Biology Exam III

Protein Ubiquitination

Ubiquitin: 1. Regulatory protein found in almost all cells (ubiquitously) with nuclei (eukaryotes) 2. Directs proteins to recycling 3. Binds to proteins & labels them for destruction

UTR

UnTranslated Region - have them at both ends; those will not be included in the protein - purpose: going to be contributing to identifying where the beginning & end of protein is - WATCH YOUTUBE VIDEO

Is UTR = intron?

- NO; UTR is just a region in mRNA that's not going to be translated. - An intron is a region that exists between an exon & another exon. The spliceosome will not act on the UTRs & will only act on the introns by the function of the snRNPs

G-protein-coupled receptor (GPCRs)

- 3 components needed: G protein, G protein receptor, & effector - signal received by receptor - activated receptor recruits G-protein - G-protein undergoes conformational change - G-protein releases GDP allowing for GTP to come in activating the G-protein - when activated, it then leaves the receptor & moves toward effector enzyme activating the enzyme which leads to cellular response - when we no longer need cellular response, we're going to hydrolyze GTP on G-protein & go back to inactive state - more than 50% of medicines work by G-protein-coupled receptor ------------------- 1. Largest family of cell-surface receptors 2. A plasma membrane receptor that works with the help of a G protein 3. The G protein acts as an on/off switch: - If GDP is bound to the G protein, the G protein is inactive

tRNAs have a specific *anticodon* & bring amino acids to ribosomes

- 3D structure, 2D structure, Cartoon - anticodon & amino acid binding site

Types of Receptors

- 4 ways a cell can receive a signal known as receptors. Membrane Bound Receptors: 1. Ion channel receptors: respond to ions 2. G protein coupled receptors: require G protein 3. Receptor tyrosine kinases Intracellular Receptors

Split Genes & RNA Splicing cont'd pt. 2

- 5' Cap, Exon 1, Intron, Exon 2, Poly A tail - first thing that's going to happen is that the introns are recognized by one of components of the spliceosome known as snRNPs - snRNPs is a molecule that's made of protein & an RNA molecule. It is the RNA molecule that's capable of recognizing where the intron begins & ends - once it does that, then more molecules are going to come together forming the larger molecule known as the spliceosome & as a group then the ribosomal subunits are going to cleave the introns off & join the exons together - WATCH YOUTUBE VIDEO

Operons can have multiple levels of gene expression regulation

- Catabolite repression & the lac operon

Protein kinases & cyclins regulate most of the events in the cell cycle

- Cyclin D belongs to a series of proteins known as cyclins. - as name suggest, their concentration is going to cycle as the cell cycle progresses - so, we start with Cyclin D which was produced by the Ras/Map kinase pathway & cyclin D is going to be present throughout the cell cycle until the end - the events of cyclin D is going to lead to the initial expression of the protein Cyclin E - when we have the max amount of Cyclin E, we are then going to transition from the G1 phase of the cell cycle into the S phase of cell cycle - as Cyclin E starts working, we're going to accumulate Cyclin A - when we have enough Cyclin A present, we're going to transition from S phase to G2 Phase - as cyclin A is working, we have accumulation of Cyclin B - When Cyclin B is at the cusp, we transition from G2 phase to mitosis - at end of mitosis, we need to get rid of all the cyclins...they need to be completely gone of cell cycle or else we're going to continue cell cycle over and over again. - important to recognize which cyclin is associated with which phase of cell cycle; order matters & note that when we have the highest concentration that we transition from one phase to another - cyclins do not act on their own; always in association with another protein known as CDKs (protein kinases)

Generals on Eukaryotic DNA replication cont'd

- DNA replication occurs during the S phase. It is the main event of S phase in cell cycle - semiconservative = both strands of DNA are going to serve as templates to make new strands of DNA - at the end of the day, we should have two exact copies of the DNA = goal of DNA replication - DNA replication is a very complex process & is going to require many proteins in order to achieve DNA replication

Mismatch Repair (MMR) cont'd

- DNA replication took place but that doesn't mean mistakes weren't left behind - going to happen right after DNA replication & where majority of DNA replication happens - going to have a complex of enzymes that are going to read the DNA (instead of structure) & when they find a mismatch then they're going to stop & mismatch mechanism is going to have to make a decision. Which is the correct information? Is G or T the correct information? - way it determines which of the 2 nucleotides is correct is by looking at which strand is methylated. The old/template strand is methylated but newly synthesized strand is not methylated. - this mechanism is going to determine which methylated strand has the correct sequence & therefore going to determine that G is correct and T is incorrect - Going to remove T & introduce C & also going to methylate the newly synthesized strand & therefore keeping track of areas of DNA that has already been checked

Eukaryotic genes are usually *monocistronic* & contain coding & non-coding regions cont'd

- Genes/G1 includes the exons & introns - Unit of transcription includes promotor & terminator sequence - RNA processing includes multiple steps including RNA splicing - Primary transcript includes both exons & introns

Elongation of the Polypeptide Chain cont'd

- In order to start the process of elongation, we need to bring the appropriate tRNA to the A site - elongation requires 3 specific steps: 1) we need to recognize the codon, 2) we need to be able to attach the new amino acid with the growing amino acid chain (so, we're going to make peptide bonds), & 3) keep moving the ribosomes so that we can keep reading the mRNA - if you have the mRNA, ribosome keeps moving & mRNA stays put - the tRNA doesn't come by itself, we have other proteins that are going to help us bring the tRNA to the right place & those are known as *elongation factors*

Problem: 20 tRNA but 64 codons.

- Multiple codons can code for the same amino acid. Degenerate part of DNA - How do we make sure the right tRNA matches if we have multiple options? Low level specificity

Translation: Initiation

- Now that we have a charged tRNA & ribosome, how do we start translation? - we have initiation step, elongation step, & termination step - to start translation, we need to recognize the mature mRNA which is the job of the small subunit - the small subunit along with other components is able to distinguish the 5' cap from the poly- a tail & will orient itself where the 5' cap is - when small subunit comes to the mature mRNA, it's going to orient itself to occupy components of 5' cap, 5' UTR, & start codon - once small subunit binds to mRNA in appropriate orientation, the start codon is going to be recognized by appropriate tRNA - note that there's only going to be one tRNA for this start codon which is the tRNA that codes for methionine. so all proteins as their first amino acid, methionine. - once we have small subunit bound to mRNA & have initiator tRNA attached to the start codon, now we can bring the large subunit which will require energy that will come from hydrolysis of GTP - we're going to have that the large subunit comes in which is going to create along with the small subunit all the sites A, P, & E site - when we form the complex, notice that the first tRNA will be occupying the P site leaving the A site exposing the next codon available - now that we have this complex put together, next thing to do is to elongate the peptide

Regulating Transcription in Bacteria cont'd

- Now we're going to be talking about how we are going to be regulating transcription in bacteria. And we have options 1. Negative control = stopping transcription - using repressor protein which is going to bind to operator & prevent RNA polymerase from properly binding to the promoter which prevents transcription from happening - regulated via feedback regulations 2. Positive control = promote transcription - use activator which will bind to activator site & is going to assist the RNA polymerase in binding to the promoter & allowing for transcription to happen

Transcription: ELONGATION cont'd

- RNA polymerase will continue to unwind DNA & start to read the template strand & add complimentary ribonucleotides - RNA polymerase will continue to unwind DNA & can start from scratch adding ribonucleotides & will not need a primer & is going to be joining the ribonucleotides with phosphodiester bonds - RNA polymerase will work quickly; can add 40 nucleotides per second - it is also important to recognize that as long as the transcription factors are bound to the promotor region, then another RNA polymerase can come behind the first one & then make multiple copies of the same mRNA so that at the end of the day we're going to have multiple proteins at the same time being made

Overview

- RNA processing & transcription happens in the nucleus - once we have mature mRNA, it can leave nucleus into cytoplasm where translation happens - WATCH VIDEO

Split Genes & RNA Splicing cont'd

- RNA splicing job is to get rid of the introns & join together the exons - done by spliceosome; humongous molecule & is a combo of proteins & RNA molecules - the catalytic activity of the spliceosomes is going to come from the RNA molecules & not form the proteins, so we're going to call the spliceosome a ribozyme - ribozyme = a molecule that has catalytic activity but instead of being a protein, it's a ribosomal RNA - further evidence that RNA was the original material - WATCH YOUTUBE VIDEO

1) Alternative RNA splicing

- RNA splicing: cell can use the same gene/mRNA to produce different products; very effective. Efficient & reduces the space in the DNA for different products - example of a gene that undergoes alternative RNA splicing is: Troponin T gene - Troponin T gene is a component of muscle cells & depending on developmental stage, might need different versions of troponin T & we can achieve the different versions of troponin T via alternative RNA splicing - going to have version of Troponin T gene when in developing in utero & another version of Troponin T gene once you are born - way you achieve this is by cutting the introns in a different way - when we look at primary transcript, during process of RNA splicing, what we have is that we're going to cut this intron between exons 1 & 2, cut intron between exon 2 & 3, but then cut intron between 3 & 5 & remove exon 4 = how you get product #1 - in order to get product #2, you need to remove the introns in a different way by cutting in different places; cut this intron between exons 1 & 2, cut intron between exon 2 & 4, & cut introns between exon 4 & 5 = product #2 - only in alternative splicing are we removing introns & exons. So, how can we do that? Done by spliceosomes - how do we distinguish between where to start intron & end an intron? SNRPs - we're going to have different versions of SNRPs during development than the ones when we're born. Depending on which SNRPs are expressed, we can recognize different introns therefore cutting them in different places - in addition to alternative RNA splicing that allows us to translate our mRNA in different ways, we can actually stop translation by using mRNA degradation

Start Codon & Stop Codons

- Start Codon: AUG - Stop Codons: UAA, UAG, UGA

Accurate translation requires 2 steps cont'd

- part of the accuracy of translation depends on matching the appropriate tRNA with the appropriate amino acid - second process that needs to happen is we need to match the anti codon with the appropriate codon in the mRNA - there are 64 codons - there are 20 tRNAs

Translation is the RNA-directed synthesis of a polypeptide (protein)

- translation is going to convert the information in mRNA into amino acids - the final product of translation is the formation of a protein - so a series of amino acids join together via peptide bonds = the final product of translation

Different cells respond differently to the same signal

- This is a real example with adrenergic receptors. - there's many of them. Three examples: alpha 1, alpha 2, & beta - these receptors are going to bind to adrenaline & noradrenaline; so they are all capable of binding to the same signal - when alpha 1 binds to the signals, it's going to lead to the activation of phospholipase C which will lead to the cleavage of PIP2 which will produce IP2 & DAG which will in turn release calcium ions & lead to the contraction of smooth muscles - in alpha 2, it will inhibit the downstream pathways so we do not activate adenyl cyclase & do not release calcium chloride so therefore we activate the release of NT & stop muscle contraction - when same signal interacts with Beta adrenergic receptors, it will activate adenylyl-cyclase, lead to the formation of cAMP, which will lead to heart muscle contraction, smooth muscle relaxation, & glycogenolysis - recap: these are three different receptors interacting with the same signal (adrenalin/noradrenalin) & having completely different outcomes as they interact with the same signal

Comparison of Translation in different cell types

- Transcription & translation both happen in cytoplasm in prokaryotes. So, as soon as you have a mRNA made you can immediately translate it bc you don't need further processing. Transcription & translation are coupled in prokaryotes & happens much faster in prokaryotes than eukaryotes. - in eukaryotes, transcription happens in the nucleus & translation happens in cytoplasm so they are physically separated. - The prokaryotic mRNA is not going to require processing--no poly-a tail, 5'cap, or introns so it's going to be quickly translated. - in eukaryotes, in order to initiate translation, we need to identify 5'cap - in order to orient where start codon is in prokaryotes, bc we don't have 5' cap we're going need particular sequence in mRNA known as the Shine-Dalgarno sequence which will be before the start codon & that's how the small subunit can orient itself as to where it should bind. & remember that in eukaryotes, the 5'cap tells us where the start codon is. - Ribosomes in prokaryotes are smaller than the ones in eukaryotes - Process of translation is much more complex in eukaryotes than in prokaryotes

Control elements that regulate Transcription cont'd

- Transcription factors: proteins that are going to recognize the promoter region & going to recruit RNA polymerase for transcription to happen - in addition to transcription factors, some genes & some promoters are going to be requiring additional regulatory regions which include proximal control elements, enhancers, & activators - proximal control regions: nearby the DNA & therefore going to promote transcription - enhancer regions: regions in DNA that are far away from the promoter; can think of enhancers as activator regions of operons. So they can behave exactly the same way. Enhancers are the activators in eukaryotes - what binds to enhancers are activators - now discussion role of enhancers & activators & not proximal control elements. - let's talk about how enhancers & activators are going to be promoting transcription

Review of Transcription

- Transcription is when we make mRNA molecule - starts when transcription factors bind to promoter region. Notice that the DNA is not unwound at this point. - Transcription factors recognize the TATA box promoter region then transcription factors are going to recruit RNA polymerase which in this case is RNA polymerase II - RNA polymerase II can unwind DNA & stabilize the transcription factor & can also add the appropriate ribonucleotide that's complimentary to the DNA template strand. Also forms phosphodiester bonds. - so you keep adding nucleotides after nucleotides after nucleotides until you reach polyadenylation signal. RNA polymerase goes past 10 to 35 nucleotides after polyadenylation signal & stops transcription. - once we stop transcription, our mRNA is not ready to be translated. We need further processing known as mRNA processing. - that RNA processing is going to include addition of 5' cap, poly-A tail, & removal of introns & joining of exons in a process known as RNA splicing - RNA splicing is going to require a much large molecule known as the spliceosome which is a mixture of proteins & ribosomal RNA. Spliceosome is considered a ribozyme bc the catalytic activity of this molecule comes from the RNA molecules. - Spliceosome recognizes & removes the introns & joins together the exons - after that, we have what is known as a mature RNA & is now a molecule ready for the next process which is translation

Molecular Components of Catabolite Repression cont'd pt. 2

- WATCH VIDEO

Questions to consider when studying

- What if the promoter can't be recognized? What if termination signal can't be identified? What would happen if introns can't be removed?

