemical role do RNA primers play in making this possible?

- RNA primers with a free 3' hydroxyl group allow elongation of the polynucleotide chain, RNA serves as the primer, short segment of RNA (10-12 nucleotides long) first synthesized on DNA template, synthesis of RNA directed by RNA polymerase called primase, this short segment of RNA allows DNA Pol III to add DNA nucleotides intiating DNA synthesis Discontinuity of the lagging strand - continuous addition of RNA primers allow Okazaki fragments to be subsequently added to the strand, allows movement to still progress in the 5' to 3' direction

What is telomerase and how specifically does it help fill in the gap left on 5' chromosome ends in Eukaryotes? What kinds of cells have high telomerase activity? Why can't all of our cells have high telomerase activity (Why can't we use telomerase to make us immortal?)?

- Telomerase - enzyme that adds short tandemly repeated DNA sequences to the ends of eukaryotic chromosomes eukaryotic enzyme, ribonucleoprotein (RNA serves as template for synthesis of DNA complement/reverse transcriptase), once RNA primer removed on lagging srtand no free 3'OH to elongate, telomerase adds repeats of six-nucleotide sequence to 3' end to fill gaps 1. telomerase binds to 3' G rich tail (caused by removal of primer at linear ends of chromosomes) 2. tolmeric DNA is synthesized on G rich tail 3. telomerase is translocated and steps 1/2 are repeated 4. telomerase released, primase/DNA polymerase fill gaps 5. primer removed and gap sealed by DNA ligase High telomerase activity is in stem cells and malignant cells (immortalized) Not all cells can have high telomerase activity because it is largely linked with mutations and this would lead to many mutations/cancers in these cells, would be even more dangerous in cells that are replicating and replacing frequently (ex. Skin cells and other epithelial cells) We can't use telomerase to make us immortal because it would lead to the presence of mutations over time which would lead to many malignant cells ultimately forming and then death (cancerous cells would overtake body as telomerase would likely have mutations/problems within it

What role do the 3 phosphates play for free, unincorporated nucleotide-triphosphates? Why must these phosphates be attached and not separate?

- The energy stored within the tri-phosphates is required to overcome the binding threshold necessary for binding between a phosphate and hydroxyl group (between nucleotides) Must be attached so that energy is not released unnecessarily

-Three main post-transcriptional modifications (what are they?) These lead to mature mRNA, which is then stable enough and marked for transport outside of the nucleus.

1. Addition of 5' cap (7-mG cap) 7-methylguanosine (7-mG) cap, added before synthesis of the initial transcript is complete and appears to be important to subsequent processing within the nucleus, cap stabilizers the mRNA by protecting the 5' end of the molecule from nuclease attack, thought to facilitate the transport of mature mRNAs across the nuclear membrane into the cytoplasm and in the initiation of translation of the mRNA into protein 2. Cleavage of 3' end and addition of 3' tail (poly-A-tail) 3. Excision of introns

Know what constitutes a "cycle" (the three steps, the temperatures and what is going on in the reaction during each step)

1. HOT 95º - 30 seconds, denaturing 2. LOW 50ºC - 30 seconds, annealing/reforming 3. INTERMEDIATE 72ºC - 60 seconds, DNA polymerase extends The three steps make up one cycle, in total there were 35 cycles

-What accounts for RNA's versatility as a molecule? I'm looking for biochemical explanations.

2'OH RNA's versatility due to structural diversity: - Transcribed as single stranded (mRNA) - Forms complementary bonds with other nucleic acids - Creates DNA/RNA duplexes - Forms hairpin or stem-loop structures - Associates with proteins and modify their activity

-Know the general roles of initiation factors, elongation factors and termination factors (during translation).

ALL are like small tugboats Initiation factor - tug boats that allow ribosomes to coordinate self, move into position, position large and small subunits into position Elongation factor - are tugboats for tRNA, make polypeptide longer, shuttling in and shuttling out Termination factor - release tRNA from ribosome, hydrolyze last aminoacyl bond

- RNA polymerase (yet another "holoenzyme") operates in a transcription bubble that moves down a gene's length. Why not open it all up at once?

