Bio midterm 2

What are enhancers? And how do Activator proteins interact with them? Class 12

- Before the promoter region in the DNA we have enhancers which are control elements. Activator proteins interact with the enhancer sequences in the DNA and also interact with other proteins that stimulate transcription. -There are two major regions of eukaryotic transcription activators. There is the DNA binding region (so that the domain binds to a specific DNA sequence). Attached to this is the activation domain which interacts with other proteins that stimulate transcription. This allows for modular interactions. Meaning we can use a small number of activation domains and hook them up to a large and diverse array of DNA binding domains. -MASTER regulators activate the expression of other activators. So, let's say we express the myoD gene, then this makes the myoD protein, which is a transcriptional activator of itself and other transcription activators. The Master regulators kick off a cascade of gene expression that results in a muscle cell having a specific set of transcriptional activators.

In VERY simple three steps how does translation occur in the cytoplasm on ribosomes for prokaryotes. Class 11

- mRNA binds to ribosomes -tRNA molecules carrying amino acids bind to mRNA -Ribosomes catalyzes the addition of amino acids to the growing chain

After transcription of eukaryotic gene, we get pre-mRNA, what 3 things do we do to that before we export it to the nucleus? list don't describe Class 12

-5' Cap structure -3' end poly-A-tail -Introns are removed

How does the ribosome translation complex select the right AUG to start with? in prokaryotes. Class 11

-A given mRNA will have many AUG, but only one is the correct site of initiation for each gene. -There is a sequence upstream of AUG which serves to help recruit the small subunit of the ribosome -An interaction between the specific sequence on the mRNA and this mRNA binding site on the ribosome can position the mRNA so that there's an AUG on the correct position.

What do we need to express a eukaryotic gene in a prokaryotic cell class 13

-A prokaryotic promoter and terminator sequence -We must only insert the coding sequence of the gene, just the exon. If we are cloning a eukaryotic DNA to put in a eukaryotic plasmid, we have to use the cDNA rather than the whole gene sequence. -Affinity tags to purify the protein from everything else in the bacteria -All the stuff in the plasmid you normally need.

Inductive cells

-As embryonic cells acquire distinct fates, they influence each other's fate by induction Spemann/Mangold experiment -Take one piece of one embryo and put it back in the opposite side of a recipient embryo - a group of cells can induce other cells to become something that maybe they would have not without this organism center. -The new structure is messed -Determination is a term referring to the eprocess by which a cell or group of cells becomes committed to a particular fate. -Differentiation refers to the resulting specialistion in structure and function.

How does long range intercellular communication work Class 14

-Cell A secrets a ligand -Specific receptors inside or on the surface of target cells bind the secreted ligand. This induces a conformational change in the receptor. The conformational change means that the receptor behaves differently in the bound vs the unbound state. Differences can mean binding different proteins, binding no proteins, turning enzyme activity on/off, etc. -Signal transduction is everything that happens between ligand binding to the receptor causing conformation of the receptor AND cell B's response

How do second messengers work? class 14

-Cells often use second messengers (small molecules that can rapidly diffuse around the cell. Second messengers bind to proteins and induce a conformational change) -The first signal messenger ligand, epinephrine, binds to the receptor. Then the GPCR exchanges GDP to GTP on a G protein and activates an enzyme called Adenylyl cyclase. Adenylyl cyclase generates the second messenger molecule cyclic AMP by converting ATP to cyclic AMP. This can now diffuse around the cell and act as a ligand for other molecules. -Cyclic AMP can be turned off and converted to AMP by phosphodeiterase. Large amounts of cAMP can be turned on/off in the cell. -Cyclic AMP from the Adenylyl cyclase binds to a protein kinase called protein kinase A. Protein kinase A is only active when it is bound to cyclic AMP. When it's active, it can phosphorylate other proteins to change their activity. Why do we have such a complicated way of doing this? -This allows a huge amount of signal amplification to come from a small signal. Example: during a fight of flight scenario, we need to take a small signal and break down a bunch of glycogen to glucose to use for energy. From the bidning of ` epinephrine to GCPR we get 1 billion glycogens broken down into glucose.

Describe a mis-sense substitution Class 11

-Changing a single nucleotide in the template DNA causes a different codon to come from the mRNA, and that codon gives rise to a different amino acid than the initial template strand would have. -This is problem, but usually is not terrible -But it can be. Sickle cell hemoglobin is a result of a mis-sense substitution

Describe a silent mutation Class 11

-Changing a single nucleotide in the template DNA causes a different codon to come from the mRNA, but that codon gives the same amino acid as the initial template strand would have. -Sequence of the protein if unaffected

Karyotype and Chromosome number class 15

-Chromosome number is constant within a species, but variable between species. The karyotype is the comple complement of chromosomes present in the cell. -Euploid cells have the correct number of chromosomes -Aneuploid cells do not

Nurse experiment

-Cloning by complementation: using simple organisms with specific mutations to identify the equivalent human gene. -Say you have a strain of yeast that does not have the leusine gene, the cells die, they cannot grow. -How can we make them survive in the media with no leucine? Provide the Leu2 gene from a plasmid. -Take that same cell but with a version of cdc2 that unfolds at 37 degrees, causing the cells to die. -How can we make them survive at 37 degrees. Provide the human cdc2 gene from a plasmid. What can we use to complement the yeast Cdc2? Human cDNA.

Describe crosstalk specifcity and scaffolding proteins Class 14

-Crosstalk between signaling pathways allows for nuanced/integrated responses. -Scaffolding proteins makes the process of signal transduction really fast and specific by tethering protein kinases onto scaffolding proteins. This makes the activation of one always lead to the activation of the other, which will always lead to the activation of the toher, etc.