Regulation at the Protein Level

- affect level of product/protein - so once you have a protein, you can manipulate amount of protein - we've already discussed some of the way swe can then regulate the activity of a protein: competitive inhibitors, noncompetitive inhibitors, allosteric regulators - but there's one more way we can regulate protein activity = through ubiquitination

Translation requires multiple components

- amino acids - ribosome; made up of a large & small subunit - tRNAs - messenger RNA

Proofreading activities of *DNA polymerases* cont'd

- as they're adding nucleotides, they're also going to check that they added the correct nucleotides which is the job of the proofreading activity (checking for correct formation of hydrogen bonds) - if we find out that wrong nucleotide was added, DNA polymerases will have activity known as *exonuclease activity* - exonuclease activity is the ability to cleave a phosphodiester bond from & removing nucleotides from it - nuclease activity can happen in different directions; can cut from hydroxyl group to phosphate aka 3' to 5' or you can cut from phosphate to hydroxyl group aka 5' to 3' - DNA polymerase III & I have 3' to 5' exonuclease activity . As either of them is adding a nucleotide & realize they've made a mistake so they're going to cut from 3' in previous nucleotide & cut 5' in newly coming nucleotide - other type of activity is cutting on other end known as 5' to 3' exonuclease activity. Only DNA polymerase I has this capability

A single signal transduction pathway can lead to the regulation of one or more cellular activities

- it is common that once a receptor is activated, we not only trigger a single response, we can trigger multiple responses which is shown in the image. - shown is a receptor in the center & when bound to its ligand, a growth factor, it can trigger 3 pathways - one pathway is going to lead to gene expression which will lead to motility of cell - a second pathway is going lead to cell proliferation - a third pathway will lead to protein synthesis which is going to prevent cell death (apoptosis) - *& it all happens with activation of a single receptor. So, this receptor is capable of doing multiple things.* - What kind of receptor is shown in this figure? tyrosine kinases; can dimerize & there's a bunch of K's (kinases) which can trigger phosphorylation cascade --> activation --| inhibition

Transcription: TERMINATION cont'd

- as we keep elongating, we eventually need to stop bc we only want to copy a portion of the DNA--not the entire DNA so we need to recognize the termination signal - in eukaryotes, the termination signal is the *polyadenylation signal*. - so, the RNA polymerase is going to keep adding nucleotides & identify stop signal, polyadenylation signal, is going to include polyadenylation signal in the mRNA & is going to continue 10-35 nucleotides after the termination signal before it completely comes off & then stops transcription - important to recognize that the polyadenylation signal will be included in the mRNA - at the end of transcription, notice that we still don't have a mature mRNA. we have what's knowna s a primer in transcript or the pre-mRNA - so this transcript is not ready for translation bc we still have a combo of exons & introns so we're not ready to make a protein out of this. - we're going to require further RNA processing before we're able to use mRNA for translation

Poly A tail cont'd

- at the end of mRNA on 3' end, we're going to add poly a tail which is the job of an enzyme called poly A polymerase - job of poly A polymerase is to add a series of adenosines forming the poly A tail - important to not confuse the poly A tail with the polyadenylation signal; polyadenylation signal was the termination signal for transcription. But poly A tail happens during mRNA processing

Matthew Meselson and Franklin Stahl's 1958 experiment cont'd pt. 1

- back in the 1950s, structure of DNA was solved by Watson & Creek. Then many questions came to be. One of them was: how do we make copies of this molecule? - we know that DNA organized as a double helix, anti-parallel fashion. What ways is it possible for this molecule to replicate? - 3 potential hypotheses: A. Semiconservative replication: at the beginning of the first replication, we have mixture of old & new DNA but after the second replication you started to have more new DNA & less of the hybrid. (H:H,N;N,H) B. Conservative replication: old DNA stayed old & you synthesize new DNA from scratch. So, you never had a combination of old & new in the same band but had very distinct groups of just old DNA & just new DNA (O,N:O,N;N,N) B. Dispersive replication: from the old strands you're going to have a mixture of new & old. So after the first DNA replication, you should get a hybrid of strands that contain both old & new DNA (H:H,H;H,H)

Gene Expression

- based on the information that's found in the DNA. - now discuss how the information in the DNA is organized

*Housekeeping genes* are always expressed cont'd

- bc of the function that they have, they will always be expressed in the cell - housekeeping genes aka constitutive genes bc they're always on - going to be performing important functions for the cell; that could be genes that are involved in ATP synthesis, translation, breakdown of molecules, genes that code for ribosomal components, genes that code for RNA polymerase, etc. All of these genes are always going to be on bc we constantly need them. - we can't afford to have them on & off. We always need to have them constantly on.

More on Calcium Ions, Inositol Triphosphate (IP3) and Diacylglycerol can act as second messengers

- calcium ions are at high concentration outside of cell, inside endoplasmic reticulum, & mitochondrial matrix (all extracellular fluid) - calcium ions at low concentration inside cytosol - calcium, IP3, & DAG are going to be associated with G-protein-coupled receptors. - when G-protein-coupled receptors is activated by presence of a signal, activates G-protein, which in turn activates effector enzyme (phospholipase C)

Negative Control: Induction cont'd

- catabolic processes are under negative control induction - catabolic processes = breakdown of molecules - if your genes are involved in breaking things down, they're probably going to be under negative control induction; we didn't make lactose, we broke lactose - looking at activity of enzyme B-galactosidase & checking whether or not it is under negative control induction or repression - when we added lactose, we had an increase in activity of the product suggesting that it's induction

The Cell Cycle is Regulated by Cell Signals

- cell cycle = dependent on signals from outside & inside of cell which is shown in the image - cell cycle is going to be responding to signals from the outside known as mitogens aka growth factors but can also respond to signals usually coming from the mitochondria - depending on the signal, the cell cycle will be allowed to continue & allowed to replicate OR if the signal is indicating that the conditions are not right, then the cell cycle will be stopped & the cell will not replicate - turns out that there's a particular signaling pathway that's going to be very important in regulating whether or not the cell cycle will e moving forward or not.

long-distance signaling communication

- cells have to be very far from each other - ex. hormones in body - hormones produced in pituitary gland & released through circulatory system & their receptors are going to be far away like your reproductive system - note that only the cells that have the receptors for the signal is going to be receiving the signals

DNA Repair

- changes happen, they become permanent, & we need to have a way to repair these changes - DNA repair is going to be an important process that needs to happen so that we can retain the cell to be functional

Ligand-gated ion channel

- channels are proteins that are going to carry ions down their concentration gradient - type of receptor: ligand-gated ion channel in that the signal is going to be received by the channel (so the signal is the ligand). Once the signal is received, then the channel opens allowing the ions to move down concentration gradient which leads to particular cellular response 1. Receptor changes shape when ligand binds 2. High specificity - specific ions, such as Na+, Ca2+, K+ (specific to charge, size, shape of ion) 3. Important in the nervous system - very common in nervous system

Gene Expression cont'd pt. 3

- converting the genetic information into a gene product - when we talk about the gene product being a messenger RNA, then we're talking about using 2 specific processes known as transcription & translation. - idea = when the gene product is going to be mRNA, we're then going to have that the info in the DNA is going to be transcribed, the product is going to be messenger RNA which can then be further processed through process of translation to make a protein. - we call this the central dogma - talking about the genes that have the info to be transcribed into mRNA molecule that can later be translated into a protein

Replication Fork cont'd

- created by separating the 2 strands of DNA which is done by the job of an enzyme, helicase - helicases are going to break the hydrogen bonds that are holding the nitrogenous bases & in doing so are going to separate the 2 DNA strands - the 2 strands are going to want to naturally come back together & form hydrogen bonds so we're going to need another protein to keep those strands separate from each other which is done by the job of single stranded binding proteins (SSBs) - the single stranded binding proteins are going to stabilize the 2 strands of DNA so that they don't come back together. Now, we have created the replication fork. - now, we're going to try to open more parts of the DNA but as we keep trying to do this we're going to find resistance ahead of the fork. this is bc the DNA is intertwined so as you try to open this you're going to find resistance ahead of the fork so you need to loosen the resistance so you can separate the strands of DNA which is the job of the topoisomerases - topoisomerases are going to work ahead of the fork & loosen the tension that is being created ahead of the fork so that the helicases can continue their job of opening the DNA. topoisomerases are going to nick the backbone of the DNA--so they are breaking phosphodiester bonds & in doing so are relieving some of the tension that's happening ahead of the fork & the helicases can continue opening the DNA as it's trying to allow for copy of the entire DNA. - once we have replication fork stabilized, we can now begin process of making a copy of the DNA

Protein kinases & cyclins regulate most of the events in the cell cycle cont'd

- cyclins do not act on their own; always in association with another protein known as CDKs (protein kinases)

Replicating the Ends of Eukaryotic Chromosomes cont'd

- dark blue = parental strand/template strand - shown lagging strand. - Red = RNA primer - light blue = newly synthesized DNA - at the end of replication is that DNA polymerase is going to come & is going to remove both primer & ligase is going to join both newly synthesized DNAs together - cannot add anymore nucleotides here bc it's going to have exposed phosphate as opposed to exposed hydroxyl group - if we don't fix this, after each replication the newly synthesized DNA is going to get shorter and shorter and shorter. This is a problem bc we're going be losing essential DNA/information!!! NOT desirable! - we need a mechanism to fix this & that is the job of telomeres

by the time we finish RNA processing

- end up with 5' cap, 5' UTR, info where i'm going to start & stop making protein, only have exons & no introns, have 3'UTR which will have polyadenylation signal, then finally poly A tail = finally ready to leave nucleus & undergo translation we no longer have introns in coding region & only have exons

Mutations are random cont'd

- environmental & chemical factors are not guiding mutations--not "ooh this would be a good spot to introduce a mutation bc this new thing is going to happen" - RANDOM places will be affected - mutations are not going to happen bc a cell was placed in a situation where mutation will be useful; if i'm placed in radioactive environment, a mutation that takes place isn't going to say "ooh let me change you so all of your cells will be radioactive resistant" NOT HOW IT WORKS!!! - important that unless the chemical is mutagenic, the exposure to the chemical or environmental factor is not what causes the resistance. It is a selective pressure that's choosing for that resistance. - the resistant mutagens was already there before they were selected

Chromatin-modifying Enzymes cont'd

- enzymes that depending on their function are going to promote histone acetyltransferases (HATs) or histone deacetylases (HDACs) 1. histone acetyltransferases (HATs) are going to transfer acetyl groups to the histones & are going to promote euchromatin formation 2. histone deacetylases (HDACs): remove acetyl groups from histones & promote heterochromatin formation - image shows cross section of interaction between histones & DNA. - How many histones does DNA wrap around? 8 - histones = purple spheres & have extensions known as histone tails - histone tails have specific amino acids usually lysines to which you can add acetyl groups. If tails have acetyl groups attached to it, going to loosen up the DNA but if you don't have acetyl groups attached to it, you're going to tighten the DNA

Gene Expression in Eukaryotes

- even though we have taken some time to understand the different processes involved in gene expression--so we talked about transcription, RNA processing, & translation in eukaryotes, we're going to be talking today about how exactly & what mechanisms are available for the cell in order to regulate these processes. bc we know that the genes are going to expressed under certain conditions/times as opposed to being constantly expressed. - so that is known as regulation of gene expression

Telomeres cont'd

- extensions that exist in each chromosome. - we're going to find that this portion of the chromosome is where the essential information is & from that we have extensions known as telomeres that are basically just a series of repeats & the specific repeats varies from species to species. In humans, the specific repeats 5'TTAGC3' exists in humans - these repeats do not encode for anything functional. & just repeat continuously & extend away from chromosome/essential information. The idea of these telomeres is to postpone the erosions of the essential genes - not only are telomeres important. we need to have something else

5' methylated CAP cont'd

- first step in mRNA processing is the addition of 5' methylated CAP which is going to require 2 enzymes: capping enzyme & methyl transferase - capping enzyme adds guanine nucleotide to the 5' end of the mRNA - methyl transferase adds series of methyls into the guanine; bound on top of the phosphates & know we've methylated the 5' cap

Termination of the Signal cont'd

- just as important as it is to trigger a signal response & trigger a cellular pathway, it is also very important to be able to stop it when needed (terminate signal) - the cell has multiple ways of stopping the cell signaling pathway: - reducing ligand concentration - trigger phosphorylation cascade then we're going to have to remove those phosphates to stop the phosphorylation cascade - if second messenger was produced, we have to be able to remove the second messenger - if transcription was triggered, we're going to have to be able to stop/block transcription

Matthew Meselson & Franklin Stahl's 1958 experiment supported Watson & Crick's hypothesis that DNA replication was semiconservative

- took sample at time zero & waited 20 min to get first replication then 40 min is second replication - sample zero showed old DNA which was expected - after first replication saw hybrid (dismisses conservative hypothesis) - after second generation, light band started to emerge which meant that DNA was replicating in semiconservative fashion (not dispersive)

Replication begins at special sites called *origins of replication* cont'd

- first thing that needs to happen is we need to find a place to start DNA replication & this is not a random place. It is a very specific place known as origins of replication - origins of replication = specific regions of DNA known as ori regions - these ori regions are high in AT content bc it makes it easier to break 2 hydrogen bonds than 3 hydrogen bonds--makes it easier to separate DNA - in a human chromosome, you can have as many as 100,000 origins of replication spots & this is so that we can speed up the process of DNA replication - once origins of replication are identified, it is in these regions that we're going to begin separate the 2 strands of DNA & going to form what is known as a replication bubble - replication bubble is where we're going to separate the 2 strands of DNA so that we can use 1 strand for DNA synthesis & the other strand for DNA synthesis - in a replication bubble, there's 1 strand of DNA shown in gray & the other strand of DNA shown in gray. The colored bands show are the new strands of DNA - notice that when you form the replication bubble, you form 2 replication forks - one fork moves in 1 direction & the other fork moves in the opposite direction. The events flip in 1 fork. In one fork, the leading strand is on the top & ligand strand is on bottom & the opposite is true on the other fork.

gene expression in prokaryotes & eukaryotes

- gene expression is going to happen in different parts of the cell depending on the cell type. - In prokaryotes, all of gene expression (transcription & translation) is going to happen in cytoplasm. - In eukaryotes, transcription is going to happen inside of the nucleus & translation is going to happen outside of nucleus in cytoplasm.