Allows for stability of the genetic information being opened up, prevents mRNA from rebinding to the template strand, want DNA stability

DNA Pol IV

DNA repair that has been damaged by external forces

DNA Pol V

DNA repair that has been damaged by external forces

Be familiar with these enzymes and their role in Prokaryotic DNA Replication: DnaA initiator, Helicase, Gyrase, SSBP, Primase, DNA Polymerase III (this is an enzyme complex with various jobs), DNA Polymerase I and DNA Ligase drawing a picture makes this easier.

DnaA initiator - opens up the double strands, specific initiator protein, responsible for initiating replication by binding to a region of 9mers (five repeating sequences of 9 base pairs), newly formed complex then undergoes a slight conformational change and associates with the region of 13mers which causes the helix to destabilize and open up exposing single stranded regions of DNA binds to ORI causing conformation change Helicase subsequent binding, consists of multiple copies of DnaB polypeptide, assesmbled as a hexamer of subunits around one of exposed ssDNA molecules, recruits holoenzyme (holoenzyme - for proteins with multiple subunits the complex formed by the union of all subunits necessary for all functions of the enzme) to bind to newly formed replication fork to formally initiate replication and the proceeds to move along ssDNA opening up helix as it progresses, helicases require energy supplied by hydrolysis of ATP which aids in denaturing the hydrogen bonds that stabilize the double helix Gyrase - as unwinding proceeds coiling tension is created ahead of replication fork (often creating supercoiling), supercoiling can be relaxed by gyrase (member of larger group of enzymes referred to as DNA topoisomerases), makes either single or double stranded cuts and also catalyzes localized movements that have the effect of undoing the twists and knots created during supercoiling SSBP - once helicase has opened up helix and ssDNA is available base pairing must be inhibited until it can serve as a template for synthesis, accomplished by proteins that bind specifically to ssDNA Primase - synthesis of RNA is directed by a form of RNA polymerase called primase, recruited to replication fork by DNA helicase and which does not require a free 3' end to initiate synthesis DNA Polymerase III - attaches to RNA primer, begins to add deoxyribonucleotides initiating DNA synthesis, synthesizes in only the 5' to 3' direction DNA Polymerase I - clips out RNA primer and replaces the primer with DNA nucleotides DNA ligase - joining of Okazaki fragments together, capable of catalyzing the formation of the phosphodiester bond that seals the nick between the discontinuously synthesized strands

-ncRNA can be subdivided into many categories. We covered small non-coding RNA (regulate gene expression in bacteria) and CRISPR RNAs (an adaptive immune system in bacteria) in Prokaryotes. Be able to draw or explain how each of these work.

EXAM: Know how this works in bacteria, and how we might be able to use it in biotechnology 1. Acquisition (can be done synthetically) 2. Creation of guide RNA 3. Interference (targets second time infected with virus) Do this through the neighboring cas genes, become little guide RNAs How does it know not to cut the storage sequence? The repeats don't exist in the viral DNA, want to find something that does not have repeats so only cuts the invader To edit a human genome - simply have to change the guide RNA, cut one particular place in the genome, only around 30 base pairs long Every time cut at DNA (double stranded break), will likely not go back together perfectly Double strand break repair sRNA (small noncoding RNAs) - Regulatory functions still being discovered - sRNAs have between 50 and 500 nucleotides - Involved in gene regulation (response to change in environment, bacterial sRNAs have positive and negative regulation) - Critical role in bacterial cell communication - Quorum sensing: bacteria sense their population density and adjust behavior o At low cell density, pathogenic bacteria are harmless; do not express virulence factors o At high density, launch attack of virulence factors

-What are a few biochemical or cellular challenges that Eukaryotes face in terms of transcription that Prokaryotes do not?

Eukaryotes have membrane bound organelles therefore the transportation of RNAs is important Need 5'cap and 3'poly-A-tail Excision of introns (eukaryotic mRNAs require processing to make mature mRNAs) Eukaryotes: occurs in nucleus, mRNA must leave nucleus for translation, chromatin remodeling (chromatin must uncoil to make DNA accessible to RNA Pol), RNA polymerases rely more heavily on transcription factors to scan/bind DNA, enhancers and silencers are DNA sites whose presence influence transcriptional regulation, post-transcriptional processing DNA must be uncoiled from histones RNA polymerase requires TF only in eukaryotes

-Why is methionine a convenient amino acid to be the first one on every polypeptide chain (think about translational initiation)?