Describe how cyclins and cyclin-dependent kinases (CDKs) control the cell cycle Class 15

-Cyclins act in concert with Cyclin-dependent kinase (CDK) -The cyclin: CDK phosphorylates a wide range of proteins using a phosphate group from ATP. This phosphorylation changes properties of proteins. Could activate some and inactivate others. -MPF is a complex of cyclin and Cyclin dependent kinase CDK -CDK is always expressed and present at a constant level, but cyclin oscillates, so the amount of MPF oscillates with the cell cycle -Cyclin is not present immediately after mitosis. Starts to be expressed in G2, and accumulates through G2. MPF becomes active whent the concentration of the cyclin reaches the critical threshold. MDF can phosphorylate its target protein, pushin the cell past the G checkpoint and into mitosis. At the end of mitosis the cyclin is degraded and we have the empty CDK again. -MPK is not the only cyclin CPK complex. Cyclins are complex!

Describe how cell cycle deregulation is involved in the onset and progression of cancer (watch lecture) class 15

-Defective checkpoints allow cell cycle to progression despite errors -Gene expression is regulated based on two copies of each gene -More copied leads to more expression, so coordinated expression is imbalanced -Chromosomal rearrangements can move genes so that they are correctly expressed

Describe how signal transduction is relevant for human health and disease, using examples related to cancer (e.g. Herceptin®/Trastuzumab, Zelboraf®/Vemurafenib) and erectile dysfunction (Viagra®/Sildenafil) (from lecture) class 14

-Deregulation of cell signaling is fundamentally important in many diseases -There are many ways to lock this pathway in the 'on' state If a cell believes that ligand is there all the time, it will grow way too fast and divide way too fast. -Let's say we have a cell with some activated RAF, you block it and the cells don't grow anymore. Then, they figure out how to bypass that block you put up there. How would the cells bypass raf inhibition? -Activate MEK or activate ERK -Viagra, increased vasodilation affects more than one part of the body.

How can a repressive environment be created? Class 12

-Different histone modification can repress transcription as well as DNA methylation. -HDAC is a histone modification that removes acetyl groups and closes chromatin structure. DNA methylation in eukaryotes closes chromatin structure as well. -Both of these are examples of epigenetic changes, a change where something doesn't change the DNA sequence, but the behavior is changed.

Like prokaryotic cells, eukaryotic cells need to recruit a RNAP to the promoter. In eukaryotic cells in order for this to happen we need the gene to be in a permissive chromatin environment in order for transcription factors to recruit RNAP to the specific gene. What does a permissive chromatin environment mean? And, how can we make such an environment? Class 12

-Eukaryotic chromosomes exist as chromatin (a 1:1 complex of DNA and histone proteins(positively charge)). 8 histone proteins come together to form an octamer. DNA wraps around histone octamers twice to create a nucleosome. Nucleosomes can then be coiled. Chromatid is the maximum state of condensation (creates a visible chromosome). -This condensed structure of nucleosomes makes it difficult for proteins that would bind it like RNAP2, so we need a way to open it up. Some of the histones have tails that stick out through the double helix. These tails can then be chemically modified by various enzymes in the cell and can change the properties of the nucleosome. -Often this modification is the addition of acetyl groups by histone acetyltransferases. This can open up the chromatin structure -Another modification could be the use of ATP-dependent chromatin remodeling enzymes -These use energy from ATP hydrolysis to remodel chromatin. They move nucleosomes around and expose the DNA sequence to allow proteins like RNAPOL2 to bind.

Describe the structure of a typical eukaryotic gene Class 12

-Eukaryotic genes are not organized into operons. RNAP2 transcibes one gene at a time. -Eukaryotic cells have nuclei, so transcription (occuring in the nucleus) is separated from translation (occuring in the cytoplasm. In prokaryotes there is no separation, so translation and transcription occur pretty much simultaneously. -This separation allows the transcript mRNA to be further processed in the nucleus before being exported to the cytoplasm for translation.

Why is an affinity tag needed? class 13

-Even if we induce expression of our protein to a very high level, there are many other proteins in the cell. We have to get rid of those by using an affinity tag. -We have something that binds very tightly and specifically to the affinity tage that we can put on some beads. We put the lysate onto the beads and we take everything that doesn't bind to the beads and we throw it away. Wash it a few times. Then, we can disrupt the interaction between the protein of interest and the beads and elute(remove) our protein of interest from the beads. If our beads are specific enough and we have washed enough we should end up with a very purified protein

What is fertilization? Class 16

-Fertilization is the formation of a ziploid zygote from a haploid egg and sperm(fusion of two gametes) -Sperm(n) and egg(n) come together to zygote (2n). That is a happy zygote. But, we know that there are many sperm per egg, so what would happen if two sperm fertilized the egg? It would make a 3n zygote that will not develop -Molecules and events at the egg surface play a crucial role in each step of fertilization Sperm penetrates the protective layer around the egg. -Receptors on the egg surface bind to molecules of the sperm surface -Changes at the egg surface prevent polyspermy(the entry of multiple sperm nuclei into the egg)

Describe the G1 checkpoint

-G1 is the commitment point checkpoint -Reminded that cells need to be exposed to PDFG to grow otherwise they will leave the cell cycle. -Passage through G1 commits a cell to division and requires a cell to be a certain size and to be exposed to growth factors. Without growth factors instead of proceeding through G1, the cell actually leaves completely and enters this quiescent state called Go. They can come in and out of this state, but most of our cells are in this state at a given time.

