RNA Rearrangement PDF

Okay, it is one o'clock. Are we ready to get started on this happy Friday? started on this happy Friday? No, I mean it's Friday Good news all of the content for this block is now available The last lecture which was the gene code lecture, which is our flipped classroom that is up and available to y...

Okay, it is one o'clock. Are we ready to get started on this happy Friday? started on this happy Friday? No, I mean it's Friday Good news all of the content for this block is now available The last lecture which was the gene code lecture, which is our flipped classroom that is up and available to you I recommend that you not watch that one until after we've done splice his own work But if you feel do you want to work ahead? Just at least make sure you take a look at mRNA processing before you go into translation Okay, so Everything is available everything that we are going to cover The only things that are not Things that you can see in advance before more like an hour beforehand are the learning activity Which is Monday and the flipped classroom, which is the following Monday? Both of these dates are intended to be practice We are going to work with the material live and in person together in groups Your groups for the learning activity are already posted They're small groups. There's like 18 of them. I May bring signs But if you can at least reach out to your group in advance that'd be great That way you at least know who you're looking for make sense Because that seems to be a good five minutes at the start of each of these learning activities starting to fight each other Okay, so let's start with our questions Hopefully we can see that I'm doing some of these open-ended questions But also providing you with an example of what this could look like on a quiz or an exam To sort of give us a better sense What about this first one? These aren't the same. We got to find it remove it replace it Seal the backbone How we find it what we find those are the things that differ the actual steps of repair differ a little But at the end we've got a gap in the DNA backbone what fills that in DNA like this. DNA like this. So what is unique about each form what they repair and how they do it Do I need a template to be able to fill in the missing bases am I just taking two pieces of material and cramming them together What is the damage and what am I using to see the damaged is the biggest area of difference? damaged is the biggest area of difference? What about this next one? No Why does it change and certain repair mechanisms correlate with very specific Cancers because those repair mechanisms are important to that cell type BRCA1 mutations correlate with breast and ovarian cancer because Those are repair mechanisms important in those tissues That makes sense Can repair cause problems I attempt to fix something but I do it wrong That's gonna cause a problem. My effort to fix something changes the sequence That's gonna cause a problem. My effort to invade another strand causes a rearrangement That's mitotic error and we know at the chromosomal level that can be catastrophic That makes sense. Oh No Those were supposed to clickable. I am sorry You want to just read through So I am gonna post all these summaries of these ones up there But DNA and RNA, please do not tell me double-stranded versus single-stranded because that is not accurate focus on the sugar focus on the higher order structures that are formed How are higher order structures formed Complementary-based pairing hybridization Structural non-coding RNAs are the ones that play a structural role to either an enzyme or to a molecule itself tRNA rRNA SNO RNAs SN RNAs They play structural roles Regulatory RNAs Regulate those other processes SI RNA which can break down other mRNA molecules can interfere Not long non-coding short non-coding anything that's going to Alter the activity of something else is going to be a regulatory RNA So yes, we do have some overlap in some of those, but these are the key groups RNA sequence changes if I change the RNA I Change how it can fold because RNA folding is based on Complementary-based pairing if I have different bases in there. I can't make the same hairpins loops Folds if you will Make sense and then we talked about polycystronic mRNA, which will come up multiple times in multiple lectures But why does that make sense for prokaryotes, but not for us? What's our big difference? Membrane-bound organelles our processes are separated and so they don't need to function together Their processes are existing together in time and space So having coordinated regulation like this is essential It makes sense for them All right moving back into our transcription All of these processes we're doing from that categorization of initiation, elongation, and termination Initiation for DNA replication involved origin recognition, helicase activity, creation of the pre-initiation complex to open the material and then fire those origins where we need our polymerase to have a primer to land We finished with the summary, can I? RNA polymerase has it all in one So controlling transcription is all about controlling landing at the start Your promoter is a critical sequence for landing the polymerase when we land at the promoter We are essentially at the transcription start site. This is upstream of the actual code It determines as part of this which strand will tell us the code and which strand We will actually use as a template to synthesize this material the strength of a promoter is Tied to its sequence and ability to land that polymerase The better you are at landing, like you are just a nice open field That polymerase helicopter can come straight in every time does so with absolute efficiency That's going to be what's called a strong promoter. We're really good at our job Because our job is to be the landing pad of the RNA polymerase weaker promoters Would be say a more closed Confirmation, so a little bit of material in the way not as open of a field a little bit harder for the pilot to land Those weaker promoters are going to have lower levels of transcription because it's harder for that material to land Changes in the basis of a promoter have that impact We also have sequences and sites that we refer to as termination sites for transcription Transcription has a start and