It structurally fits perfectly into the P site for initiation, linear in shape Small and uncharged, conformational fit

-Organismal complexity (as measured by total number of distinct cell types) appears to be positively correlated with the presence of non-coding DNA. This suggests complex organisms have evolved non-coding RNA to modulate gene expression enough to account for an increased diversity of cellular phenotypes. This is still a theory, but is gaining support. Be able to tell me how humans (~20-24K coding genes) have so much more cellular complexity despite having about the same number of coding genes as nematodes (~21,000 coding genes).

Non-coding regions or genes are still able to code for products other than proteins that are important DNA and transcription, transcription makes all the types of RNAs, makes ncRNAs which though are coming from genes, non-coding genes do not code for proteins, play important role in gene expression, are able to fine tune processed involving coding DNA and coding RNA that will ultimately make proteins and phenotypic expression REVIEW

-What is a nonsense mutation? Missense mutation? Silent mutation?

Nonsense mutation - a mutation that changes a codon specifying an amino acid into a termination codon, leading to premature termination during translation of mRNA Missense mutation - a mutation that changes a codon to that of another amino acid and thus results in an amino acid substitution in the translated protein, such changes can make the protein nonfunctional Silent mutation - mutation that does not result in an amino acid change, often occurs in the wobble position

-Know what an "open reading frame" is and what its presence suggests about a region of DNA.

ORF (open reading frame) - nucleotide sequence organized as triplets that encodes the amino acid sequence of a polypeptide, including an initiation codon and a termination codon DNA sequence produces RNA with start and stop, series of triplet codons specify amino acids to make polypeptide; in some viruses initiation at different AUG positions out of frame with another leads to distinct polypeptides 6 potential locations, key factors are where is ATG (followed by multiples of 3) then concludes with a stop codon Has a beginning, middle, end; can be variety of lengths

-What types of chemical bonds are catalyzed by the ribosome?

Peptide bonds

-To the best of our ability, we can predict brown or blue eye color 91% of the time. What is happening the other 9% of the time (why can't we get it 100% correct)?

Phenotype is wrong, when trying to predict may have correct genetic information, subjectivity 9% can be attributed to subjectivity in humans determining eye color and the fact that we are only using 3% of the genome and there are likely many other factors influencing the phenotype Most likely reason - only using 3% of genome, likely other areas that are affecting it that we are not able to identify

-Know the names of the core promoter sites in Prok. And Euk. And know what proteins bind to them: general Transcription Factors, TBP, Sigma, etc.

Prok - -35, -10, sigma, RNA polymerase, DBD transcription factor serves as an enhancer Euk - TATA box (Goldberg-Hagness) is core promoter site, activators = enhancers, silencers = repressors, TBP and gTFIID enter first, H goes in and serves as a crowbar, FAEB then bind, PIC (pre-initiation complex), TBP is subunit of TFIID, activators are binding at enhancer site and activator is a TF, TF bind to DNA then activate other/bind to other things, activators are proteins from mRNA transacting factors can move around, up regulator - activator, down regulator - repressor All cells have general transcription factors but enhancers/silencers repressors/activators is what differs between cells, PIC is same in all cells

-Know the roles of each ribosomal subunit. The small subunit of the ribosome locates and positions the ribosomal A, P and E sites correctly on the mRNA. The large subunit houses the catalytic subunit of the ribosome, the 23S ribosomal RNA in Prokaryotes (28S rRNA in Euk.).

Prokaryotes - 23S, 16S Eukaryotes - 28S, 18S 28S rRNA is most important rRNA in eukaryotes, is actual enzymatic piece of ribosome that is forming peptide bonds 18S helps recognize the Kozak sequence (smaller subunit is 18S) DNA codes for rRNA - 4 different kinds in humans, made from polycistronic rRNA (one long piece of rRNA and gets cut into 3 pieces) 5 and 5.8S in euk (5S in prok)

->90% of all RNA by volume is tRNA and rRNA. All other RNA is either messenger RNA (mRNA, coding for proteins) or non-coding RNA.

REVIEW

-The 20 amino acids have common backbones (carboxyl and amino groups) with various 'R' groups that give each one unique chemical properties. In sequence, these create limitless shapes and therefore function. These aren't codes in the way DNA sequences are codes, rather unique manifestations of folding and spatial chemistry.

REVIEW

-What functional forms might a given polypeptide take? >structural, enzymatic, signaling molecule, etc. RNAs can be functional in similarly diverse ways.