How do G-protein-coupled receptors work Class 14

-GPCRs interact with G-proteins and span the plasma membrane 7 times. -When ligand binds to the GCPR it causes a G-protein to associate with the receptor. -Association with the receptors causes the G protein to release GDP and bind GTP. This now active G-protein diffuses away from GPCR and binds to other proteins/enzymes causing them to change their properties. G proteins are GTPases (they hydrolyze GTP to GDP) so overtime G protein hydrolyzes GTP to GDP returning itself to the inactive form. -This means there is a full cycle of activation and deactivation so we dont need an enzyme like protein phosphatase to turn off the signal

Gastrulation

-Gastrulation, the movement of cells from the blastula surface to th einterior of the embryo. -rearranges the cells of a blastula into a three-layered embryo called gastrula -We start with a ball of cells after cleavage, and these cells start moving in the surface and coming in through a blastopore lip. As the cells are entering and moving, the final structure becomes layered in cells that are now different. -The three layers produced by gastrulation are called embryonic germ layers: -The ectoderm forms the outer layer -The endoderm lines the digestive tract -The mesoderm partly fills the space between the endoderm and ectoderm. -Each of these layers contributes to specific structures in the adult animal, they form different types of tissues. So the cells are nont differentiated into these tissues, but they are committed to become a specific type of tissue. So the trophoblast forms the embryo side of the placenta.

So we definitely need this certain type of environment in eukaryotic cells for transcription, but that's not enough. How we we actually recruit RNAP2 to the promoter? Why is this STIll not enough regulation? Class 12

-General transcription factors (GTF) land on a specific sequence (TATAAAA) and bind to the core promoter and recruit RNAPT. -BUT, General transcription factors recognize short DNA sequences (4,6,8bp), so if this is all that was needed for transcription, we would get the expression of a lot of genes too frequently.

Cytoplasmic determinant

-Genetic content is the same after mitotic cell division, but the two cells may have different cytoplasmic determinants. Helps determine their commitment and influence what the cells will be. Leads to different gene expression. -P granules are distributed throughout the newly fertilized egg and move to the posterior end before the first cleavage division. With each subsequet cleavage, the P granules are partitioned into the posterior-most cells. P granules are involved in the specification of germ cell fate. P granules act as cytoplasmic determinants, fixing germ cell fate at the earliest stage of development.

What is growth factor signaling? Class 14

-In the absence of growth factors, mammalian cells exit the cell cycle -In the precense, they continue through the cycle. Too much growth factor signaling causes cancer.

describe a Frameshift mutation Class 11

-Insertion or deletion of nucleotides. If the number of nucleotides is not a multiple of 3 we will change the reading frame of the mRNA downstream. -Could result in a premature STOP codon -If the frameshift is late (towards the 3' end of the gene) then most of the protein is normal, there is a good chance it will be functional -If the frameshift happens early (toward the 5' end of the gene) Very little normal protein sequence-most likely non-functional

Describe the necessary components of an intercellular communication system Class 14

-Intercellular communication is not just one big came of telephone where info is transferred from cell to cell. -Cells involved in sending the signal secrete specific ligands (proteins or small molecules that can diffuse away from the signaling cell and then bind to receptors on distant cells and elicit a response there. -Cells involved in receiving the signal express appropriate receptors (proteins capable of recognizing the signaling molecule)

Regular PCR is often a waste of energy when we really want to look at specific RNA. Describe RT-PCR Class 12

-It is often a waste to amplify the whole genomic DNA sequence when we are looking at a specific RNA, because through the process of transcription and pre-mRNA processing we get rid of a lot of the info in the original DNA anyway. So what we can do is make a cDNA copy by reverse transcription, degrade the RNA so it doesn't interfere, and then amplify it by PCR (this is called RT-PCR). We are left with the single strand of DNA we want in amplification. -Then, we can use gel electrophoresis to figure out where the RNA is and how much there is.

What happens when we put the plasmid into a bacteria class 13

-Keeping and replicating a plasmid costs the bacteria energy, so we need to convince the bacteria to keep the plasmid. -So we put an antibiotic resistance gene on the plasmid. And then we put it in an agar plate with ampicillin (an antibiotic that kills bacteria) so all the bacteria will die except the ones that have the antibiotic resistance gene which is on the plasmid. This is our way of forcing the bacteria to want to keep the plasmid.

Transcription initiation can be regulated so DNA isn't just constantly being transcribed to mRNA. Describe how this process work in the lac operon. And now the lac operon can be repressed. class 10

-Lactose is composed of glucose and galactose. E.coli can use lactose as a carbon source, but prefers to use pure glucose if available. -Lac operon combines positive and negative regulation of gene expression. -Very similar to the way trp operon is regulated -When lactose is absent the lac repressor is in the correct shape to bind to DNA, block RNA polymerase and turn transcription off. When lactose is present, it binds to the repressor, changes its shape, DOESN't bind to DNA, and transcription proceeds. -But what is both glucose and lactose are present? How do we know to block transcription? - When glucose is scarce, and E. coli needs to use lactose as a sugar, E. coli generates cyclic AMP (cAMP). cAMP binds to a protein called CAP. When bound to cAMP, Cap act activates transcription of the operon by binding to the promoter. So if we have a lot of glucose, no cAMP will be made and therefore we won't waster energy transcribing lactose.