The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals

- genetic code is nearly universal with some exceptions; 99.999 is universal meaning that the genetic code present in bacteria is exact to the same genetic code present in complex organisms - this allows for ability to exchange genes - you can take green fluorescent protein & put that into bacteria & now bacteria can fluorese...can take gene from completely unrelated organism & put it into a different organism & that gene can be expressed & functional in that organism - uses: dog breeds (big, small, color)

Nucleotide Excision Repair cont'd

- happens before DNA replication takes place - looking for differences in the topography/structure of DNA - particularly good at detecting damages/mutations due to radiation - usually when we are exposed to UV radiation or other types of radiation, what happens is that instead of thymines interacting with adenines, they're going to interact with each other in same strand of DNA which forms *thymine dimers* - thymine dimers are produced when we expose ourself to UV radiation. - thymine dimers produce a bump in DNA & these abnormal structure will be detected by nucleotide excision repair. - once bump is detected, we're going to bring a particular set of enzymes, *endonucleases* - endonucleases are a category of enzymes that can cut the DNA in the middle of the DNA (endo = middle/inside) - will cut before & after structural damage & then we're going to recruit polymerase I to fix & put correct sequence - then we need DNA ligase I to go ahead & ligate the new piece we are now newly synthesizing

How can gene expression be regulated?

- how & what mechanisms are going to be available for a cell to regulate gene expression? We're going to find taht we can regulate gene expression at multiple levels of this entire process. - we can regulate transcription by having changes directly in the DNA preventing the process of transcription - we can also have changes in the mRNA. So, preventing translation. - we can even have modifications to the product. So, we can no longer have the activity of that product. - so we can really have control over every step of the gene expression process. - so let's discuss how this process can work

Specificity: Different kinds of cells have different collections of proteins

- how is the specificity of a cell achieved? The specificity has nothing to do with the signal. Specificity of a response is going to be dependent on what receptor is receiving the signal and the proteins downstream of that receptor. - *specificity & type of response is dependent on the receptor & downstream proteins & is not dependent on the signal* - Cell A: has the same receptor. It's the same, it's just we're in different cells - Cell C: We have receptor 1 and receptor 2 - Cell D: completely different receptor

Histone acetylation

- if histone deacetylases are acting on the histones, they're going to remove acetyl groups & promote heterochromatin conformation - if histone acetyl transferases are acting on histones, they're adding acetyl groups to histone tails & therefore relaxing DNA around histones promoting euchromatin conformation - where we have euchromatin conformation, we have DNA accessible so gene expression can happen - if you have acetyl groups, that region of DNA will be expressed, if you don't have acetyl groups, that region of the DNA will not be expressed

Proteins in cytoplasm

- if the protein is going to have its final destination in the cytoplasm, this whole process of translation occurs in the cytoplasm & as soon as the protein is made it's going to then be residing in the cytoplasm - so, if i'm a protein residing in the cytoplasm after this process ends, I'm going to be residing in the cytoplasm - but, not all proteins are going to be residing in the cytoplasm - other proteins belong in the ER, golgi, or plasma membrane - if a protein belongs somewhere besides the cytoplasm, then a different event has to happen

Most genes encode proteins, but some code for RNAs

- important to recognize that there's a lot of information in every gene & that information is different depending on the gene - at the end of the day, when we talk about gene products that the info is available in the DNA, we're talking about RNA molecules - so, in the image we have 3 different types of genes: Gene I, 2, & 3 - each of these genes can produce a different RNA molecule - the product of a gene is an RNA molecule - we are going to be talking today about mRNA which can then be used for translation to make a protein - but other genes like gene 2 & gene 3, the product is going to just be an RNA molecule & that RNA molecule will never be translated into a protein (includes genes that code for regulatory RNAs, ribosomal RNA, & transfer RNA)

Regulation at the Transcriptional Level

- in addition to being able to manipulate the structure of DNA to whether or not we're going to allow a particular region of the DNA to be accessible for recognition & therefore initiation of transcription or not, we can also regulate at the transcriptional level - so even when the DNA is accessible for recognition for transcription, we can also regulate what's going to happen to that exposed DNA & whether it'll be allowed to transcribe or not - in order for that, we're going to have to introduce you to regions of DNA that have different roles in allowing for transcription to happen or not

DNA Methylation cont'd

- in addition to changing whether the DNA can be found in the heterochromatin or euchromatin, you can also manipulate gene expression by the methylation patterns present in the DNA. - it is the job of the DNA methyltransferases (DNMTs) to add those methyl groups to the DNA. Usually these enzymes are going to add methyl groups to either adenine or cytosine. For most part, when methyl groups are present to these nucleotides, they're going to suppress gene expression - also remember that your DNA methylation patterns are inherited & your DNA methylation patterns can be affected by the environment (stress, nutrition, trauma) which can be transmitted to offspring. So, any changes in methylation patterns that your parents had, you inherited those methylation changes - way this works is, you have your particular nitrogenous base (cytosine) & have DNA methyltransferase enzyme that's going to take methyl group from molecule (SAM = methyl donor) & adds it to cytosine = methylated cytosine - can have different areas of DNA sequence being methylated - if one of those sites is a promoter region, then usually a methylated promoter will not be expressed in the gene. Methylated promoter will not be recognized by transcription factors & therefore we will not have transcription of those methylated promoters

Ribosomes cont'd

- in addition to needing tRNAs, we're also going to need ribosomes - the ribosomes are very large molecules & are a mixture of proteins & ribosomal RNA - ribosome is made up of large & small subunit - small subunit recognizes mRNA - when large & small subunit come together, they're going to form very specific regions in that molecule which are known as *binding sites* - P binding site = where growing protein will be (p for protein) - A site = where anticodon allows the next amino acid to be added (a for anticodon/amino acid) - E site = where we have end tRNA leaving (e for exit) - ribosome is considered a ribozyme bc the catalytic activity is going to come from the ribosomal subunit. It is the ribosomal RNA that's going to form the peptide bond. It is the ribosomal RNA that's going to join one amino acid with another amino acid. So therefore ribosome is another example of a ribozyme.

Bacterial genes are organized in *operons*

- in addition to the regions just mentioned in previous slide, we're also going to find that prokaryotes are going to organize their genetic info differently than eukaryotes - so, prokaryotes organize their genes in *operons* - in an operon, there's multiple genes working together (ex. G1, G2, G3) which are going to be under the action of a single promotor. When transscription is initiatied, the promotor is going to include the information for three different genes. When mRNA is made, it's going to have info from multiple proteins known as polycistronic mRNA - polycistronic mRNA: has info for more than 1 protein - there's other proteins upstream the promotor that are going to regulate the process--regulatory genes

Matthew Meselson and Franklin Stahl's 1958 experiment cont'd pt. 2

- in order to distinguish between these three models, Meselson & Stahl designed experiment & decided to label nitrogen bc you had the nitrogenous bases - they went ahead & started the cells in a medium that was made with heavy nitrogen. So, the DNA will have heavy nitrogen - heavy nitrogen represents old DNA (N15) - new DNA will be made with the lighter nitrogen, nitrogen 14 (N14) - bc we have different weights when you spin these molecules around then they're going to settle in different places. so the old DNA is going to settle in the bottom & new DNA going to settle on top & hybrid DNA will settle in the middle. with that, you can distinguish between the three models. - semiconservative = see old, hybrid, then hybrid & new - conservative = see old, old & new, then old & new throughout - dispersive = see old, hybrid, hybrid throughout

Gene Expression happens in different places depending on the cell type

- in prokaryotes, both transcription & translation happens in cytoplasm - in eukaryotes, transcription happens in the nucleus & translation happens in cytoplasm - in addition to differences in location, we are also going to find that the eukaryotic mRNA is not going to be ready after transcription to be transferred from the nucleus into the cytoplasm & the mRNA of eukaryotes requires an additional step known as RNA processing before it can then be transferred from the nucleus into the cytoplasm

Schematic of a Prokaryotic Operon cont'd

- in prokaryotes, we traditionally have the genes organize in what's known as an operon. An operon is going to be a situation where we have one promoter regulating the expression of multiple genes. The multiple genes are known as structural genes. - In the image, there's 1 promoter shown in green & 3 structural genes in dark, medium, & light blue. - Depending on the operon, the structural genes can be coding for enzymes, components that are going to lead to the structure of the cell, etc. There are just multiple genes that usually are working in activities that are related to each other. So, gene 1 is related to the activities of the activities of gene 2 which are then related to the activities of gene 3. So they tend to be genes related in similar functions. - there's going to be additional regulatory components that are present in an operon. So, let's discuss what are these components present in an operon. - the first one is the operator. The operator is the region in the DNA that's always present between the promoter & the structural genes (shown in yellow). - if nothing is binding to the operator (or is free), we are going to have usually transcription of the structural genes. - there are specific proteins called repressors which are the ones that are able to bind to the operator region. When repressors bind to the operator region, then we stop transcription. - In contrast to operators, we have activators. As name suggests, activators are going to promote transcription. So, when an activator binds to the activator site, we're going to promote transcription. So the things that bind to the activator site are going to be activators. Notice that the activators are far away from the promoter & operator. The two bands represent that they are far away from the promoter region or where the gene regulating is present. So, when something binds to the activator, it is called activators. When the activators are bound to the activator site, then we are going to be promoting transcription. - so the gene that's going to code for the activators or the repressors are known as the regulatory gene. The regulatory gene is even far away from this whole process & whose product is going to be an activator that's going to bind to the activator site or the product is a repressor & is going to bind to the operator region. In some cases we have 2 regulatory genes, one that codes for activator & one that codes for repressor

Regulating Gene Product Activity

- in the event that the product is already made, you can then regulate the activity of the product & therefore prevent the product from converting a substrate to a product & therefore prevent that activity.

Combinatorial Control of Gene Activation cont'd

- in this image, have 5 genes which are all present in the same cell but each of the genes have a different combination of enhancers shown by different colors. So, different genes have different enhancer regions. - Can have situation where in this particular cell, only a certain type of activators will be present in the cell. In this example the particular activators that are present in this cell is blue, black, green, & orange. When these activators are present, which genes will be expressed? 1, 2, 4 - Genes 3 & 5 will not be expressed bc they need different activators - in this example, you can activate 3 genes at once & not allow 2 genes to be expressed = combinatorial regulation

Genetic instructions are written in a series of *non-overlapping, three-nucleotide code* cont'd

- information in DNA is organized in 3 nucleotides at a time known as codons (3 nucleotides together is known as a codon) - you read three nucleotides & move on to read three more nucleotides; non overlapping - not only is DNA organized in 3 nucleotides at a time, but each codon is going to code for the same amino acid so that's called non ambiguous; if we're talking about codon coding for UCA, UCA is always going to code for serine - genetic code can also be degenerate meaning you can have more than one codon coding for the same amino acid; UCU, UCC, UCA, & UCG can code for serine

3) Noncoding RNAs play multiple roles in controlling gene expression cont'd

- lastly, we have a very specific way by which we can control translation through use of *noncoding RNAs* - noncoding RNAs are usually very small RNA molecules that are going to be able to interfere with translation process - remember that not all genes will code for proteins but can also code for rRNAs or tRNA. Majority of genes in our body are going to transcribe for what's called *noncoding RNAs*; RNAs that are not going to be rRNAs, tRNAs, or mRNAs but known as noncoding RNAs - noncoding RNAs regulate a series of processes in your cells including the process of transcription & translation - noncoding RNAs can affect the chromatin structure & will promote heterochromatin conformation & can also affect the translation of mRNAs - now going to be talking about how noncoding RNAs affect mRNA translation

Mature mRNA is transported to cytoplasm for Translation pt. 1

- mature mRNA has a 5' cap, poly-a tail, 5' UTR, 3' UTR, start codon, stop codon - the region between the start codon & the stop codon is where it's going to be used for translation

Amplification in Signal Transduction

- means that you're only using a single signal but then you magnify the response to that one signal - using phosphorylation cascade is an effective way of amplifying that signal - so we get one signal, but that one signal is going to activate a 1,000 kinases which in turn activates 100,000 kinases which in turn activates 10,000,000 kinases which activates billions of proteins leading to a huge response...& you only need one signal to do this - ex. immune responses to viruses

Eukaryotic genes are usually *monocistronic* & contain coding & non-coding regions

- monocistronic: one gene for one promotor - there are some operons present in eukaryotes but majority of genetic info is organized monocistronic - have promoter region (green), single gene, G1 (blue area) but this gene is going to have *discontinuous* info--have regions that's going to have info to become a protein (exons/coding) & have regions that doesn't have info to become a protein (introns/non-coding) - at the end of transcription in eukaryotes, we find that the mRNA that's produced after transcription is not ready to be translated into a protein bc we have combo of exons & introns so we're going to have to further process the messenger RNA so that we can remove the introns & keep the exons together & form a continuous series of exons known as RNA processing. - & it's not until then & a couple other processes that we will see then that we'll see a mature RNA that's going to be then be translated

Cell Cycle

- most of cells are functional in G1 phase--only have one copy of DNA, DNA is completely unwound & not condensed & available for processes to take place - S phase = all the DNA replication process & making sure DNA is free of errors bc at the end of the cycle we're going to end up with two cells & want to make sure both cells have correct information - so a lot of effort is taken by the cell to avoid mutations to accumulate - now going to be discussing the events that happens in G1 phase...gene expression

Mutations are responsible for introducing diversity in the genetic material cont'd

- mutations are going to happen but the effect of that mutation is dependent on two factors: 1) Where mutation takes place; 2) What type of mutation it is 1. in DNA, the mutation can happen on coding & non-coding regions; regions in the DNA specifically in eukaryotic DNA - coding regions known as exons; has information that's used to code for a protein - non-coding region known as introns; regions of the DNA that's not going to have code for a protein - so depending on whether the mutation occurs in coding or non-coding regions we will have different outcomes - whether or not the mutation occurs in a somatic vs. gamete cell - somatic = body cells, gamete = sex cells; whether the mutation occurs in a somatic or gamete cell it will have different consequences 2. type of mutation plays a role

Local Signaling Communication

- nearby but not touching each other 1. Paracrine regulation - cell #1 releasing signal which is received by cell #2 - cell #1 & cell #2 do not have to be the same cell type - *cell #2 able to receive signal bc it has appropriate receptor for that signal* 2. Autocrine regulation

Antiparallel DNA affects replication cont'd

- not only is the formation of the phosphodiester bonds important in DNA replication but also the fact that the 2 strands of DNA are oriented in an antiparallel fashion meaning that one runs from 3' to 5' & the other one runs in the opposite direction. - so this is going to have an effect on how the DNA is replicated & will have an effect on where the primers are placed - so, we're going to see that when we talk about the leading strand, we're going to require only one primer bc once we add a nucleotide, a hydroxyl is available so then we can add another nucleotide & we'll have another hyrdoxyl available... - but, if we were to put the primer in the same place as we did in the other strand, what happens is the 3' is here which wouldn't be useful for me so you don't put the primer there but put primer close to the fork. So, in the leading strand, the primer is away from the fork. In the lagging strand, the primer is close to the fork. - What happens then is we're going to have multiple primers in the lagging strand & therefore going to have multiple pieces of DNA synthesized in a fragmented fashion & those fragments are known as: okazaki fragments - we're going to see that on one side of the fork, we have a continuous synthesis of DNA & on the other side of the fork we have a discontinuous synthesis of DNA & those fragments are known as okazaki fragments after the scientist that discovered them.