REVIEW - Primary: Sequence of amino acids - Secondary: α-helix and β-pleated sheets (Figure 14-18) - Tertiary: Three-dimensional conformation (Figure 14-19) - Quaternary: Composed of more than one polypeptide chains (Figure 14-20) Examples: immunoglobulins, transport proteins, hormones, histones, transcription factors, enzymes

- Know why RNApol II in Eukaryotes must be able to interact with more numerous and diverse TFs and promoter regions than RNApol I or RNApol III.

RNA Pol I - codes for rRNA (makes up ribosomes, four subunits) RNA Pol II - involved in multicellularity, involved in making mRNA and snRNA which make tens of thousands, requires TF diversity at promoter regions because fine tuning genes; responsible for transcription of wide range of genes in eukaryotes, activity of RNAPII is dependent on cis-acting elements and trans-acting transcription factors, core-promoters corresponding to RNAP II determine where RNAP II binds to DNA to begin transcription RNA Pol III - transcribes ssrRNA and tRNA (but only 20 amino acids)

three types of chemical remodeling

RWE readers writers erasers

-Know the difference between "reading frame" and codon. What type of information is stored in a single codon?

Reading frame - always 6 reading frames possible for double stranded DNA, always 3 reading frames possible for single stranded DNA; a linear sequence of codons in nucleic acids Codon - a triplet of messenger (mRNA) nucleotides that specifies a particular amino acid or a start or stop signal in the genetic code. Sixty-one codons specify the amino acids used in proteins, and three codons, called stop codons (UAG, UAA, UGA), signal termination of growth of the polypeptide chain. One codon (AUG) acts as a start codon in addition to specifying an amino acid A single codon stores information for an amino acid consecutive non-overlapping triplets

Recombinant DNA is genetic information from one organism placed (physically or otherwise) into cells of another organism. This process helps us understand the downstream phenotypic effects of those specific DNA sequences (transgenes). This works because DNA, RNA and protein is common to all living things (6 billion years of evolution stemming from common ancestors). Every living thing has the machinery to use DNA. That is why we can take DNA from Jellyfish cells corresponding to glowing proteins and insert it into any other cell and see glowing proteins. Though the enzymes involved may be slightly different, DNA is "read" the same way by our cells and jellyfish cells.

Recombinant DNA technology: segments of eukaryotic DNA corresponding to specific genes isolated and spliced into the bacterial DNA, insert insulin gene (DNA) into bacterial cell and you see insulin protein in all progenitor cells, DNA encoding human Beta-globin gene microinjected into fertilized mouse egg, now this protein present and expressed in mouse and transmitted to progeny

-In general terms, what is involved in translational initiation? For example, what binds to the consensus sequence (Shine-Dalgarno/Kozak) on the template mRNA first? How does translation proceed: A, P and E sites inside ribosome? What portion of the Ribosome actually catalyzes the peptide bond between amino acids in the growing chain? Explain how this catalytic piece harkens back to the "RNA world hypothesis."

Small subunit: mRNA, initiation factors (little tugboats), smaller subunit of ribosome Shine-Dalgarno sequence attracts the small subunit in prokaryotes, initiation is involved in the small subunit Methionine goes to P site, subsequent tRNA go from A to P to E Portion that catalyzes is the P site RNA world hypothesis - essential for all life; a hypothesis describing life based on RNA acting in information (genomes) and catalytic (ribozymes) roles that predates current life forms that utilize DNA genomes and proteins catalysts Elongation factors are tugboats for tRNA, make polypeptide longer Release factors release tRNA from ribosome, hydrolyze last aminoacyl bond Fact that acting like an enzyme Initation factors attract small subunit of ribosome to mRNA, Shine-Dalgarno sequence places AUG into correct start position within the ribosome

-Splicing of introns may or may not require enzymatic assistance (spliceosome). In lower Eukaryotes, self-splicing sometimes occur (auto-catalytic process).

Spliceosomes - splice introns and exons REVIEW

-What happens when a stop codon moves into the A-site of the ribosome?

Stop codon leaves a big hole, brings in release factors, doesn't code for tRNA rather leaves a hole so release factor destabilizes and hydrolyzes Now small unit of ribosome can look for another Kozak sequence Recruits termination factor, which cuts the aminoacyl bond and hydrolyzes, destabilizes the ribosome, when last tRNA is in the P site Aminoacyl bond is between the tRNA and the polypeptide

-The minimal preinitiation complex (PIC) in Eukaryotes includes RNA polymerase II and six general transcription factors:.

TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Additional regulatory complexes (co-activators and chromatin-remodeling complexes) could also be components of the PIC Because transcribing from DNA must move the histones around DNA to mRNA, Co-activators, chromatin-remodeling complexes to move around histones

Know how to describe primers and how they lead to the product of PCR.

The primers are for both Forward (5' to 3') and Reverse (3' to 5'), can chose primers for those regions, DNA polymerase protein is adding nucleotides to growing chain that is on either side of the primers, must add nucleotides to the 5' to 3' ends, builds strands on both sides though eventually runs into stop point of other primer Primers used in PCR lab: F and R for 12s rRNA, 16s rRNA, cytochrome c, NAD

-Explain the wobble hypothesis. Know how to interpret figure 13-7. How does an amino acid chain change if I introduce a frame-shift mutation? Be able to identify an open reading frame in a DNA sequence.

The wobble hypothesis: - Initial two ribonucleotides of triplet codes are often more critical than the third - Third position o Less spatially constrained o Need not adhere as strictly to established base-pairing rules Initial two ribonucleotides of triplet codes are often more critical than the third member in attracting the correct tRNA, he postulated that hydrogen bonding at the third position of the codon-anticodon interaction would be less spatially constrained and need not adhere as strictly to the established base-pairing rules, wobble hypothesis proposes a more flexible set of base-pairing rules at the third position of the codon Reasoning for wobble position - large space between second and third sites, not spatially constrained and does not need hydrogen bonds so it is considered promiscuous Frameshift mutation - mutational event leading to the insertion or deletion (indels) of a number of base pairs in a gene that is not a multiple of three, this shifts the codon reading frame in all codons that follow the mutational site An amino acid chain would change likely to be an entirely different amino acid, could result in a change to the entire protein First nucleotide of anti-codon corresponds to first nucleotide of mRNA

-How is transcription initiated and terminated in Prokaryotes? Be able to draw or describe accurately, including relevant details, e.g., role of Sigma Subunit or Rho Factor. Which DNA strand is the template for transcription?

Transcription initiated - -35, -10 sigma factor where RNA polymerase binds, initiation Transcription terminated - can be terminated by either passive hairpin (more likely) or active (rho factor mediated thing), enzyme traverses entire gene until a termination nucleotide sequence is encountered, in bacteria termination transcribed into RNA causes newly formed transcript to fold back on itself (hairpin), at times termination depends on the rho termination factor Template strand is the 3' to 5' end TTGACA and TATAAT (Pribnow box), positioned at -35 and -10 with respect to transcription initiation site REVIEW Rho factor - specific sequence signals for Rho factor (enzyme) that cleaves sequence off

-In addition to these core promoter regions, there are other islands of DNA sequence further away from the actual gene: these are enhancers or silencers. Eukaryotes have greater numbers and diversity of these enhancer and silencer sites per gene. Proteins called transcription factors (activators or repressors) that recognize these secondary consensus sequences are usually present in some unique conformation per gene for transcription. The transcription factors interact with RNA polymerase to modulate transcription in a specific way. Eukaryotic genes are often controlled by hundreds of these Activators and Repressors (collectively called transcription factors or TFs). Prokaryotic genes employ much fewer TFs for gene expression control. (They are unicellular, so they have not evolved such fine tuning of gene expression)

Two types of TF: general and enhancer/silencers REVIEW

-How is RNA chemical structure related to its niche in the central dogma?

Use of Uracils and Thymines because Us require less energy to make than Ts 2'OH keeps it from being double stranded, which is fine because only transcribing one side of gene, 2'OH is exposed and reactive which makes mRNA reactive which allows it to be degraded, don't want it to always be around because it is produced/used so often in the body and only want it to be transcribed/expressed once, temporary-ness allows perfect fit into the central dogma, RNA is incredibly essential in all processes Good messenger Times folding on self include - ribosomes, tRNA, hairpin structures, introns, spliceosomes Niche - can be a messenger in the central dogma, outside of the dogma it can be non-coding and fulfill other central roles

-What is meant by the term "variant"? Explain how a DNA variant is a very different concept than a DNA mutation. At minimum, how many single nucleotide polymorphism identities are necessary to accurately predict blue, brown or intermediate eye colors?