Describe how alternative splicing can be used to generate multiple distinct mRNAs, encoding multiple distinct proteins, from the same gene Class 12

-Not every mature mRNA has to contain every exon. We could combine exon 1, 2, and 3. Or, we could skip exon 2 and just do 1 and 3. -There are many ways that alternative splicing can occur which allows for a single mRNA transcript to give rise to loads of different protein sequences.

Describe how restriction enzymes work? Class 13

-Nucleases that cut DNA at specific DNA sequences. They protect bacteria from bacteriophage injection by cutting the DNA of invading phage. -They recognize palindromic DNA sequences. They have the same 5'-3' sequence on both DNA strands (generally 4, 6, 8). They cut at the same nucleotide base on both strands. If that nucleotide is in the middle of the palindromic it will result in blunt ends. If it is off center it will have sticky ends. Sticky ends can then be annealed to other things which will be useful. -Different restriction enzymes recognize different sequences. Sometimes one sequence can be recognized by more than one reaction enzyme (but maybe one leaves sticky ends and one leaves blunt ends). -Restriction enzymes have short recognition sequences so an enzyme with an 8bp recognition sequence will cut at over 45,000 sites in the human genome.

Explain holistically how all cells in multicellular organisms contain the same genome, but that different cell types express distinct subsets of genes Class 12

-Only a fraction of the 20,000 genes in the human genome are active in a given cell type. -Virtually every cell in your body contains completely identical DNA, but only a given fraction is expressed in a given cell type, giving rise to the variety of functions. Gene expression can be regulated at many stages. This regulation can occur at both transcription and translation.

Open mitosis Closes mitosis Class 15

-Open: Most eukaryotes The nuclear envelope breaks down and is re-formed after cytokinesis -Closed: - diatoms and some yeasts Nuclear envelope remains intact. Centrosomes form within the nucleus, which is divided alogn with the rest of the cell during cytokinesis

What 3 things are needed for a plasmid class 13

-Origin of replication -A region with lots of restriction enzyme sites where we can put our DNA (so a place on the DNA with lots of palindromic sequences) -A gene to confer an advantage bacteria who get and keep the plasmid

describe a Nonsense substitution Class 11

-Pretty much the same as mis-sense substitution, but the different amino acid that the new codon gives rise to is actually a STOP codon. This will truncate the protein. -This is pretty bad

Prometaphase Metaphase Anaphase class 15

-Prometaphase Nuclear envelope fragments Kinetochores appear Spindle attaches to kineotchores -Metaphase (chromosomes align) Longest phase Chromosomes gather Centrosomes are at opposite poles Microtubules attach to kinetrochores at centromeres -Anaphase (chromosomes move to poles) Shortest Kinetochore microtubules shorten

What are protein kinases and protein phosphatase? Class 14

-Protein kinase is an enzyme that will specifically phosphorylate (transfer a phosphate from ATP) to serine, threonine or tyrosine. Phosphorylation causes a conformational change and importantly adds a negative charge. -Protein phosphatase is an enzyme that removes a phosphate

Describe how introns are removed and why Class 12

-RNA splicing occurs from the pre-mRNA to remove intron from the genes. -Spliceosome is complicated but basically pinches the pre-mRNA and cuts out the introns. -The mature mRNA has multiple exons which altogether code for a protein . Frequently, one or two exons code for a specific domain of a protein, but this doesn't have to be the case. -Really just a fraction of the human genome actually codes for proteins (1.5%). More for less complex organisms but still there is a good amount of non-coding DNA in every organism.

How do we add DNA to a plasmid class 13

-Restriction enzyme cuts the sugar-phosphate backbones at the same nucleotide. -Cut a DNA molecule by the came restriction enzyme so it has compatible ends to be inserted into the plasmid. -The sticky ends can anneal the plasmid DNA to the inserted DNA. -DNA ligase covalently links nucleotides together to seal up the plasmid.

S-phase G2-phase Prophase Class 15

-S-phase (DNA synthesis) -G2-phase (a gap with a checkpoint to ensure DNA synthesis is complete) -Makes sure DNA synthesis went smoothly -Prophase (chromosomes condense) Chromosomes condensa nd sister chromatids align Nucleoli disappear Asters and spindle form Centromeres move towards opposite poles

Sister chromatids Microtubules Centromere Class 15

-Sister chromatids After replication but before mitosis, the two identical copies of chromosomes remain associated. -Microtubules attach as the centromere through a structure called kinetochore.

How do intracellular steroid hormones work? Class 14

-Steroid hormones are derived from cholesterol and are hydrophobic, they can pass through the plasma membrane. -Although steroid hormone receptors are transcription factors, they are held in the cytoplasm so they do not act as transcription factors until they bind to their ligand (hormone) causing a conformational change which results in the receptor moving to the nucleus and allowing it to activate expression of specific genes. -A steroid hormone diffuses into a cell, binds to its receptor and causes the receptor to move to the nucleus and act as a transcription factor and activates gene expression

Describe holistically how gene expression can be regulated at several stages Class 11

-Synthesis, degradation and localization can be independently regulated. But often we observe 'coherent' regulation -Both mRNA and the protein can also be degraded overtime. New rate of production of mRNA is a function of both how frequently that mRNA is synthesized and how frequently it is degraded. -Cells can regulate synthesis of both RNA and protein and the dehydration of RNA and protein and the localization of these molecules.

Telophase Cytokineses Class 15

-Telophase (nuclei re-form) Nuclear envelope reforms Chromosomes decondense Nucleoli reform -Cytokinesis (cell division and separation) Occurs simultaneously with telophase Occurs after the daughter chromosomes have moved to the daughter cells Different in plant and animals cells because plants have cell walls.