DNA Synthesis cont'd

- once we have replication fork stabilized, we can now begin process of making a copy of the DNA known as DNA synthesis - now we're going to require multiple proteins to do this - first protein is primase. It's job is to introduce an RNA primer to create a complementary primer of the template strand. So, the primase can read the template strand & create a complimentary RNA molecule from that & produce an RNA primer. So, the RNA primer is going to be a piece of RNA & can be anywhere from 5-10 nucleotides long & the importance of RNA primer is that its going to provide us a free hydroxyl group that we need in order to make phosphodiester bonds & to continue adding nucleotides as we start the process of DNA synthesis. - the enzyme that's going to add the new nucleotides is going to be the DNA polymerase III. It's going to use the 3' that's available in the primer to add the first nucleotide. DNA polymerase is going to add the first base in the complimentary nucleotide so if we have an A here, it's going to add a T there & will allow those hydrogen bonds to make & then join the nucelotides by making the phosphodiester bonds. - remember that when a DNA polymerase grabs a nucleotide, it can only hold it in a position where the phosphate is facing out so this is why we can only synthesize DNA from the 5' to the 3'. Hence, why we need the RNA primer so that we can have a free hydroxyl group so that we can make the first phosphodiester bond.

Catabolite Repression cont'd

- positive = activator we made - negative = repressor we made - going to have that. under a type of gene expression known as catabolite repression, we're going to have a combo of both positive & negative control - turns out that if you're a bacteria, most of your life revolves around eating, finding nutrients, or replicating - it's going to be incredibly important for bacteria to recognize & divert its energy to the best source of carbon & energy - for bacteria, their best source of carbon is glucose. Even though there's other sources of carbon such as maltose, lactose, fructose, when glucose is present, bacteria are going to prefer glucose over any other available sugar. - under catabolite repression, is going to be able to distinguish between "do i have glucose or do i have something else. If i have glucose, I'm going to stop everything & just be interested in glucose but if i don't have glucose, I'm going to have to settle for the sugar that i have so that I can make energy from that."

Receptor tyrosine kinases (RTKs)

- present in plasma membrane of cells - require 2 signals to bind to them & when that happens, they dimerize (come together) & become active - as kinases, they're going to phosphorylate themselves in tyrosine residues (amino acids) & hydrolyze ATP - once phosphorylated, it's going to recruit different proteins which will lead to different cellular responses - RTKs can trigger multiple cellular responses, one for each phosphate group attached to it; one receptor can illicit 6 different responses. --------------------------- 1. Membrane receptors that attach phosphates to *tyrosines* 2. Can trigger multiple signal transduction pathways at once *3. Abnormal functioning of RTKs is associated with many types of cancers*

Gene Expression cont'd

- process in which you convert the DNA info into a gene product - there's multiple gene products including proteins, rRNA, tRNA (note that not all gene products are proteins) - two processes: transcription & translation which we call the *central dogma* of biology; idea that there's information in a particular region of DNA that we know as a gene & that information is going to be transcribed into a messenger RNA which in turn is going to be translated into a make a protein

Reception

- receiving the signal - has 4 ways it can receive the signal (any cell) - *the ability fo a cell to respond to a signal depends on whether or not it has a receptor specific to that signal.* - if you don't have receptor, it doesn't matter if you're flooded w/ signals, you will never be affected by the presence of that signal. - doesn't matter if there's a presence of a signal. cellular response depends on presence of receptor

All Organisms Follow the Same General Steps during Transcription cont'd

- regardless of how you organize genetic info, (if you organize with operons or monocistronic,) the process of transcription is the same - divided into three stages: Initiation, Elongation, Termination - Initiation: determines where we start process of transcription - Elongation: where we put all the ribonucleotides together - Termination: we get signal that says, "this is the end of transcript. stop transcription"

The three stages of signaling

- regardless of whether the signaling is via direct contact, local or long-distance, receiving cell is going to process that info in a very specific way that's always going to require 3 steps: 1. Reception 2. Transduction 3. Response

The Unit of Transcription cont'd

- regions in the DNA that's going to have specific molecular components that's going to allow that particular region of the DNA to transcribe into a messenger RNA & further translate it into a protein - promotor region: yellow; always found upstream (before) of RNA-coding region. Used for initiation of transcription - RNA-coding region: information to become a protein - terminator region: used as the site where transcription is going to be stopped

Nuclear Responses cont'd

- response that happens in the nucleus - when signal is received by receptor, a series of transduction event takes place that eventually lead to the activation/phosphorylation of a molecule known as a transcription factor - transcription factor is a molecule that when activated is going to activate/deactivate gene expression

Connecting Okazaki fragments and removing RNA primers cont'd

- so as we progress, we're going to find that one strand is continuous & the other is fragmented so we're going to need more proteins to help finalize the process which is going to be: DNA polymerase I & DNA ligase - DNA polymerase I is going to remove all the primers that we have added including that one in the leading strand & in the lagging strand it will have to remove multiples. Once it removes those primers it can fill in the place with correct nucleotides but then we have to join the fragments which is the job of the DNA ligase - DNA ligase is going to join the okazaki fragments

Molecular Components of Catabolite Repression cont'd

- so what we're going to need to distinguish between whether I have glucose or whether I have another type of sugar - when it comes to the lac operon, we're still going to have all the regulatory components that we just discussed; we're going to have the promoter, operator, & structural genes - in addition to that, we're going to have an activator site & going to need an activator protein. The activator protein is known as CAP. - also going to need cyclic AMP (second messenger); in this case cyclic AMP is going to be found at low levels when glucose is present & is going to be found at high levels when glucose is low or not present - in image we have lac operons (structural genes), operator region, promoter, activator site, lac I is representing regulatory gene that codes for repressor - lac operon is going to be under negative control induction meaning that when repressor protein is made, it's made as an active form so in absence of lactose we have that this particular structural genes is being prevented from being transcribed. - 1) under condition of absence of glucose & presence of lactose: we have high levels of cyclic AMP which is going to bind to activator protein known as CAP which binds to activator site. bc we have lactose in environment, we have that the repressor will be inactivated bc that's the job of lactose - recall that of that is going to become allolactose & bind to the repressor inactivating the repressor & therefore preventing repressor from binding to operator region & therefore RNA polymerase can find promoter with the help of transcription factors & will allow for transcription to happen & allow for expression of genes that are going to then breakdown lactose. 2. Presence of glucose & presence of lactose: have low levels of cyclic AMP therefore CAP protein cannot bind to activator therefore RNA polymerase will not be able to bind properly to promoter. So, even though we have an inducer present (lactose), bc the RNA polymerase cannot find the promoter very well in the absence of the activator, we are not going to have proper transcription of the lac genes - goes back to question, what if both are present? So, even though activator is present, bc the level of cyclin AMP are low, the activator cannot bind to the activator site, RNA polymerase cannot actively find the promoter, even though nothing is blocking the operator we still cannot have transcription.

More on cyclic amp

- the other way we can do transduction is by using second messengers - example of second messenger is cyclic amp - cyclic amp is going to be usually associated with activation of a G-protein-coupled receptor - cyclic amp is going to require that the effector protein is adenylyl cyclase - we're going to have G-protein-coupled receptor that receives the signal that in turns activates G-protein - activated G-protein binds to effector enzyme which is adenylyl cyclase - activated adenylyl cyclase convert ATP into cyclic AMP - activated cyclic AMP is going to behave as a second messenger which can activate multiple pathways or phosphorylate multiple cascades that can then lead to cellular response - the way to turn off a pathway activated by cyclic AMP is by the activity of enzyme phosphodiesterase - phosphodiesterase is going to act on cAMP which will convert that into AMP & now whole system is turned off and there's no cellular response

Accuracy in translation requires that the appropriate amino acid is present on a tRNA

- so, how do we add the appropriate amino acid to the tRNA? That is the job of an enzyme, *aminoacyl-tRNA synthetase* - the process of adding the appropriate amino acid to the appropriate tRNA is known as *aminoacylation* also known as "charging" the tRNA - so when we say that a tRNA is charged, it means that it has its amino acid attached to it & the enzyme the attaches the appropriate amino acid to the tRNA is the aminoacyl-tRNA synthetase enzyme - this enzyme has 3 binding sites: one binding site recognizes the anticodon in the tRNA, second binding site recognizes the amino acid, last binding site will bind to ATP - the first step to charge a tRNA is the binding of amino acid & ATP to the appropriate aminoacyl-tRNA synthetase enzyme - ATP hydrolysis will happen so we can join the amino acid with AMP forming the AA-AMP complex (amino acid-AMP complex) - once AA-AMP complex is formed, we bring appropriate tRNA & we know we have appropriate tRNA bc we have to match the anticodon in the tRNA with the anticodon region on the enzyme. When those two match, we use last bit of energy present in AMP to then join the amino acid with the tRNA & we end up with a charged tRNA - tRNA that has an amino acid attached to it = end of charging process - there are 20 amino acids, we're going to need 20 aminoacyl-tRNA synthetases, we're going to need 20 tRNAS

Cross-talk occurs between pathways cont'd

- sometimes, pathways can interact with each other. Known as "cross-talk" - one pathway is triggered by a particular signal, & a completely different pathway is triggered by a different signal but they both converge at some point in the cell pathway - depending on the interaction, they can enhance the activity of each other OR they can counteract the activity of each other (inhibit function) - figure is an example of different pathways that all converge in the activation of protein known as CREB - so there's multiple ways a cell can activate CREB; when calcium enters the cell or have different molecules in the environment - Wnt pathway: Wnt receptors receive Wnt & get activated & multiple pathways are activated which all converge in the activation of CREB - there are second pathways that are responding to completely different signals & when they get activated, they can either enhance or inhibit the activation of CREB. --> = activation --| = inhibition - this is crosstalk when one pathway is affecting the actions of another pathway that was triggered in a completely different way. - this crosstalk is very common in a cell. multiple ways to inhibit/enhance CREB

Telomerase cont'd

- specific enzyme whose job is to lengthen the telomeres. - an enzyme known as the reverse transcriptase; an enzyme that is going to use an RNA molecule as a template to make DNA - it is also important tot recognize that in humans, telomerase is active only when we are in utero; only when fetus is developing is the only time when telomerase is active. As soon as we're born, the telomerase is no longer active.

tRNAs have a specific *anticodon* & bring amino acids to ribosomes cont'd

- tRNAs are RNA molecules that have a specific structures & specific regions - those regions include the anticodon region - regardless of which tRNA figure presented (next slides), there's always an anticodon at the bottom of the tRNA & at the top of the tRNA there's the amino acid binding region at the 3' end - *so, every tRNA has an anticodon region & an amino acid binding region* - so, how do we add the appropriate amino acid to the tRNA?