Variant - variants are expected and common differences for a nucleotide Mutation - not common and not expected 6 SNPs are needed to accurately predict blue, brown, or intermediate eye colors Multifactorial nature of the genome that there is so much that cant be accounted for

"chromatin is dynamic in eukaryotes"

able to change/likely to change, in instance of DNA the structure is changing, chromatins and histones go back and forth between closed and opened, chemical modifications to histone tails, net charges are changed

acetylation

addition of an acetyl group to the positively charge amino acid lysine effectively changes the net charge of the protein by neutralizing the positive charge, linked to gene activation enzyme: histone acetyltrasferase (HAT), addition of acetyl group neutralizes (+) charge

gyrase

as unwinding proceeds coiling tension is created ahead of replication fork (often creating supercoiling), supercoiling can be relaxed by gyrase (member of larger group of enzymes referred to as DNA topoisomerases), makes either single or double stranded cuts and also catalyzes localized movements that have the effect of undoing the twists and knots created during supercoiling

DNA polymerase III

attaches to RNA primer, begins to add deoxyribonucleotides initiating DNA synthesis, synthesizes in only the 5' to 3' direction, assembly/proofreads/holds together

hydrogen bonds

between base pairs (very weak electrostatic attraction between a covalently bonded hydrogen atom and an atom with an unshared electron pair, hydrogen atom assumes a partial positive charge while unshared electron pair characteristic of covalently bonded oxygen and nitrogen atoms assume a partial negative charge, opposite charges result in hydrogen bond), greater number provide stability

DNA polymerase I

clips out RNA primer and replaces the primer with DNA nucleotides

chromatin

complex of DNA, RNA, histones, and nonhistone proteins that make up uncoiled chromosomes, characteristic of eukaryotic interphase nucleus at interphase eukaryotic chromosomes uncoil and decondense into a form called chromatin, during interphase chromatin is dispersed throughout nucleus, during cell division chromatin coils and condenses back into visible chromosomes

-What are the main components of the CRISPR RNA system that scientists can inject into cells from any organism on the planet to stimulate a targeted DNA alteration? Explain how this might be useful in a broader context. (The "Kurzgesagt" Youtube video explains this nicely)

crRNA containing repeats and spacers, target viral DNA, Cas protein, Guide RNA, fragments - Acquisition: Integration of invading phage DNA into CRISPR loci - crRNA biogenesis: Transcription of CRISPR loci, processing of crRNA biogenesis; requires Cas proteins - Interference: Targeting and cleavage of phage DNA sequences complementary to crRNAs Challenge is controlling delivery cas protein is essential for the cutting

DNA Pol II

elongate existing DNA strand, DNA repair that has been damaged by external forces, encoded by a gene activated by disruption of DNA synthesis at replication fork

DNA Pol I

elongate existing DNA strand, remove RNA primer and replace with DNA, demonstrates 5' to 3' exonuclease activity

E

express (coded information) concept of information flow, initial event in flow of information is transcription of DNA (three main types of RNA: mRNA, tRNA, rRNA), mRNA translated into proteins by process mediated by tRNA and rRNA, translation where chemical information in mRNA directs construction of a chain of amino acids called polypeptide, central dogma

DNA ligase

joining of Okazaki fragments together, capable of catalyzing the formation of the phosphodiester bond that seals the nick between the discontinuously synthesized strands

-tRNAs are special RNAs that physically connect the nucleotide triplet code (mRNA) and the amino acid code (polypeptide chain). Know the 2 key features of the tRNA that allow for this translation ability (anticodon and amino acid binding site [another example of a 3' OH]). tRNAs can only match an amino acid to one codon. The ribosome complex is responsible for the enzymatic addition of that amino acid to the growing chain of amino acids.

key features of tRNA that allow translation ability - anticodon - amino acid binding site REVIEW

2. methylation

methyl groups can be added to both arginine and lysine rediues in histones and this change has been correlated with gene activity enzyme: methyltransferase, adds methyl groups to arginine and lysine residues in histones

methylation

methyl groups can be added to both arginine and lysine rediues in histones and this change has been correlated with gene activity enzyme: methyltransferase, adds methyl groups to arginine and lysine residues in histones

M

mutate (then would have no built in variation and no ability to adapt to new scenarios and environmental conditions), variability among organisms through the process of mutation, alteration is reflected during transcription and translation affecting the specific protein, if mutation is present in a gamete may be passed to future generations and be distributed to population, leads to evolution