How does receptor tyrosine kinases activate gene expression? Class 14

-The 2 signaling molecules, in this case PDFG which bind to the extracellulas region of two different receptor tyrosine kinases. When the two molecules of the receptor tyrosine kinase are both bound to a growth factor then these protein molecules come together to form a dimer. The RTK are both a receptor and a tyrosine kinase so it can phosphorylate tyrosine residues. The two RTK in a dimer phosphorylate eachother. This can cause a conformational change that allows the binding of a variety of proteins. -Now that we have the activated RTK that can bind to a relay molecule and activate the relay molecule this kicks off a cascade of protein phosphorylation. This is the phosphorylation of many protein kinases in a line in the intracellular region. The third and final protein kinase can phosphorylate other proteins, converting them to inactive or active states. Often it is a transcription factor that this final protein kinase activates

Describe how chromosomes are accurately partitioned between two daughter cells

-The anaphase checkpoint (M checkpoint) Until all the chromosomes are correctly attached, stop signal is received. Once all chromosomes are bi-oriented at the metaphase, anaphase is induced -More detail: Chromosomes are lying on the metaphase plate before anaphase begins. Then during anaphase, the chromosomes are pulled to opposite poles of the cell to produce two new nuclei with identical DNA content. In order for anaphase to proceed every single chromosome has to have one sister chromatid attached to one pole and other to the other pole.

How do sigma factors help prokaryotes initiate transcription? class 10

-The consensus sequences work in complex with a protein called the sigma factor to recruit RNA polymerase to the promoter -Note: sigma factor is not necessary during transcription, BUT it is necessary to recruit RNA polymerase. -7 different sigma factors exist for varying sets of environmental conditions (levels of available nutrients, temperature, etc.). They can make a less suitable environment more suitable by activating or repressing the transcription of specific genes.

How is translation initiated? Class 11

-The small ribosomal subunit and the large ribosomal exist separately and come together on an mRNA. -The small ribosomal subunit binds to mRNA -Initiator codon is AUG, so there's a special initiator tRNA with the codon CAU, which recognizes AUG. This comes in and is covalently coned to a methionine, which is what is encoded by AUG -Hydrolysis of GTP allows the recruitment of the large ribosomal subunit. -Now the initiator tRNA is at the P site, and A site is ready to accept tRNA with an anticodon that recognizes the next codon.

Short-range communication vio ligand/receptor communication Class 14

-The two adjacent cells can communicate by one cell exposing a ligand on its surface and another cell expressing the receptor for that ligand on its surface. -The binding of one cell to another will elicit a signaling response downstream of that receptor.

Describe amino acid and protein polarity Class 11

-The variable R sidechain determines the amino acid and polarity in a sense -The C terminus (carboxyl group) is negatively charged. The N terminus (amino group) is positively charged. And two amino acids can come together in a dehydration reaction to form a peptide bond and build a protein.

Describe the mechanisms of translation within the ribosome Class 11

-The whole recognition of a codon and moving it from A to P to E site cycle is repeated to extend the protein -There are two moments of GTP hydrolysis. -The first allows the P site to give the growing chain to the tRNA at the A site which is moving into the P site -The second shifts the whole ribosome by 3 nucleotides along the mRNA.

How does the ribosome translation complex know to stop? How does this create a full circle? Class 11

-There is no tRNA that recognises the stop codons, UAA, UAG, or UGA. -Instead release factors recognize these codons(like tRNA recognizes codons) and promote hydrolysis of the bond between the tRNA and the completed polypeptide. -Ribosomal factor binding causes the protein to be cleaved from the 3' end of the tRNA by hydrolysis and therefore releases the protein. -Now we have the mRNA, large subunit, and small subunit all separate again. SO the small subunit can find another AUG.

describe the 3' end poly-A-tail and its function Class 12

-This is many consecutive adenosines (A) added by poly(A) polymerase using ATP. This protects the 3' end of the transcript. AAUAAA signals for poly(A) polymerase to occur and the cleavage there allows the RNA transcript to be released from RNA polymerase. -Both the cap end and poly-A-tail are required in order for mRNA to be translated so we can be positive that we have the full mRNA not just a fragment.

Describe the 5 key properties of the genetic code Class 11

-Triplet-based Each codon in the mRNA comprises three nucleotides: a triplet -Redundant There are 64 possible codons for 20 amino acids. Therefore, the code is redundant. There are some amino acids that are encoded by more than one codon. -Unambiguous Each codon specifies one (and only one) amino acid or STOP BTW, AUG is for start 61 codons encode amino acids and then there are 3 codons for stop which terminate protein synthesis at the end of the polypeptide chain. There is no punctuation between codons -Non-overlapping The 3 bases are separated never overlapping -Universal All organisms use the same genetic code. Prokaryotes and Eukaryotes alike. The universality of the genetic code makes genetic engineering possible

How can we monitor gene expression? Class 12

-We can use complementary RNA molecules to visualize how much RNA is present, where in an organism the RNA is present, and even where in a cell the RNA is present. -We take a gene that codes for our region of interest, and we can change that gene for that instead of having a stop codon at its end, it has the coding sequence of GFP and then add a stop codon.

Describe how CRISPR/CAS9 work Class 13

-can cut DNA at essentially any sequence guided by a 20bp sequence complementary with an RNA molecule. -Since this is such a long recognition sequence, unlike restriction enzymes, we can be certain it is unique in a genome and will only cut ONCE and at the specific site we want it to cut at.