Regulation of Gene Expression

- talking about the different molecular mechanisms that will be available for the cell in order to either turn on or off a gene when necessary - now, we'll talk about the different types of genes that are present within a cell

Telomerase function cont'd

- telomerase = purple circle - within telomerase is going to have a piece of RNA & this piece of RNA is complementary to telomeric region. So, this telomerase can use this piece of RNA to find the telomeric regions by complementation & can use the same piece of RNA to extend/make longer the telomeric regions. - telomerase is acting on the parental DNA & is extending the parental DNA - at the end of the work of the telomerase, we can still add our primer as usual, we can then elongate - even as the primaries are removed, we're still going to have a shortening of the newly synthesized DNA but at least now we're losing the telomeric information & not essential information. - yes, we are shortening but we are shortening the telomeres not essential information - reason why telomerase is active during our development in the utero is bc during that time most of our cells are going to be heavily dividing so we need to make sure that those cells have enough lifetime in order to create a functional organism; significant increase in DNA replication - once born, activity of telomerase is gone, lost & now every time we replicate, we shorten telomeres & shorten the telomeres...& at some point we're going to lose all the telomeric regions & infringe in the essential information...process of aging

*Facultative genes* are only expressed when needed

- telomerases - when we eat - changes in humidity/temperature - injured - replication of the cell

MicroRNAs (miRNAs) cont'd

- the noncoding RNAs we're discussing are called MicroRNAs (miRNAs) - MicroRNAs (miRNAs) initially start as small double-stranded RNA molecules that are going to require further processing before they are able to interfere with very specific mRNAs - out of all the mRNAs that are available & present at one time in a cell, this microRNA can identify a particular mRNA & do this bc they have the complimentary sequence to that mRNA - so, this is a highly specific process. Can say, "I want that and that mRNA to be stopped & leave the rest" - as a whole, every time we use RNA molecules to interfere with gene expression we call that *RNA interference (RNAi)* - RNAi is the process by which you interfere with gene expression using RNA molecules Discuss how microRNAs work: - there's going to be a gene in your DNA that codes for this microRNA & this gene is going to be expressed & produce a product. - Product is known as *primary miRNA transcript* which is a very complex molecule that has multiple functional regions - These functional regions are hairpin regions - There's multiple versions of Drosha; Drosha is going to recognize specific functional regions in this pre-miRNA transcript & everything else is going to be destroyed by Drosha; so Drosha is going to destroy everything except the one functional region of the miRNA - all of this is happening in the nucleus. So, the role of Drosha is to narrow down the functional region of the RNA & to leave it as a double-stranded miRNA - double-stranded miRNA is going to leave the nucleus & will be found in the cytoplasm. Once found in cytoplasm, going to be recognized by protein called Dicer. - Dicer is going to further process miRNA & make it single-stranded & will be roughly 20-25 base pairs long - once we have mature miRNA (single-strand), now going to interact with large complex known as RISC complex (RNA-induced silencing complex) - combination of RISC with mature miRNA together are going to be surveying all the mRNAs in the cell until they find the one that has complimentarity to the miRNA - when the one that has complimentarity is found, they're going to join together via hydrogen bonds & going to form a double-stranded RNA molecule - this double-stranded RNA molecule is then going to be recognized by component in RISC known as argonaute which will degrade the double-stranded RNA preventing translation from happening

Wobble hypothesis explains the degenerate nature of DNA code

- the way it works is if we were to focus on leucine (next slide). Notice that when we code for leucine, we have CUU, CUC, CUA, or CUG so it's only the last nucleotide that changes so then we can use the same tRNA for this particular leucine - If codon is CUC, you can use this tRNA. If the codon is CUU, you can use the same tRNA. - *in most cases, if we change the last nucleotide in a codon, it usually leads to a silent mutation.*

Termination of Translation

- translation stops when we encounter a stop codon - as we elongate it & keep adding, we adding, & adding, we finally reach stop codon. When we reach stop codon, we will not add a tRNA bc there's no tRNA for a stop codon. Instead, we're going to add a release factor - release factors are proteins that recognize stop codons & 3 types of stop codons: UAA, UAG, UGA - once we find a stop codon, a release factor binds to that position & separates the polypeptide from the tRNA by adding a water molecule. Now, we have a free protein & free tRNA. This process will require some energy - Lastly, complete disassembly of entire complex

Cyclins bind & activate cyclin dependent kinases (CDKs). CDKs phosphorylate target proteins

- understand how cyclins & CDKs are going to work together to have an effect on the cell cycle - the cyclins are going to appear once we activate the Ras/Map kinase pathway but the CDKs are always present in the cell as are the target proteins just waiting for cyclin to show up - cyclins will only show up when Ras/Map pathway gets activated - cyclin will bind to appropriate CDK & form a complex known as cyclin/CDK complex which will have specific target proteins. - Target proteins are going to bind to CDK complex - when complex is formed with target protein, target protein is going to be phosphorylated by CDK & then target protein once phosphorylated depending on target protein the phosphorylation can lead to activation/deactivation of target protein. - real example shown in next slide

Retinablastoma protein (pRb) prevents excess cell proliferation cont'd

- using Retinablastoma as target protein of CDK/cyclin complex - Retinablastoma = very important protein in cell cycle; it's function is going to be when it transitions from G1 to S phase. Binds to protein known as E2F & E2F is a transcription factor. when bound, no gene expression. - When Ras/map kinase gets activated & cyclin D is produced, cyclin D is going to bind to appropriate CDK & this complex is going to be at the target protein. - when cyclin D & CDK complex binds to 'Rb, they're going to phosphorylate Rb & in this case the phosphorylation inactivates Rb. Rb comes off E2F - E2F is now able to work as transcription factor allowing for gene expression to happen & the specific genes activated are going to be associated with events in the S phase

Protein Ubiquitination cont'd

- we can add ubiquitination to a target protein which actually tags that protein for destruction - so adding a series of ubiquitins to a particular protein is basically sending signal that we're going to destroy this protein & we're going to recycle the components - image shows structure of ubiquitin - ubiquitin is going to have important regions: - *C-terminus*: region binds to target proteins (we add ubiquitin to a particular protein via c-terminus of ubiquitin) - adding one ubiquitin is not enough, we need to add multiple ubiquitins before the cell is convinced that this particular targeted protein is going to be degraded. So, the way we can add more ubiquitins to each other in a chain is by adding them to *Lysin 48*

Negative Control: Repression cont'd

- we can identify whether certain genes are under negative control repression by doing an experiment - turns out that most of the genes are involved in an anabolic process; anabolic processes build things up - most genes that are involved in anabolic processes are going to be under negative control repression & you can actually check if the genes are going to be under negative control repression through experiment shown in the image - we have couple of positive controls; red = cell # (nothing in the experiment is killing the cells) black = total protein production (machinery for cell production is working just fine); so these two are working as positive controls (whole process working fine) - then we can test condition of interest shown in blue; checking whether the genes of interest are working under negative control repression - genes looking at are arginine biosynthesis - we should expect that in absence of argine, we should have expressions of these genes are active (enzymes made just fine in absence of arginine). - then if you have excess of arginine, immediately see that we will stop production of enzymes - when you see stalling of production of enzymes = strong indication is that these genes expressed are under negative control repression - add more arginine = line goes back to zero

Regulating Gene Product Amount

- we can regulate the amount of gene product; how much gene product we will have - we can then have mechanisms that can be regulating transcription. So, if I don't allow transcription to happen, I won't have a product so that's zero amount of product. - we can regulate at the DNA level to prevent transcription - we can also regulate at the mRNA level & prevent translation - both of these will result in no product whatsoever

Determinants of DNA Replication Fidelity

- we have many mechanisms of DNA repair & occur at different stages in the cell cycle - some DNA repair mechanisms are going to happen before we start S-phase; so pre-replication - DNA repair mechanisms occurring during the DNA replication process - DNA repair mechanisms after DNA replication took place - we have all these checkpoints to ensure DNA mutations do not become permanent & even then they can still become permanent but do try to fix problem. 1. Nucleotide Excision Repair: happens in pre-replication stage; before we start DNA synthesis 2. Exonucleolytic Proofreading: happens during DNA replication 3. DNA Mismatch Repair: happens after DNA replication is finished

Eukaryotic Gene

- we have our promoter region, coding region, termination of gene transcription region - nearby, we have proximal elements - far away, we have enhancer regions - enhancer regions are going to be recognized by activators - activators recognize enhancer regions in DNA. When activators bind to enhancer regions, they're going to recruit large protein known as DNA-bending protein - DNA-bending protein will bend the DNA putting the enhancer regions & activators right on top of the promoter region of interest - in addition to bending the DNA so the enhancers are right next to the promoter, we're also going to recruit mediator proteins which will bind to enhancer regions & promoter & going to recruit transcription factors. So, mediator proteins are going to interact with the activator/enhancer portion of DNA as well as with the promoter region of DNA & are going to recruit transcription factors - when transcription factors are bound to promoter region, they're going to recruit RNA polymerase & allow transcription to happen - this type of gene that requires so many steps & so many proteins is going to be a facultative gene - only under very specific circumstances are we going to have activators present so they can bind to the enhancers, so they can bring DNA protein, that in turn will bend DNA allowing for mediators to recognize enhancers & promoters, allowing for transcription factors to be recruited, & finally RNA polymerase can do its job - some facultative genes will use this regulation bc under very specific circumstances are we going to be expressing them. So, we want to be absolutely certain that those specific circumstances have happened in the cell before we actually express those genes

Protein Degradation

- we have our target protein & going to keep adding ubiquitin to our target protein - C-terminus is purple - Lysine 48 is green - once added enough ubiquitins, then the cell is convinced that the targeted protein is sent for degradation & that happens in the proteasome - proteasome is a structure inside cells that receives ubiquitin & the proteins & destroys them & the components/amino acids are recycled back so we can make more proteins from it

Negative Control: Induction

- we have situation where repressor is active & therefore binding to operator preventing genes from being expressed - in this example, looking at genes involved in breakdown of molecule known as lactose (lac operon) - as cell in environment is reaching nutrients, we see that lactose is going to start to enter the cell & more lactose will enter until we have a lot of lactose inside of the cell & therefore have to produce a signal saying "hey we have a lot of product here. we're going to have to have something to start breaking down the product." - as amount of lactose starts to accumulate inside the cell, some of that lactose is going to become the isomer of lactose which is *allolactose* - we're going to have formation of isomer called allolactose & when it's made it's going to bind to the repressor so know allolactose is behaving in different way than we've seen before & when allolactose binds to repressor, it makes repressor inactive & therefore allowing transcription to proceed - in this example, allolactose is behaving as an inducer; inducers are molecules that when they bind to repressor, they're going to inactivate the repressor allowing for transcription to happen - once transcription happens, the genes that are going to be expressed are going to include a gene known as B-galactosidase. - when B-galactosidase is made, job of it is to cut lactose & when you cut lactose it creates glucose & galactose & you can produce energy from that glucose - after all the lactose has been cut & no longer have lactose available, the same enzyme B-galactosidase comes & is then going to cut allolactose & sending back the. operon to the original situation. - B-galactosidase removes inducer & brings back the repressor to be active preventing transcription to happen bc we no longer have product to break down

Elongation

- we start with the complex we formed in imitation & in that complex we have the tRNA with a growing polypeptide on the P site & have A site exposing the next codon - so that codon needs to then be recognized by the anticodon which is known as *codon recognition* - once codon recognition happens, then we're going to attach the incoming amino acid with the growing amino acid chain. So, we're going to form the peptide bond which is done by ribosomal RNA available in the ribosome. So that peptide bond formation is done. This whole process requires energy & comes from hydrolysis of GTP - once we have formed the peptide bond, we have situation where the tRNA on the A site is going to have the growing polypeptide chain & tRNA on the P site is empty. So, we need to move the ribosome so that we can expose again the next portion of the mRNA which is known as translocation - this step requires energy in the form of hydrolysis of GTP - once we move the ribosome, empty tRNA is going to move from P site into the E site & can now leave the complex & tRNA that has the growing amino acid can move from A site to P site. Now, exposing the next codon so that the whole thing can start again. - when do we know how to stop?

Negative Control: Repression

- we start with this operon - yellow = promoter - purple = operator - green = structural genes - in this particular case, the genes that we are controlling are genes involved in the making of arginine. So, when these genes are produced, we are going to start process of making arginine. - we start with repressor protein being made as an inactive protein meaning that transcription is going to be allowed to happen--we're going to have arginine being made & arginine is going to continue being made until we have way too much arginine (feedback). - when we have too much arginine, some of that arginine is going to come and bind to the repressor & activate the repressor & signal "we need to stop this gene. we have way too much product. we need to stop transcription of this gene bc we don't need anymore arginine". So, the same arginine that's produced is binding to the repressor, activating the repressor--so arginine is behaving as a *corepressor* - by the binding of arginine to the repressor, it activates the repressor, the repressor is able to bind to the operator region stopping transcription from happening which is known as negative control repression. - *we need formation of corepressor that activates the repressor allowing for transcription to be blocked* - corepressor is the product of whatever the genes are making. when we have way too much of arginine, it becomes corepressor & stops this whole process (negative feedback)

*Facultative genes* are only expressed when needed cont'd

- when certain circumstances happen & we need them & when those conditions are not available/present, we don't need those genes - genes that code for telomerases; telomerase gene has to be active & expressed in utero so that we can continue to extend the telomeres but that reduces/completely stops after our birth - genes that are needed when we eat; so we don't need to have the production of different genes that are involved in digestion only when we are eating is useful - when weather changes, different genes expressed in different environments/temperature/humidity. - injured; we need to trigger gene expression - genes expressed in DNA replication; only at different times do we express genes involved in gene replication - under very specific regulation to allow for the genes to be expressed/not expressed

Eukaryotic DNA is *Linear*

- when it comes to eukaryotic DNA, it's going to be linear as opposed to circular in bacteria & archea - so, prokaryotic DNA is circular but eukaryotic DNA is linear which is going to have an effect on DNA replication

Gene Regulation can happen at any step of the Central Dogma

- when it comes to gene regulation, we can do this at every level of the central dogma - so, we can do changes of the DNA that are going to then allow or not allow transcription to happen - we can do changes in the mRNA that can allow or not allow translation to happen - if the protein is made, we can also have changes of the protein so that we can allow or not allow the activity of the protein to continue - today, we're going to be focusing on how bacteria organize & regulate gene expression

Cytoplasmic Responses cont'd

- when it comes to the response, we see that the response can happen either in the cytoplasm or can happen in the nucleus of the cell - when we talk about cytoplasmic responses, what we're looking at the effect of the activity of a protein, usually an enzyme - In cell A, we have a signal received by receptor which then transduces that signal in a series of steps & then we have a response which happens in the cytoplasm. - We're either activating/deactivating a protein & usually an enzyme being affected in this process or we can have multiple enzymes being affected by a signal. - effects seen in the cytoplasm so gene expression is not involved in this type of response

DNA *epigenetic* changes regulate gene expression con't

- when we are changing the structure & not the sequence of DNA, we call this epigenetic changes - epigenetic changes = modifications to structure that are not going to affect the sequence of DNA (not acting on A, C, G, T; acting outside those areas) & these epigenetic changes can have an effect on expression - two examples of epigenetic changes = histone acetylation & DNA methylation - usually, histone acetylation will promote gene expression whereas DNA methylation for most part will suppress gene expression 1. Histone acetylation: act on histone (protein where DNA is wrapping around); adding an acetyl group to particular regions of the histones allows the histones to come unwound from DNA exposing regions of DNA that can now be accessible for expression 2. DNA methylation: adding methyl group to specific regions of DNA (outside of sequence) - epigenetic changes = acting on structure of DNA which can change gene expression now, lets talk about the events that happen in either histone acetylation & DNA methylation...