a stop So talking about promoters we do have some critical elements This is a sequence of DNA promoters are DNA primers are They are RNA So our DNA polymerase needs RNA to function our RNA polymerase needs DNA to function we cannot We cannot get transcription without a promoter Because our polymerase is not going to land. It's not gonna have anywhere to go and do its job If I lose a promoter entirely, I have a structural rearrangement that takes me away from the promoter I trapped my promoter as heterochromatin No promoter no transcription Because promoters are so important. Because promoters are so important. They are evolutionarily conserved and so we have the tatabox and consensus sequences associated with our promoters these specific sequences can be found in multiple species multiple genes and alterations Can have impact on ability to associate that polymerase? These are upstream of our actual gene code We have similarities to our prokaryotic friends, but ours are a lot more complicated Because our promoters also have regulation tied to other critical gene sequences We're gonna do that with the regulation piece, but our longer promoters will still have Similarities we will still have sequence similarities space between critical sequences. It's just the distance can be much further Why does that make sense? What would we expect our polymerases in general to be compared to our prokaryotic friends? be compared to our prokaryotic friends? Bigger have more components? Be more of them So it would make sense that our landing pad would need to be a little bit longer There's a difference between a drone and a helicopter A tiny drone, big helicopter, different space needs so to initiate Transcription we must get to the promoter polymerase must land This is where I think enhancers are probably the coolest sequences in your DNA Because they can be anywhere Anywhere in three- dimensional space their job is to help land that polymerase at that promoter and so the same transcription factor can bind multiple enhancer elements or multiple sequences within the genome for enhancing transcription and Get really complicated patterns to promote that polymerase association So what I'm showing here is the nice RNA example where we've got the two sequences the tata box and the consensus sequence association of our polymerase and the ability to Transcribe the material versus our eukaryotic example where we have a lot more other players helping us orient that material and we can even have Very far away elements bending the genetic material to get that polymerase in place so We have tons of RNA polymerases Each of them has a different function and so I say tons We're gonna break it down to these three main categories, right? Paul one, which is all about our RNA Paul two which is going to be our main genes, right? These are our protein coding mRNA, but also our SN RNA because turns out our mRNA and SN RNA can be very closely associated in sequence Where our SN RNA pieces can be right after our main mRNA genes What does that sound like a polycystironic mRNA, right And then of course we have our Paul three which is going to be other components of regulatory RNA Or I'm sorry structural RNA Transcription is Really amazing Because everything we needed to do to replicate took so many different pieces and yet here we are all self- contained Once we land and open the material we can start laying in bases These bases are going to have a different sugar. Of course, it's the same three prime hydroxyl need that we had before and We are going to create a transcript that as we make it we are going to process it Just like in our prokaryotic friends as they made it they began translational stuff We are going to make it and begin processing it and the processing lecture will be next week, but All of those steps are important Remember that in every Sequence of DNA, it's double stranded for us. We have a sense and an anti-sense strand The anti-sense strand is our template our Sense strand makes sense. It tells us the code we are going to be creating So the Coding strand the sense strand matches what we are making see See we see that so if you're Looking to do this analysis and say okay which sequence matters which base pair change matters You've got to figure out which one is giving me the code Which one is the template? All polymerase is synthesized material in what direction? Five prime to three prime so our Coding strand is going to match that because our material needs to move What direction to achieve that synthesis? three prime to five prime How do we stop this if it was all about getting it started? How do we end it? We have to be told to write there. The whole point of this was I am controlling when this starts Eukaryotic regulation is predominantly at the level of transcription. I am controlling when things are on when I am making transcripts I Have multiple polymerases. So my stopping them is going to be polymerase specific my first one has termination factors My second one can move past the point of stop and do more work So it's going to have a little bit more regulation and a few additional options to consider We can prevent The transcription machinery from falling off so that we can make those SNRNAs We can essentially block the material from falling so it can move on and make this other piece we need In order to actually get that off then we actually do end up needing Exonucleus activity we have to cut material away at that point Does that make sense a? lot of this material is going to be controlled by The domains within the polymerase itself. I actually have to have Certain features to be able to do this extra activity. So this is all Paul to This polymerase is a lot of remnants of our prokaryotic friends Incredibly complicated very carefully regulated But it gives us the ability to make all these other RNAs that we really need to carry out critical functions and Like many of the things we do in genetics. There's lots of stuff. We still don't understand But all of this material regarding these ability of Paul to to do more work the ability of Paul to to Actually encode for these SNRNAs and other pieces that's really new science the last 10 years So we look at our yeast friends. We can see that we have lots of additional Factors called in as part of this, but essentially we are At an