What are the three different kinds of RNA that are universal for both prokaryotes and eukaryotes that we talked about? class 10

-mRNA (codes proteins), rRNA (integral components of ribosomes), and tRNA (required for decoding translation)

Describe Quorum sensing class 14

-occurs in both eukaryotes and prokaryotes. Cells sensing how many identical cells are sound them to control behaviors from happening at low densities when such behaviors are only advantageous in high densities -Example antibiotic production: Bacteria will wait until there is sufficient density and then all simultaneously switch on genes responsible for producing antibodies, creating a large amount of antibodies that can actually kill off other bacteria (whereas if the gene was switched on when there was a low density of the bacteria it would have not worked/been worth it) -Collective behavior is dependent on communication. Every cell in an organism has to have the capacity to send and receive signals.

Describe the structure of tRNAs, how they are charged with the correct amino acid, and how they interact with mRNA during protein synthesis Class 11

-tRNAs recognize specific codons in the mRNA -They are charged with amino acids by enzymes called tRNA aminoacyl synthetases. They choose the correct one because tRNAs have an anticodon that recognizes the correct amino acid by base pairing. The amino acid is then covalently linked to the 3' end of the tRNA. NOTE: There are fewer tRNAs than there are codons, so a given tRNA can recognize 1 or TWO codons. -tRNAs that have an amino acid banded can convey this info from the mRNA to the active site of translation.

How many RNA polymerases do eukaryotes have? which one are we focussing on?

3 RNAP1 (transcribes rRNA), RNAP2(synthesizes mRNA), RNAP3 (synthesizes tRNA). We will focus on RNAP because that is where the regulation and variation come from

Describe how telomeres and telomerase allow the ends of linear chromosomes to be maintained through repeated rounds of replication Class 12

A telomere is the end of a chromosome. Telomeres are made of repetitive sequences of non-coding DNA that protect the chromosome from damage. Each time a cell divides, the telomeres become shorter. Eventually, the telomeres become so short that the cell can no longer divide. BUT, Telomerase, also called telomere terminal transferase, is an enzyme made of protein and RNA subunits that elongates chromosomes by adding TTAGGG sequences to the end of existing chromosomes.

Describe Northern blot Class 12

After gel electrophoresis we can open up a cell and purify the RNA away, then we can transfer RNA to the nitrocellulose membrane. Then, we put a stack of paper towels and books sitting in liquid. The liquid gets sucked up and brings the RNA with it. RNA follows the water, but sticks to the membrane. We can then use a labeled RNA molecule with the reverse complement sequence and look for signals. Northern blots are also good for looking for alternative splicing events because we can look at length unlike PCR.

Holistically, What must happen to go from a zygote to an adult

All starts with one cell, a zygote. This zygote has to do two things, it has to grow in cell number. Second, the cells need to be specialties with diverse functions. The cells come together to form tissues and then tissues come together to form organs and organs come together to form organ systems. Form multicellular organisms

Describe the overall mechanism by which RNA is synthesized from a DNA template by RNA polymerase in prokaryotes class 10

At the start of a gene, there is a promoter. The promoter determines where transcription starts(starts downstream of promoter but upstream of coding region). RNA polymerase is used to synthesize RNA in the 5' to 3' direction. It is recruited to the promoter of the gene. It unwinds the DNA and catalyses the formation of an RNA molecule which is the reverse complement of the template strand(in other words the same as the coding strand but U instead of t). DNA becomes overwound ahead of RNA polymerase and under-wound behind it. This problem is overcome by topoisomerase enzyme which releases tension. Finally RNA polymerase falls off the DNA when it hits the terminator and the completed RNA transcript is released. Transcription will end at the terminator

What is the experiment that lead to the discovery that each gene encodes a specific protein? Describe it. class 10

Beadle and Tatum's experiment. -A fungus, which can normally take up arginine from the environment or synthesize itself, was exposed to radiation with x-rays to cause mutations (which inactivates genes). These 'mutants' were then grown in the absence of specific nutrients and researchers looked for strains that appear to be deficient for the synthesis of arginine. If the mutants were grown in the presence of arginine, it didn't matter if they had a broken biosynthetic pathway, but if the mutants were grown in the absence of arginine then the biosynthetic pathway needed to be functional. They had the wild type, and class I, II, and III mutants. The wild type could grow without any of the supplements. Class I, II, and III mutants all did not grow with minimal medium. Class I mutants could grow on arnithine, citrulline or arginine. Class II mutants could only grow on citrulline or arginine. Class III mutants required arginine to grow. From this we could conclude that Class I mutants had a mutation in the enzyme A. Class II mutants had a mutation in enzyme B. Class III mutants had mutations in enzyme C. This means that each enzyme in the arginine biosynthesis pathway is encoded by one gene.

Describe how end joining and recombination can be used in concert with CRISPR/Cas9 to mutate or replace genes in complex genomes, including the human genome class 13

CRISPR can cut DNA at a specific sequence. We can use HR DNA repair machinery, which takes DNA from a homologous chromosome and uses it as a template for repair(this is error free). With this we could replace the sequence with a new sequence of a gene (knock-in) of our choice (maybe GFP) and see the effect this gene has on the cell/organism.

If anaphase checkpoint fails? what happens Class 15

Daughter cells do not inherit the correct number of chromosomes and create what are called aneuploid cells. Cells that do not contain the correct amount of DNA.

How can we turn off the signal Class 14

Each of these steps in the cascade involve the activation of a protein kinase by phosphorylation. For each of these steps a protein phosphatase removes phosphate groups and turens off the signal once the growt signal factor is removed and the receptor is no longer active. This resets the entire pathway to the off state.