Only one of the two DNA strands will be transcribed cont'd

- when we do the process of transcription, we're going to use one of the two strands of DNA - separate the two strands & use one of the two strands as a template strand which will be read from 3'-5' way & then we're going to make a complimentary strand & the other strand we're not using is called the coding strand - does that mean that only one of the strands can be used for transcription? NO; both can be used for transcription - if we want to use the other strand for transcription, i'm just going to come from the opposite direction & now this strand becomes the template & the other strand becomes the coding strand

2) mRNA Degradation

- when we no longer need to translate an mRNA, we're going to destroy that mRNA by degrading it - first step is to remove poly-A tail (not clear what enzyme is removing poly-A tail but clear that poly-A tail goes out first) - then protein *Dcp1* is going to remove the cap on 5' end - once cap is removed, then enzyme *XRN1* is going to remove entire mRNA that's left - no translation of gene

Retinoblastoma protein (pRb) prevents excessive cell proliferation cont'd

- when we trigger ras/map kinase pathway, the product is the expression of the gene that codes for cyclin D - when cyclin D is present, we're going to have multiple events. One of them is an effect on a protein called retinoblastoma protein (pRb) - recall that in the absence of cyclin D, Rb is always bound to transcription factor E2F. When Rb is bound to E2F then we're not going to have any gene expression. As soon as cyclin D is expressed, it's going to bind to appropriate CDK which will lead to the phosphorylation of Rb. When Rb is phosphorylated, it's inactive; so no longer functional & comes off E2F allowing E2F to do transcription of genes that are going to be involved in the S phase. - so today, we're going to be talking about what happens in the S phase. & what happens in the S phase is DNA Replication

How we experience crosstalk?

- whenever we take medication for our thing but is also having other effects that we call side effects & that is bc that medication can affect different receptors in different ways which can lead to different responses & depending on those pathways you can have cross-talk also being affected - if you are providing someone with calcium, you need to be aware that you are going to activate a pathway that can be activated by other things as well. So you have to be aware by how the medication you're getting is activating different parts of the body which can act differently in different cells of the body

Transcription: INITIATION cont'd

- where transcription starts - transcription starts by identifying the promotor region. the info in the promotor has info for starting transcription. - promotor region = DNA region upstream of where genetic info is - in eukaryotes, promotor region is recognized by transcription factors bc it's going to have a specific repeat known as the TATA box - transcription factors bind to promoter region & recruit RNA polymerase - RNA polymerase will unwind DNA to form transcription bubble - once transcription bubble is created, ready for next step = elongation

Wobble hypothesis cont'd

- wobble hypothesis states that the last nucleotide in a codon will not affect the interaction between the codon & the anticodon; so there's low specificity in the last nucleotide of a codon - there's got to be strong interactions between the first 2 nucleotides but the last nucleotide can have low specificity

Combinatorial Control of Gene Activation

- you can use this idea of a combination of enhancers & activators to simultaneously promote the transcription of multiple genes at once - so, eukaryotes can also actually promote or prevent the transcription of multiple genes at once - they do this through process called "combinatorial control" - in combinatorial control, we're going to use a combination of activators & enhancers in order to allow for certain genes to be transcribed vs other genes - Simultaneous Transcription of the Genes

Ribosomes

1. Components: - the two ribosomal subunits - proteins - ribosomal RNA (rRNA) 2. A ribosome has 3 binding sites for tRNA: - the P site holds the tRNA that carries the growing polypeptide chain - the A site holds the tRNA that carries the next amino acid to be added to the chain - The E site is the exit site, where discharged tRNAs leave the ribosome 3. Ribosome is a *ribozyme*

*Housekeeping genes* are always expressed

1. *Constitutive* genes - Required for the maintenance of basic cellular function - Expressed in all cells of an organism 2. Genes that code for: - Ribosomes - RNA polymerases - ATPase - Lysozyme enzymes - Etc...

Replication Fork

1. *Helicases* unwind the DNA 2. *Single stranded binding proteins* (SSBs) maintain the stability of the replication fork 3. *Topoisomerases* work ahead of the replication fork & relax the DNA by temporarily nicking the DNA preventing twisting

Gene Expression cont'd pt. 2

1. *The process of converting the genetic information encoded in DNA into a final gene product* 2. Includes two stages - Transcription: where we make mRNA - Translation: where we make protein from mRNA

DNA *epigenetic* changes regulate gene expression

1. A modification in gene expression that is is independent of the DNA sequence of a gene 2. Histone Acetylation - promotes gene expression 3. DNA methylation - usually suppresses gene expression

Nuclear & Cytoplasmic Responses

1. A signal transduction pathway leads to regulation of one or more cellular activities 2. The response may occur in the cytoplasm or in the nucleus 3. Many signaling pathways regulate the synthesis of enzymes or other proteins, usually by turning genes on or off in the nucleus 4. The final activated molecule in the signaling pathway may function as a *transcription factor*

Poly A tail

1. A string of adenosine nucleotides is added to the 3' end of the RNA - Poly A polymerase 2. Always added after the polyadenylation signal

Catabolite Repression

1. A type of *positive control* 2. Regulates expression of many different genes simultaneously 3. Ensures the "best" carbon & energy source is used first 4. Affects dozens of catabolic operons

Elongation of the Polypeptide Chain

1. Amino acids are added one by one to the preceding amino acid 2. Each addition involves proteins called elongation factors & occurs in 3 steps: - Codon recognition - Peptide bond formation - Translocation

Transcription: INITIATION

1. Binding to Promoter region - TATA box (in Eukaryotes) - Recognized by transcription factors 2. Transcription initiation complex - Recruitment of RNA polymerase 3. Transcription bubble - RNA polymerase unwinds DNA

Calcium Ions, Inositol Triphosphate (IP3) and Diacylglycerol can act as second messengers

1. Calcium ions (Ca2+) act as a second messenger in many pathways 2. Calcium is an important second messenger bc cells can regulate its concentration 3. Pathways leading to the release of calcium involve *inositol triphosphate (IP3)* & diacylglycerol (DAG)* as additional second messengers

5' methylated CAP

1. Capping enzyme: adds a guanine nucleotide to the 5' end of the RNA 2. *Methyl transferase*: adds methyl groups to the guanine nucleotide

Cell Communication: An overview

1. Cells communicate with one another in 3 ways: - direct contact - local signaling - long-distance signaling 2. Target cells process the signal in 3 sequential steps: - Reception - Transduction - Response

Genetic instructions are written in a series of *non-overlapping, three-nucleotide code*

1. Codon: 3 nucleotides code for one amino acid 2. Non ambiguous code: each codon always codes for the same amino acid 3. Degenerate code: 64 codons & only 20 amino acids

Telomeres

1. Consist of many repeats of a specific nucleotide sequence (5'TTAGC3' in humans)--collectively they are called the terminal repeats 2. Do not encode functional genes 3. Postpone the erosion of genes near the ends of DNA molecules

Telomerase function

1. Contains a small RNA template with a sequence complementary to the telomere sequence 2. Starts DNA synthesis from the RNA template (reverse transcriptase) 3. Its activity elongates the 3' end of the template (parental) DNA 4. Replication can continue in the new strand in the usual way 5. The new strand will still be shorter but no information will be lost

Accurate translation requires 2 steps:

1. Correct match between a tRNA & an amino acid, done by the enzyme "aminoacyl-tRNA synthetase" 2. Correct match b/t the tRNA anticodon and an mRNA codon - PROBLEM: 20 tRNA but 64 codons

Accurate translation requires 2 steps

1. Correct match between a tRNA & an amino acid, done by the enzyme *aminoacyl-tRNA synthetase* 2. Correct match between the tRNA *anticodon* & an mRNA *codon

DNA Methylation

1. DNA methyltransferases (DNMTs) - Add methyl groups to adenine or cytosine nucleotides - Usually suppresses gene transcription 2. DNA methylation is a heritable mark

Connecting Okazaki fragments and removing RNA primers

1. DNA polymerase I - Removes RNA primer - Inserts correct DNA nucleotides 2. DNA Ligase - Joins Okazaki fragments - Seals any nick in DNA backbone

Antiparallel DNA affects replication

1. DNA polymerase add nucleotides only to the *free OH at the 3' end* 2. DNA polymerase synthesizes a *leading strand* continuously 3. The *lagging strand* is synthesized as a series of segments called *Okazaki fragments*

Mutations are random

1. Environmental factor can lead to mutations but DO NOT guide or direct the mutation 2. Mutations DO NOT occur bc the organism or cell was placed in a situation where the mutation would be useful 3. Exposure to a chemical or other environmental factor DOES NOT CAUSE resistant mutants to appear

Telomerase

1. Enzyme that catalyzes the lengthening of telomeres 2. The enzyme is a *reverse transcriptase* that carries its own RNA molecule, which is used as a template when it elongates telomeres (using RNA as template to make DNA)

Cell-cell communication

1. Essential activity of all cells; need to understand what is happening in the environment around them 2. Universal; every cell has a way of communicating either with the environment or with other cells 3. Requires proteins & chemical signals

Replicating the Ends of Eukaryotic Chromosomes

1. Eukaryotic DNA is found in multiple linear molecules 2. Replication machinery provides no way to complete the 5' end of the daughter DNA strands 3. Repeated sounds of replication produce shorter and shorter DNA molecules with uneven ends

Overview of the Cell Cycle

1. Events in a cell leading to its division 2. Three phases: - Interphase - Mitotic phase - Cytokinesis 3. Highly regulated, dependent on external signals 4. Deregulated in cancer cells

Proofreading activities of *DNA polymerases*

1. Exonuclease activity: a cleavage of phosphodiester bonds & removal of nucleotides from the end of the DNA chain 2. 3' to 5' exonuclease activity: - proofreading function - DNA pol III & I 3. 5' to 3' exonuclease activity: - Removal of RNA primer - DNA pol I only

The Responses of the MAPK/ERK pathway affect the Cell Cycle

1. Growth factor : ligand 2. Tyrosine kinase receptor: when bound to ligand it dimerizes and autophosphorylates tyrosine residues 3. Grb2: adaptor protein recruited by TKR when phosphorylated, binds to SOS and activates it. 4. SOS: guanine nucleotide exchange factor. It removes GDP from Ras and activates Ras 5. Ras: GTPase. When bound to GTP is active and recruits Raf to the cell membrane and activates RAF 6. Raf: protein kinase (serine/threonine). When activated it autophosphorylates and phosphorylates MeK. 7. Mek: protein kinase (tyrosine/threonine). When activated phosphorylates and activates ERK. 8. Erk: also known as MAPK. Protein kinase (serine/threonine). When activated phosphorylates many molecules including transcription factors. 9. Transcription factor: a protein that binds to specific DNA sequences and control their expression. In this pathway, transcription factors activate genes associated with cell proliferation.

Chromatin-modifying Enzymes

1. Histone acetyltransferases (HATs) - Adds acetyl groups to histone ends - Leads to euchromatin formation 2. Histone deacetylases (HDACs) - Remove acetyl groups from histone ends - Leads to heterochromatin formation

Molecular Components of Catabolite Repression

1. In addition to those already present in the lac operon or other catabolic operons 2. Catabolite activator protein (CAP) - is the *activator* protein 3. Cyclic AMP - Found at low levels when glucose is present - Found at high levels when glucose is scarce

Termination of the Signal

1. Inactivation mechanisms are an essential aspect of cell signaling 2. Multiple mechanisms to terminate cell signaling - Ligand concentration - Removal of phosphates - Removal of second messenger - Blocking transcription

All Organisms Follow the Same General Steps during Transcription

1. Initiation: Recognition of where to start RNA synthesis 3. Elongation: RNA synthesis 4. Termination: Completion of RNA synthesis

MicroRNAs (miRNAs)

1. MircroRNAs (miRNAs) are small double-stranded RNA molecules taht can bind to mRNA after they are processed into single-strands 2. These can degrade mRNA or block its translation 3. The phenomenon of inhibition of gene expression by RNA molecules is called *RNA interference* (RNAi)

Regulating Transcription in Bacteria

1. Negative control - Stops transcription - Uses repressor proteins A. Repressor - Regulatory proteins - Binds to the operator - Prevents RNA polymerase from binding to the promoter 2. Positive control - Activates transcription - Uses activator proteins A. Activator - REgulatory protein - Binds to activator site - Stabilizes RNA polymerase binding to the promoter

Mismatch Repair (MMR)

1. Normally happens immediately after new DNA is synthesized 2. *90% of all repair* 3. Based on the detection of methylation of the old (correct) strand 4. *Endonucleases* remove the unwanted base

3) Noncoding RNAs play multiple roles in controlling gene expression

1. ONly a small fraction of DNA codes for proteins or rRNA & tRNA 2. A significant amount of the genome may be transcribed into *noncoding RNAs* 3. Noncoding RNAs regulate gene expression at 2 points: mRNA translation & chromatin configuration

Generals on Eukaryotic DNA replication

1. Occurs in the S phase of the cell cycle 2. Semiconservative - each strand acts as a template for building a new strand in replication 3. The entire DNA molecule is replicated - two identical copies are produced 4. More than a dozen enzymes & proteins are required

Cyclic AMP

1. One of the most widely used second messengers 2. Adenylyl cyclase, an enzyme in the plasma membrane, converts ATP to cAMP in response to an extracellular signal 3 Phosphodiesterase, enzyme that converts cAMP to AMP

Cross-talk occurs between pathways

1. One signal triggers one pathway 2. A different signal triggers a different pathway 3. Pathway meet & a certain point 4. Results: - Increase activation of a target protein - Inhibition of target protein

Transduction: Cascades of molecular interactions

1. Phosphorylation & dephosphorylation - *Protein kinases:* transfer phosphates from ATP to a protein - *Protein phosphatases:* remove phosphates 2. Second messengers - Small, non-protein, water soluble moleucles or ions - Cyclic AMP (cAMP) - Ca+ - Inositol Triphosphates (IP3) - DAG

DNA Synthesis

1. Primase - begins DNA synthesis by adding an RNA primer to a DNA strand 2. Primer - made of RNA. *Provides a 3'OH group for the DNA polymerase to attach a nucleotide* - 5-10 nucleotides long 3. DNA polymerase III - Adds nucleotides that hydrogen bond with their appropriate complementary nucleotide (A with T & G with C) - Forms covalent *phosphodiester bonds* b/t nucleotides - *DNA synthesis can only happen in the 5'--> 3' direction*