-What features of messenger RNA differ from those of non-coding RNAs (ncRNAs)?

ncRNA - play important role in genetic processes, at least 75% of genome transcribed into RNA, RNAs that do not encode polypeptides (examples: tRNAs, rRNAs, and miRNAs are all examples of ncRNAs), variable size messenger RNA - mRNA, determines the amino acid sequence of polypeptides and has untranslated regions (UTRs) that can influence stability, localization, and translation (examples: human beta-actin mRNA encodes a 375 aa protein that is part of the cytoskeleton), variable size

phosphodiester bonds

nucleotides are linked by these between phosphate group at C-5' position and OH group on C-3' position (forms polynucleotide chains), each structure has C-5' end and C-3' end, two joined nucleotides form a dinucleotide, three form a tri-

SSBP

once helicase has opened up helix and ssDNA is available base pairing must be inhibited until it can serve as a template for synthesis, accomplished by proteins that bind specifically to ssDNA, single stranded binding protein

DnaA initiator

opens up the double strands, specific initiator protein, responsible for initiating replication by binding to a region of 9mers (five repeating sequences of 9 base pairs), newly formed complex then undergoes a slight conformational change and associates with the region of 13mers which causes the helix to destabilize and open up exposing single stranded regions of DNA binds to ORI causing conformation change

phosphorylation

phosphate groups can be added to the hydroxyl group of the amino acids serine and histidine introducing a negative charge on the protein enzyme: kinase, adds phosphates groups to hydroxyl groups of amino acids serine and histidine

3. phosphorylation

phosphate groups can be added to the hydroxyl group of the amino acids serine and histidine introducing a negative charge on the protein enzyme: kinase, adds phosphates groups to hydroxyl groups of amino acids serine and histidine Methylation and phosphorylation result from the action of enzymes called methyltransferases and kinases RWE: Readers, Writers, Erasers

histones

positively charged proteins associated with chromosomal DNA in eukaryotes, contain large amounts of lysine and arginine, makes electrostatic bonding to negatively charged phosphate possible (five main types H1, H2A, H2B, H3, H4)

R

replicate (create multiple of information) once genetic material replicates and is doubled in amount then partitioned equally via mitosis into daughter cells, genetic material also replicated in gametes but only half designated to each cell, both mitosis and meiosis are part of cellular reproduction

-RNA differs from DNA in three critical ways:

single stranded presence of a 2'hydroxyl on the ribose Uracil bases in place of Thymine bases (T). Hydrogen bonds are free so can interact with itself, proteins, ribosomes

S

store (coded information) storage of information requires molecule to act as repository of genetic information that may or may not be expressed by the cell in which it resides, clear that while most cells contain complete copy of the organism's genome at any point in time they express only a part of this genetic potential, in bacteria many genes can turn on and off, ex. Humans - skin cells might activate melanin gene but never digestive genes (vice versa for digestive cells)

helicase

subsequent binding, consists of multiple copies of DnaB polypeptide, assesmbled as a hexamer of subunits around one of exposed ssDNA molecules, recruits holoenzyme (holoenzyme - for proteins with multiple subunits the complex formed by the union of all subunits necessary for all functions of the enzme) to bind to newly formed replication fork to formally initiate replication and the proceeds to move along ssDNA opening up helix as it progresses, helicases require energy supplied by hydrolysis of ATP which aids in denaturing the hydrogen bonds that stabilize the double helix recruits RNA primer to replication fork

primase

synthesis of RNA is directed by a form of RNA polymerase called primase

chromatin remodeling

to accommodate DNA-protein interactions chromatin structure must change, to allow replication and gene expression chromatin must relax compact structure/expose regions of DNA to regulatory proteins/have a reversal mechanism for inactivity Chemical modifications are important for genetic function, histone tails provide potential targets along chromatin fiber for chemical modifications: 1. acetylation - addition of an acetyl group to the positively charge amino acid lysine effectively changes the net charge of the protein by neutralizing the positive charge, linked to gene activation enzyme: histone acetyltrasferase (HAT), addition of acetyl group neutralizes (+) charge

heterochromatin

usually always condensed areas are mostly inactive, appears stained during interphase, genetically inactive (lacks genes or contains repressed genes), replicates later in S phase than euchromatin, telomere maintains chromosome integrity, centromere involved in chromosome movement

euchromatin

usually uncoiled and active, appears unstained during interphase