Use the trp operon to describe feedback inhibition.Describe the fast and slow way gene expression can be regulated. class 10

Fast: Regulating enzyme ACTIVITY, when tryptophan is made from the trp operon that build of up tryptophan actually inhibits the enzyme in the first step of the pathway. So naturally as we make more of something the rate at which we make it slows down. Slow: regulating enzyme PRODUCTION, if tryptophan is abundant for a longgg time, we actually end up degrading the enzymes that make it, reducing the expression of tryptophan.

Short-range communication in animals class 14

Gap junctions between adjacent cells in animals by mixing of the cytoplasm allows small molecules, ions, and electrical impulses to be shared.

Hunt experiment

Give sea urchins (which are a really good model for studying the cell cycle we can watch them synchronously go through early embryonic development), going through synchronized cell cycles, a short dose of radioactive methionine (an amino acid found in basically all proteins). Any newly synthesized protein will contain the radioactive methionine and be detectable. We can see a specific protein that accumulates and then goes away and then accumulated and then goes away. We are witnessing that there is a specific protein that appears and disappears in phase with cell division.

How are proteins targeted in Eukaryotes? Class 11

Growing proteins are targeted by the ER by interaction of a single sequence with the signal recognition particle SRP. -Ribosome starts translating the protein, and the synthesis foes from N to C terminus. -Once the protein reaches a certain length, there's enough protein that it's exited the exit tunnel of the ribosomes, so the protein is dangling off the back of the ribosome -This N-terminus region of the protein contains the signal peptide which is recognized by the SRP. -The SRP binds non-covalently to the signal sequence, and actively brings the ribosome and protein its translating to the ER. The SRP docks with the dedicated receptor -Now we have this complex where the ribosome is bound to the SRP and the SRP is bound to its receptor. -The SRP can then leave and the ribosome is now able to synthesize the protein and the protein is pumped into the ER. -Ultimately, the signal sequence that brought the protein to the ER can be removed. Left with the mature protein.

ZPA experiment

Hypothesis: The ZPA at the posterior tells the limb where the posterior is, so therefore, it sets a coordinate system from that posterior to anterior. If this is correct then we should be able to execute this experiment: Take a ZPA and put it into the host limb bud. Now this new limb has two ZPA: the original one, and the new one, signaling a fake posterior, but also signaling a posterior. If this would have been enough to introduce posterior identity, the limb now develops two posteriors. This is exactly what happened.

What prevents an egg from fertilizing multiple sperm

Immedietely after fertilization, there is a physical change in the egg (a bunch of sperm surrounding it). Then the outer layer of the egg separates from the inner layer of the egg. Therefore, no more sperm can fertilzie after the first one did. This change is transmitted by a wave of intracellular calcium, which restricts any more sperm from fertilizing with the egg.

Describe the 5' cap and its purpose Class 12

Modified nucleotide connected to the rest of the mRNA in an unusual 5'-5' triphosphate linkage by the capping enzyme. Now the mRNA does not have an exposed 5' end. This cap protects the mRNA from degradation by 5'-3' exonucleases. It is also necessary for a mRNA to have a cap for it to be exported. This is good because we don't want mRNA without a cap that has likely been degraded to try to be translated. In eukaryotes the small subunit of the ribosome binds to the 5' cap of the mRNA and scans to find the FIRST AUG downstream. The cap allows the correct AUG to be selected.

Morphogenesis Organogensis

Morphogenesis is the process by which cells accupy their appropiate locations -organeogenesis is the formation of organs

What happens if a gene later in the genome is expressed earlier in the embryo than we expect?

No good. The cells are confused on where they are. They differentiate into things they shouldn't. Conclusion changes in transcription factor activity in early development results in profound changes in cell identity.

Hox transcription factors

Part of a family called Homeobox genes. They code for a domain that allows a protein to bind to DNA to function as a transcription regulator. -Anterior to Posterior patterning: - In a fly, the hot genes are organized in the genome in a way that is similar to how they are expressed in the embryo. In other words, they are transcribing the embryo in the same order as they are in the genome.

Body patterning

Pattern formation is the development of a spatial organization of tissues and organs. -In animals, pattern formation begins with the establishment of the major axes -Positional information tells a cell its location relative o the body axes and to neighboring cells.

Short-range communication in plants Class 14

Plasmodesmata are holes in the cell wall that allow small molecules to move between adjacent cells.

Describe the issues with Western blot and how we solve them? Class 12

Proteins don't have base pairing. We take our protein sample. Separate it on a gel, transfer the proteins to a membran and use an antibody to the protein of interest. This will signal where the protein of interest is. -Gel electrophoresis is good for DNA and RNA because they have the same mass:charge ratio. We need to make this true for proteins -We can use SDS to keep proteins unfolded and add a consistent charge. And use B-ME to reduce disulfide bonds that hold proteins together.

Where does transcription invitation start in prokaryotes? How did we figure this out? class 10

So what researcher did is look for the recurrence of specific sequence in several prokaryotic genes. They found that there are specific sequences (consensus sequences) that determine PROMOTER location in prokaryotes. There are two sequences, and they are both important and the space between them is in important in recruiting RNA polymerase.

How do we go about determining the sequence of a polypeptide encoded by a given DNA/RNA sequence Class 11

Take a template DNA strand and do the reverse complement to get the mRNA sequence. Find the AUG that is the start. Group by 3s to get the codons for the sequence. Use genetic code to identify the amino acids for the codons

What is the blastocyst

The blastocyst is a ball of cells that contains inside a group of other cells. We see the first time of cellular differentiation The inner cell mass is a cluster of cells at the one end of the blastocyst The trophoblast is the outer epithelial layer of the blastocyst and does not contribute to the embryo, but instead intiates implantation.