Control elements that regulate Transcription

1. Proximal control elements: DNA region located close to the promoter 2. Enhancers: DNA regions far away from promoter region & gene 3. Activators: a protein that binds to an enhancer & stimulate transcription of a gene 4. Transcription factors: a protein that binds to the promoter region & can either stimulate or inhibit the transcription of a certain gene

Transcription: ELONGATION

1. RNA polymerase moves along the DNA - Unwinds double helix - No need for primer - Adds ribonucleotides - Forms phosphodiester bonds 2. 40 nucleotides pers second in eukaryotes 3. One gene can be elongated multiple times by several polymerases

Split Genes & RNA Splicing

1. RNA splicing removes introns & joins exons, creating an mRNA molecule with a continuous coding sequence 2. Spliceosomes consist of a variety of proteins & several small nuclear ribonucleoproteisn (snRNPs) that recognize the splice sites - Ribozymes are

Retinablastoma protein (pRb) prevents excess cell proliferation

1. Restricts DNA Replication - Rb is a regulatory protein 2. Binds & inhibits E2F - E2F is a transcription factor 3. Cyclin D/CDK4/6 phosphorylate Rb 4. pRB dissociates from E2F 5. E2F is free to initiate transcription of proteins required for the S phase

Retinoblastoma protein (pRb) prevents excessive cell proliferation

1. Restricts DNA replication - Rb is a regulatory protein 2. Binds & inhibits E2F - E2F is a transcription factor 3. Cyclin D/CDK4/6 phosphorylate Rb 4. pRB dissociates from E2F 5. E2F is free to initiate transcription of proteins required for the S phase

Replication begins at special sites called *origins of replication*

1. Specific region in the DNA 2. High in *AT content* 3. Multiple origins of replication in linear DNA - up to 100,000 in a human cell - speeds the process 4. Assigned as *ori* regions 5. Recognized by multiple proteins to form a replication "bubble"

The Unit of Transcription

1. Stretch of DNA transcribed into an RNA molecule 2. *Promoter region*: where transcription factors bind 3. *RNA-coding sequence*: Contains information to make an RNA molecule 4. *Terminator region*: Contains information to end transcription

Only one of the two DNA strands will be transcribed

1. Template strand - 3'-5' DNA strand - sequence of DNA copied during transcription 2. Coding strand - 5'-3' DNA strand - mRNA-like sequence corresponds to the codons that are translated into protein - strand NOT used in stranscription

Wobble hypothesis

1. The first two bases in the codon create the coding specificity 2. Base pairing of 3rd base is less-precise 3. One tRNA can recognize & bind to ore than one codon 4. Explains degenerate nature of DNA code - multiple codons for a single amino acid

Transcription: TERMINATION

1. The polymerase continues past the termination signal - *Polyadenylation signal (AAUAAA)* 2. About 10 to 35 nucleotides past the signal the RNA polymerase stops

Gene Expression

1. The process of converting the genetic information encoded in DNA into a final gene product 2. Includes 2 stages: - Transcription - Translation

More on Phosphorylation cascades

2 ways we can send the signal into the interior of the cell: 1. phosphorylation cascades - require protein kinases & protein phosphatases - in phosphorylation cascades, signal received by receptor that leads to activation of a particular molecule which in turn will activate first kinase - when kinase is first activated, it's going to phosphorylate another kinase activating that kinase which will activate another kinase & phosphorylate that kinase activating that one which will activate the target protein which is going to lead to a cellular response - series of phosphorylation events aka phosphorylation cascade & eventually leading to cellular response - once we don't need cellular response, we remove phosphates from kinases & target protein which is the job of the phosphatases & then we go back to the beginning - fantastic way of amplifying a signal which is known as amplification

How do cells in the body of a multicellular organism communicate with each other? A. By way of signaling molecules that interact with specific receptors B. Through long projections that directly connect cells to each other D. Through electrical signals passed between a cell and it's external environment D. By the transport of ions between cells in different parts of the organism. E. By the transport in water.

A. By way of signaling molecules that interact with specific receptors - going to be thru signals & receptors

What type of receptor is used in the cell signaling pathway shown below? A. Intracellular receptor B. G protein-couple receptor

A. Intracellular receptor

Matthew Meselson and Franklin Stahl's 1958 experiment

A. Semiconservative replication B. Conservative replication C. Dispersive replication - Study Figure 14.9 in your book

True or False. Each somatic cell in your body contains the same genes A. True B. False

A. True - kidney cells have exact same genes as your brain cells

Which one is the lagging strand? A. B.

B. - if we put our primers, we're going see that primer on A is going to allow for the synthesis to be continuous & with the fork making A the leading strand - & B wouldn't continue. We'd have to move the primer here making it the lagging strand

Given the following DNA, will the same protein be generated if either of the DNA strands is transcribed? A. True B. False

B. False - if you start with top strand, remember that you will always synthesize in the opposite direction & therefore our first codon will be CUG - if we use the other strand of the DNA, we will go in the opposite direction so therefore our first codon will be ACC - so, we're going to have completely different products depending on which top or bottom strand of the DNA you're using - *mRNA will always be read 5' to 3' during translation*; whatever mRNA you make, the translation machinery will always read it from 5' to 3'

The image below shows the mechanism of action of the cholera toxin is shown below. What type of receptor is the toxin binding to? A. Intracellular receptor B. G protein-coupled receptor C. Ligand-gated ion channel D. Receptor tyrosine kinase E. None of the above

B. G protein-coupled receptor

Does Transcription & Translation of every gene happens all the time? A. Yes B. No C. Maybe D. I do not know

B. No

If a missense mutation at the activator site of the lac operon makes that region constitutively active & the binding of CAP is not required any more for function, how would this affect the transcription of B-galactosidase gene? A. The gene will always be transcribed B. The gene will be transcribed every time lactose is present C. The gene will not be transcribed when glucose is present D. The gene will not be transcribed even if lactose is present E. The gene will not be transcribed only when glucose is present

B. The gene will be transcribed every time lactose is present - we have a mutation on the activator site of the lac operon that now makes it constitutively active & not requiring the CAP protein in order to function. - Role of activator site in lac operon = transcription factors to find the promoter better so that RNA polymerase can work better - we no longer require CAP protein on cAMP so we have lost the ability to recognize glucose in order to allow for promoter to be present BUT we have not lose ability to recognize lactose bc that's done in the repressor form. Even though promoter will be accessible for transcription, this gene will only be expressed when lactose is present bc that is when the promoter will be rendered inactivated & therefore allowing for transcription to happen. - question asking what is the activator site doing, what is it's role, what is it checking for (presence of glucose but also have operator region checking for presence of lactose so even if we don't have glucose we still need to have presence of lactose before this gene can be expressed)

What role is phosphorylated STAT3 playing? A. Ligand B. Transcription factor C. Second messenger D. Receptor E. None of the above

B. Transcription factor - second messengers include: cyclic AMP, IP3, DAG, & calcium - STAT3 is not a second messenger & gets phosphorylated so it means that it's involved in phosphorylation cascaded which dimerizes & crosses nucleus which lead to DAN transcription

Caffeine is an inhibitor of phosphodiesterase. Therefore, the cells of a person who has recently consumed coffee would have increased levels of A. phosphorylated proteins B. cAMP C. GTP D. adenylyl cyclase E. activated G proteins

B. cAMP - every time you drink coffee/energy drink, you inhibit phosphodiesterase therefore maintaining levels of cAMP in your body which in turn allows all the cellular responses associated with the presence of cAMP to continue to be available and one of them is to keep you alert

Based on this model of a tRNA molecule, what is the mRNA sequence that corresponds to the anticodon sequence? A. 5' UGG 3' B. 5' GGA 3' C. 5' UUC 3' D. 5' CUU 3' E. 5' ACC 3'

C. 5' UUC 3' - anticodon region bottom of tRNA - codon is going to be complimentary to anticodon region so it's going to be UUC & running from 5' to 3' - the top purple = where amino acid binds

What type of cell communication are we seeing in the figure below? A. Local; paracrine B. Local; autocrine C. Direct contact D. Long distance E. More than one type of cell communication

C. Direct contact - from cell 1 we have a receptor called, "delta" in orange - from cell 2 we have a receptor called, "notch" in purple - example of docking which is direct contact - docking of 2 receptors which is an example of direct contact - for paracrine & autocrine, you need a soluble signal; ex. cell 2 releases soluble signal & that signal binds to receptor serrate then it would be paracrine communication - soluble signal means that it's not attached to anything; it's liquid, it's in the environment

The *Hayflick limit* is the number of times a normal human cell will divide before cell division stops. Cancer cells bypass the Hayflick limit. Which of the following could explain how cancers cells are bypassing the Hayflick limit? A. The Rb protein is active B. The Helicase protein is inactive C. The Telomerase protein is active D. The CDKs are present E. More than one is correct

C. The Telomerase protein is active - there are so many cell divisions available to a cell before it stops working known as hayflick limit; named after scientist who discovered this phenomenon - if Rb protein is active = transcription that's required in S phase will be inhibited so we're not doing the cell cycle so that wouldn't be useful for cancer cells - if helicase proteins are inactive = we can't separate the 2 strands of DNA which also wouldn't be useful for cancer cells - if CDKs are present = no difference with presence or no presence of 'CDK's with cancer cells; CDKs are always present. Presence of cyclins that make a difference - if telomerase is active, then you can bypass the hayflick limit bc you can no longer going to shorten the lifespan of the cell bc you're going to keep elongating the telomeres & therefore extending the chances of a cell to divide even if you're an adult cell

Direct Contact Communication

Cells have to be touching each other & there's multiple ways they can achieve this: 1. Gap Junctions: have proteins that are going to join cells together but also going to have a gap. Through that gap, you have the different molecules that cross from one cell to the other which allows for communication to happen b/t one cell & another - cells in intestines, organ like heart or kidneys 2. Two neurons connected with each other via gap junctions allowing for different ions to cross which allows communication b/t one neuron & another - nervous system 3. Cell wall: doesn't stop you from communicating. Have region called plasmodesmata that allows different molecules to cross b/t one cell & another which allows communication to happen 4. Protein to protein interactions: receptors touching each other; direct contact leads to a particular response - T Snares, V Snares; vesicle trafficking

Point mutations

Change in a single nucleotide 1. wild type = sequence of DNA that's most common in a population 2. silent mutation = have a single change in a nucleotide but the message is unchanged 3. Missense = have a single change in a nucleotide that now changes the message 4. Nonsense = introduce a premature stop message so the message is stopped sooner than it needs to be stopped 5. Frameshift deletion = deletion in single nucleotide & message from that point on is completely changed 6. Frameshift insertion = insertion in single nucleotide message & from that point forward the message has changed

Cell D

Completely different response than we've seen in previous Cells A, B, C. So, we have a completely different receptor also capable of binding to the same signal but now we have a completely different response as compared to the ones we've had in Cells A, B, or C.

Which of the following mutations is most likely to cause a phenotypic change? A. Silent mutation B. Missense mutation is noncoding region C. Deletion after coding region D. Insertion at the beginning of coding region E. All of the mutations will cause a phenotypic change

D. Insertion at the beginning of coding region - phenotypic change: change that's going to have an effect on the appearance of an organism

The image provided is an example of what type of receptor? A. Intracellular receptor B. G protein-coupled receptor C. Ligand-gated ion channel D. Receptor tyrosine kinase E. None of the above

D. Receptor tyrosine kinase - has dimerization, phosphorylation event, & phosphorylation cascade

What role is CFTR playing in this signaling pathway? A. Ligand B. Transcription factor C. Second messenger D. Response E. Effector

D. Response - effector = adenylate cyclase which in turn activates cyclin AMP (second messenger) which in turn binds to CFTR (effector or response) - CFTR when receives second messenger is going to be open ready for release of chlorine ions

Cyclin E forms a complex with Cdk 2. This complex is important for the progression of the cell from G1 into the S phase of the cell cycle. Based on this and your knowledge on cyclins, which of the following statements is correct? A. The amount of free cyclin is greater during the S phase compared to the G1 phase B. The amount of free cyclin E is highest close to the end of the S phase C. The amount of free Cdk2 is greater during S phase compared to the G1 phase D. The amount of free cyclin E is highest close to the end of G1 phase E. The amount of free cyclin E is constant during throughout the cell cycle

D. The amount of free cyclin E is highest close to the end of G1 phase - it is the Ras/Map kinase pathway when activated will produce cyclin D - when cyclin D is produced, it triggers cell cycle - cell cycle involves multiple stages: G1 phase; cell growth (where most of our cells are at right now). Then when we accumulate Cyclin E, is then going to move from G1 to S phase where we have processes involved in making DNA replication (making a copy of DNA). Then when we complete DNA replication, continue to grow bc at the end of the day we have to separate these two cells & when the two cells separate we're going to have to have them in the right size which happens in G2 so we're just growing the cytoplasm . In mitotic stage, we separate the components of the nucleus--one chromosome for you & you...In cytokinesis, we separate the cytoplasm & now we have two functional cells. - in this whole process, cyclin E is involved in the transition from the G1 phase to the S phase - we're going to find that at the beginning of the G1 phase Cyclin E is going to be free & accumulate & accumulate & it's not until we have the max concentration of cyclin E that is going to bind to CDK & then allow for all the other events that need to happen in the S phase - answer: D. The amount of free cyclin E is highest close to the end of G1 phase - by the time we're in the S phase, cyclin E needs to be joined together with CDK (concentration of CDK doesn't change throughout cell cycle)

In eukaryotic cells, transcription cannot begin until A. The AUG (start) codon is identified B. The two DNA strands have completely separated & exposed the promoter C. The DNA introns are removed from the template D. The transcription factors bind to the promoter E. More than one answer is correct

D. The transcription factors bind to the promoter - A. is for translation. It's not required for transcription. - B. You don't need to separate the DNAs to find the promoter - C. introns are not removed from the molecule until much later.