How are prokaryotic genes structures and organized? class 10

They are organized in operons. And operon is a 'A functioning unit of genomic DNA containing a cluster of genes under the control of a single promoter, which is used to recruit RNA polymerase' -The RNA polymerase will then transcribe an RNA molecule that contains the coding sequence for the genes in that operand.

Where do transcription and translation occur in the prokaryotic cell. How many copies of a protein come from one gene?(not an actual number) class 10

They both occur in the cytoplasm as prokaryotic cells do not have a nucleus. This means there is no separation of transcription and translation. The mRNA that is being transcribed from an operon can be translated by ribosomes while its still being transcribed. The one gene gives rise to many copies of mRNA and each mRNA is being translated by the ribosomes to proteins.

Rao and Johnson experiment

Took cells at different phases of the cell cycle and fused their cells. The cytoplasms fuse and mix, but the nuclei don't. So, they looked at the nucleus of each cell. They take a cell in s-phase, it's replicating DNA, and fuse it to a cell that's in G1 phase. What they observed is that the G1 phase cell immediately enters s phase. Similarly, if you take a cell in M phase and fuse it to a cell in G1 phase, the G1 cell immediately attempts to go through mitosis, even though it hasn't replicated DNA. Conclusion: the cell cycle is regulated by soluble factors in the cytoplasm.

Transcription initiation can be regulated so DNA isn't just constantly being transcribed to mRNA. Describe how this process work in the Trp operon. And now the trp operon can be repressed. class 10

Tryptophan is an amino acid that is essential for cell growth. -E. coli can synthesize tryptophan or take it up from the environment, but obviously would prefer and be more cost effective for it to take it from the environment. -Transcription of the trp operon, followed by translation, produces the enzymes necessary to synthesize tryptophan. - In order to make these enzymes, the e.coli has to express the genes encoding these proteins. -Just by the binding of a Single RNA polymerase will allows the whole trp operon to transcribe. Making a mRNA that will code for all 5 proteins. -The default state of the promoter is ON, but it can be repressed by the trp repressor. -When tryptophan is available in the environment it will bind to the trp repressor causing a conformational change and allows it to slot into the DNA at a specific binding site of the trp operon preventing RNA polymerase from binding to the promoter and transcribing the gene. This ensures that if tryptophan is abundant, and e.coli can just just take up tryptophan rather than synthesizing it, it will stop wasting energy on transcription of the operon to make the enzymes.

Describe how CRISPR-mediated gene knockout can be used to determine gene function class 13

Use CRISPR to cut DNA at a specific sequence. Results in a double strand break. NHEJ DNA repair machinery occurs, and the ends are immediately glued back together, but sometimes sequences are gained or lost. If we do this many times eventually we will get a frameshift mutation leads to the gene being knocked out and we can observe the function that gene had based on what happens to the cell/organism without that gene

Cleavage

We get the ONE cell from fertilization and then right after that, inside the structure in the zygote, we see cleavage. The physical size of that embryo is not changing, but what is changing is the cell number within that space. So the egg is a huge cell. That cell contains all the energy and materials to make many cells. So cleavage, by definition, is the rapid growth in cell number without growing the actual size of the organism. Generate the first group of cells and then can be allocated into different development trajectories. During Cleavage, cell cycle is shortened, and it goes very very pahse. MSMSMSMS. Mitosis, S phase, Mitosis, S phase. Cleavage partitions the cyctoplasm of one large cell into many smaller cells called blastomeres. The blastula is a ball of cells with a fluid-filled cavity called a blastocoel.

How do transmembrane receptors work? Class 14

We have a receptor with an extracellular region. That binds the ligand and an intracellular region whose properties change depending on whether or not the ligand is bound. This kicks off a cascade of different reactions where the signal is passed to different relay molecules and amplifies the cellular response. Often the response is a change in gene expression. But since this protein is localised in the plasma membrane it cannot directly act as a transcription factor, so it has to pass the signal to other molecules in the cytoplasm and then ultimately the nucleus.

Once we have put our eukaryotic cDNA sequence under the control of a prokaryotic promoter, we can express it. Which promoter do we use? class 13

We use the one from the lac operon, because we really understand how it works and is regulated. We can make sure we don't express our protein of interest until we have a very high density of the bacteria. We grow the bacteria without glucose, but instead of using real lactose which will eventually run out we use IPTG which is a gratuitous inducer, meaning it will induce expression from the lac operon but it does not get broken down.

Describe the structure of ribosome Class 11

_There is a large and small subunit (each are composed of rRNA and proteins) -In a ribosome we have THREE tRNA binding sites, A SITE (acceptor , where tRNA bearing the next amino acid will bind), P site (contain the tRNA that is covalently bonded to the whole protein that's grown so far), and E site (Exit site, transiently occupied by a tRNA on its way out of the ribosome)

Brief overview of what plasmids are class 13

are small circular chromosomes. They can replicate in bacteria (some eukaryotes also have plasmids but we are focusing on prokaryotes). They are replicated just like bacterial chromosomes (made up of protein and a single molecule of DNA), so very low error rate. We can use plasmids to maintain these DNA sequences, and to expand these DNA sequences very accurately.

Unlike steroid receptors, most molecules used for intercellular signaling cannot pass the plasma membrane, so they need... Class 14

to detect extracellular molecules and interact with intercellular factors to transduce the signal. These receptors are what we call transmembrane proteins. They span the membrane by having part of the protein be on the inside and part of the protein on the outside.