Which of the following receptors would be inhibited by a drug that specifically blocks the addition of phosphate groups to proteins? A. G protein-coupled receptors B. ligand-gated ion channels C. steroid receptors D. receptor tyrosine kinase E. intracellular receptors

D. receptor tyrosine kinase - not A bc GDP & GTP are not proteins, they're nucleotides

In negative control of transcription, how does the presence of an inducer affect transcription? A. the inducer binds to the operator B. the inducer does not bind to the operator C. the inducer causes the repressor to bind to the operator D. the inducer prevents the repressor from binding to the operator E. the inducer binds to the promoter

D. the inducer prevents the repressor from binding to the operator - every time an inducer is present, it's going to bind to the repressor & then preventing the repressor from binding to the operator

DNA structure & packaging

DNA can exist in either tightly wound structure on a loosely wound structure - In eukaryotes we find that DNA is not found by itself, but found in close associations with proteins known as histones & DNA is going to wound to these histones - if DNA is tightly wrapped around histones = known as heterochromatin; normally don't have access for expression - if DNA is loosely wrapped around histones = known as euchromatin; have portions of DNA accessible for expression - we can affect the packaging of DNA & have other ways we can manipulate the DNA structure without having to change the actual sequence (A, G, T, C)

Gene Regulation can happen at any step of the Central Dogma

DNA, RNA, translational, & even when protein is made - what mechanisms in eukaryotes are they able to regulate gene expression at all of these points?

Which of the following is NOT a function of RNA polymerase? A. Initiating a polynucleotide strand B. Opening the double-helix to expose the template C. 3' to 5' exonuclease activity D. 5' to 3' exonuclease activity E. All are functions of the RNA polymerase

E. All are functions of the RNA polymerase - Initiating a polynucleotide strand = occurs during elongation process; by itself can add first ribonucleotide & can keep adding them - able to form transcription bubble by separating double stranded DNA which occurs in initiation process; it is the RNA that makes the transcription bubbble= Opening the double-helix to expose the template - has proofreading activity & can happen in both directions = 3' to 5' & 5' to 3' - all of these are present in an RNA polyymerase molecule which is why transcription happens a lot faster than DNA replication ever occurs - further evidence that RNA was the first genetic material bc if you're going to replicate it you're going to simplify things & RNA polymerase can do that

In the figure below, how is the signal being transduced? A. Phosphorylation cascade B. Dephosphorylation cascade C. Using cAMP as second messenger D. Using calcium ions as second messenger E. More than one is correct

E. More than one is correct - A. Phosphorylation cascade - D. Using calcium ions as second messenger

Regulation of Gene Expression

Eukaryotes

What kind of receptors are Adrenergic receptors?

G-protein-coupled receptors using second messengers

If each somatic cell in your body contains the same genes, why is a skin cell different from a brain cell?

GENE EXPRESSION!!! - even though each cell has exact same genes, some of those genes are going to expressed in certain cell types & other genes will be expressed in certain cell types therefore distinguishing different cell types - gene expression is what makes that difference

Given the following observation: In the U.S. where people have access to shampoos with chemicals that kill lice, there is an increase in lice that are resistant to those chemicals. Which of the following is the most likely explanation for this? Hypothesis A: Resistant strains of lice were always there--and are just more frequent now because all the non-resistant lice died a sudsy death. Hypothesis B: Exposure to lice shampoo actually caused mutations for resistance to the shampoo

Hypothesis A: Resistant strains of lice were always there--and are just more frequent now because all the non-resistant lice died a sudsy death. - shampoo itself is not causing the mutation. the lice shampoo selecting for pre-existing mutations (role of selective pressure)

What would you expect about this particular genes being different in terms of transcription or genetic components of these genes compared to genes that may not be turning on or off? What sites would be constantly available?

If we focus on transcription, what's the first step in transcription? Binding to the promoter. So, the promoter regions of constitutive genes are going to be different than the promoter regions of the facultative genes (genes that are not always expressed). There also might be something that inhibits the prevention of the gene.

Activator site

It is at this site in the DNA that molecules called activators can bind to & allow RNA polymerase to transcribe the DNA, thus switching the operon on. - In contrast to operators, we have activators. As name suggests, activators are going to promote transcription. So, when an activator binds to the activator site, we're going to promote transcription. So the things that bind to the activator site are going to be activators. Notice that the activators are far away from the promoter & operator. The two bands represent that they are far away from the promoter region or where the gene regulating is present. So, when something binds to the activator, it is called activators. When the activators are bound to the activator site, then we are going to be promoting transcription.

Operator

It is at this site in the DNA that molecules called repressors can bind to & block RNA polymerase from transcribing the DNA, thus shutting off the operon. - the first one is the operator. The operator is the region in the DNA that's always present between the promoter & the structural genes (shown in yellow). - if nothing is binding to the operator (or is free), we are going to have usually transcription of the structural genes.

Telomerase activity

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Regulation at the Translational Level

Mechanism that allow a cell to fine-tune gene expression rapidly in response to environmental changes - once mRNA is made, we can manipulate not only the product but whether or not the product will be made - what mechanisms are available in the cell that can then regulate at the translational level (going to be acting on mRNA)

Processing Pre-mRNA into Mature mRNA in Eukaryotes

Overview of mRNA processing: - first thing that needs to happen is the addition of a 5' cap & a poly-A tail - after that, we're going to remove all the introns & join together all the exons

The Bacterial Operon

Regulatory Features

Mutations are responsible for introducing diversity in the genetic material

The effect of the mutation will depend on 2 factors: 1. Where in the DNA the mutation took place - Coding vs. non coding regions in the DNA - Somatic vs. Gamete cells 2. The type of mutation

Cell B

The same signal with the same receptor but now in a different cell type is now going to have a completely different response to that interaction. So, the same signal interacts with the same receptor but now we have different downstream proteins of that receptor so then we're going to have different responses. In this particular case, the signal is triggering 2 cell responses which are going to be different from Cell A.

Gene Expression cont'd pt. 1

Transcription - all of our somatic cells have the exact same genetic information however some of them can become kidney cells, pancreatic cells, spleen cells, brain cells, etc. - what allows for that differentiation between the different somatic cells is the different patterns of gene expression

Nucleotide Excision Repair

Triggered in response to normal intracellular metabolism or physical & chemical insults from the external environment 1. Detection of damage: recognition of abnormal structure 2. Excision of damaged region: *endonucleases* 3. Repair damaged region: DNA polymerase I fills the gap 4. Sealing of the nick between new & old DNA: DNA ligase

Negative Control

Two options: - Negative Control Repression - Negative Control Induction

Point mutations are chemical changes in just one base pair of a gene

Types of point mutations: - Wild type: THE BIG RED DOG RAN OUT - Silence: THE BIG R*E*D DOG RAND OUT - Missense: THE BIG R*A*D DOG RAN OUT - Nonsense: THE BIG RED - Frameshift deletion: THE BGR EDD OGR ANO - Frameshift insertion: THE BIG RED *Z*DO GRA

Cell A

We have receptor 1 which is able to interact with the triangle signal. When this interaction happens, receptor 1 gets activated & is going to act on the different proteins that are downstream from that receptor & we're going to get type of receptor called Response 1.

mRNA Processing

after transcription, mRNA is not ready to be translated so multiple things are going to need to happen

Polyadenylation is present in

both DNA & mRNA

exonuclease activity

can only cleave DNA from the ends

tRNAs Cartoon

cartoon

Endonucleases

category of enzymes that can cut DNA in the middle of DNA sequence

Overview of the Cell Cycle cont'd

cell cycle leads to replication of a cell. Cell cycle divided into stages: interphase, mitotic phase, & cytokinesis - interphase = longest phase which includes growth phase 1, S phase, & growth phase 2 - mitotic phase = very short - cytokinesis = when you divide the cytoplasms into the 2 cells - cell cycle is highly regulated as making 2 cells - too much cell proliferation is usually a sign that something is going wrong in body - deregulation = usually a sign of cancer cells (not all deregulated cells are cancer cells but all cancer cells are deregulated) - cell cycle = dependent on signals from outside & inside of cell

tRNAs 3D structure

cloverleaf shape

Regulatory gene

controls the expression of one or more genes. May code for a protein or RNA. Often codes for repressor or activator proteins. - so the gene that's going to code for the activators or the repressors are known as the regulatory gene. The regulatory gene is even far away from this whole process & whose product is going to be an activator that's going to bind to the activator site or the product is a repressor & is going to bind to the operator region. In some cases we have 2 regulatory genes, one that codes for activator & one that codes for repressor

Targeting Proteins to their Final Destination

cytoplasm, ER, golgi, plasma membrane - if a protein belongs somewhere else besides the cytoplasm, as soon as the protein is made, at the beginning of that protein sequence, we're going to have what's known as a signal peptide--a series of 3-4 amino acids that are going to tell another protein, "hey we don't belong in the cytoplasm, we belong somewhere else". So, that signal protein is going to be recognized by the signal recognition particle. The signal recognition particle is going to bind to the signal peptide & is then going to recruit the entire complex into the membrane of the ER. The signal recognition particle is going to bind to the SRP receptor protein. The SRP receptor is a translocation complex (job is to translocate things; to move things from one side to the other). As soon as the SRP protein binds to the SRP protein, it's going to open the translocator. Translocating will continue & as the polypeptide grows, it's going to move through he translocator proteins & when we're done, we're going to see that the protein is now found on the inside of the ER. If this is the final destination of the protein, it stays there. if destination is golgi, then we're going to form a vesicle & that vesicle is going to transport that protein to the golgi. If the destination is the plasma membrane, it's going to go from golgi to the plasma membrane using vesicle trafficking process.

tRNAs 2D structure

flat structure

Intracellular Receptors

found inside cell nor embedded in membrane - in cytosol, receptor is available therefore the signal has to cross the membrane in order to find the receptor - molecules involved are small, nonpolar, noncharged molecules which can cross membrane, bind to receptor forming a complex which will then cross nuclear membrane & directly bind to DNA allowing for gene expression to take place 1. Found in the cytosol or nucleus of target cells 2. Usually activated by small or hydrophobic chemical messengers - steroid - hormones

Cell Communication affects the Cell Cycle

how cell signaling is associated with a very specific cell response. discuss how cell communication is going to affect the cell cycle

Promotor region only exists

in DNA & not present in mRNA

Monday, October 12

mOrE nOtEs!

5' Cap & Poly A tail exist in

mRNA

Transcription

making RNA from DNA - focus on eukaryotic cells

Structural Genes

may code for a structural protein, an enzyme, or an RNA molecule not involved in regulation. *The products of these genes are needed for the morphological or functional traits of the cell* - Depending on the operon, the structural genes can be coding for enzymes, components that are going to lead to the structure of the cell, etc. There are just multiple genes that usually are working in activities that are related to each other. So, gene 1 is related to the activities of the activities of gene 2 which are then related to the activities of gene 3. So they tend to be genes related in similar functions.

DNA replication is a

multistep process

Purpose of 5' methylated CAP

needed for multiple things; needed when mRNA is going to try to leave the nucleus (need to make sure 5' is there before it can leave the nucleus), once outside of cytoplasm both the 5' cap & poly-a tail are need to protect the mRNA from being destroyed

Mature mRNA is transported to cytoplasm for Translation pt. 2

note that transcription in eukaryotes happens in the nucleus & not until mRNA is mature is able to leave the nucleus & enter the cytoplasm which is where translation will take place

Cell Communication

notes

Friday, October 16

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Monday, October 19

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Monday, October 5, 2020

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Wednesday, October 14

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Wednesday, October 7

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Friday, October 9

notes!!!

DNA replication is the only

process where the DNA strand is completely unwound which is why it requires the topoisomerase

Transcription

producing the first RNA molecule; doesn't include mRNA processing

Translation

protein synthesis - goal of translation: produce a protein from that mRNA

The Responses of the MAPK/ERK pathway affect the Cell Cycle cont'd

referred to either RAS/MAPkinase pathway - signaling pathway that's then going to have an effect on the cell cycle - presence of growth factor (ex. insulin) in enough concentration outside of cell, it's going to bind to it's appropriate receptor, receptor tyrosine kinase which will lead to dimerization & activation of RTKs. - Autophosphorylation will happen & then recruit Grb2 which gets activated & leads to recruitment of SOS - When SOS gets recruited by Grb2, it gets activated - when SOS gets activated, it will bind to Ras leading to conformational change in Ras which will release GDP & will bind to GTP - when Ras binds to GTP, it's activated & activates Raf - Raf is a kinase which will autophosphorylate & will become active - Raf phosphorylates Mek, Mek phosphorylates Erk - Erk phosphorylates transcription factor, & transcription factor will happen - once transcription factor gets activated, one of the genes that is going to be expressed is the gene that's going to code for cyclin D - when cyclin D is expressed, it's going to trigger the events of the cell cycle - the signaling pathway of Mek-Erk pathway is going to lead the activation of the gene that's going to code for Cyclin D. When Cyclin D is expressed, we're going to start process of triggering the cell cycle.

Autocrine Receptor

same cell that is releasing the signal is also receiving the signal bc it has the receptors - ex. if a cell finds itself invaded by a virus, the cell sends signal saying, "I have a virus" which is going to be received by a receptor & that receptor is going to trigger a signaling pathway that's going to lead to cell death

Mutations

some errors always happen - Radiation: UV radiation, X-rays - Mutagenic Chemicals (chemicals that can produce mutations): cigarette smoke, preserved food (specifically nitrate & nitrite), barbecuing (mutagenics present when burning your food) - Infectious Agents: Human papillomavirus (HPV), helicobacter pylori - DNA replication itself (errors only 1 in 10 billion nucleotides) & those mistakes can become permanent

What is the solution to the shortening of the ends of linear DNA molecules?

that is the job of multiple components: telomeres & telomerase

Cell C

there's 2 different receptors & are interacting with 2 different signals. We're going to have the same receptor interacting with same signal we've been discussing & we're going to have a different receptor interacting with a different signal. Receptor 1 is going to interact with signal & is going to have a particular response. Receptor 2 is going to interact with a completely different signal but the pathway activated by this process is going to have an effect on pathway 1. It can either enhance/activate the same response or inhibit the response that was triggered by receptor 1. - process known as cross-talk: when two signaling pathways converge & then have an effect on a response (can be activating/inhibiting it)

Major goal of DNA replication

trying to make 2 cells that are identical & they have the exact same genetic information - produce 2 identical copies of DNA - but the process is not perfect & mutations are always going to happen - mistakes can come from different sources