mRNA encoding region we're sort of skating along and we're going to have Factors that let us stay and do more work But for termination at that point we're going to hit the right sequence and for Other types of RNA we're going to be told it's time to leave So a lot of this is not well, what specific piece comes in and says it's time for you to dissociate We call them release factors We call them termination factors. We're not entirely sure Which one is doing the job and we're still figuring that out a lot of the time but ultimately How transcription ends is about that polymerase falling off. falling off. It is a self-contained unit. I Can land open the DNA and do my job all in one? So the only thing that's going to stop it is getting that off the DNA And so disassociation is a critical part to stopping this well It's a highly conserved process Because it is so important that we regulate it and so when it comes to additional concepts and termination We can see how We can have the ability to actually move past key points and have more complicated work So these last two slides the takeaway is just oh, hey, it can move on and do more I don't want you remembering all of these individual features because some of them have actually Changed and there's some edits to some of these pieces as we've updated Just some of the components in which parts are a part of the complex and whether there's that much overlap but remember that When it comes to stopping at an mRNA and maybe moving on to these other non coding RNAs We can continue past only if we're given the the indication we should Something binding to the polymerase that says stay and keep going Prokaryotes have a much more sequence specific Association for this stuff. We have a much stronger Factor coming in telling us what to do, but termination sequences are part of this. We need to know where the gene stops But we also have that backup system of sequences within the gene that tell us when translation should stop So that's our big takeaway We have multiple components that are going to influence when RNA Paul 2 stops its job If I don't have a need for a significant amount of processing SN RNAs are really important in processing material I don't need them right now. I'm not processing a bunch I'm kind of metabolically less active. I'm not really replicating. I'm not doing anything super special Am I gonna want to keep? all this stuff going But what if I'm having kind of a freak-out moment? But what if I'm having kind of a freak-out moment? I'm like, oh my gosh, what's going on? There's there's all this heat and there's all this cold and I'm just in all of this stress Am I gonna have the same regulation or I'm gonna be trying to respond to those stresses? And So we will find that we will impact this system based on need and specifically based on stress. I can put my system under Incredible amounts of stress because I have systems like this to help me regulate To help me deal with the fact that I'm under stress. I am I Love this particular figure because I found it more recently and it really does help us understand that there are factors They're gonna help promote termination and factors. They're gonna help suppress termination to drive desired outcomes and a lot of our Suppressing termination is very correlated with disease Preventing us from regulating this is a problem and so I think this is a really nice summary of how stress can affect transcription termination We predominantly regulate at the level of transcription. So it makes sense that there would be times where stress impacts this Makes sense. I Will repeat this material quite a bit. It comes up in virtually every lecture. I Can affect function by changing more than the gene itself If I change the promoter, I change transcription if we regulate Predominantly at the level of transcription then affecting transcription affects regulation I Am only supposed to have certain products where and when I need them. I Am NOT supposed to have certain products when I don't need them when I don't want them when they're toxic And I need to clear out that toxin Changing my promoter making it less available more available affects this If I have sequences that increase affinity So if I have a single nucleotide polymorphism and insertion a deletion I move to a different promoter that makes it better for my RNA polymerase to land expressions going up Makes it worse expressions going down if I am bad as already Paul to because these are genes we're talking about that are encoding proteins RNA Paul to landing controls transcription if I land well Consistently all the time strong promoter more transcription if I land poorly Not great at it. There's stuff in the way decreased transcription If I am at the point of terminating Transcription and I am unable to do so That could also affect The ability to make SNR a it can affect the ability to actually control this process I Think it's really important that we be able to connect all of these pieces because these are all the molecular processes of genetics It's not a lot of new stuff. There's only a few new pieces here and there I think for many if you didn't have molecular you might or DNA repair you might that piece might be new But the big goal here is to really be able to categorize this stuff keywords typically Expected you know, make sure we're paying attention to those modifiers because we in science do not like absolutes. Do we? We like to be very clear on what we can demonstrate Versus what we can prove and we can't prove a lot. Can we? Because anytime I say well, here's the rule what happens There's an exception key difference between transcription and replication at No time is a living Metabolically active cell failing to undergo transcription Transcription does not stop at any time. It stops for a part Stops for a region of genetic material, but for the whole cell. Stops for a region of genetic material, but for the whole cell. Nope There is always transcription happening and the best way that we can Understand that is to think back to how we actually moved through the cell cycle What were we controlling to go from s phase to g2 to M? What were we controlling to go from s phase to g2 to M? What were we doing? Cycle independent kinase activity and how did we control it? By producing cyclins and Phosphorylating other proteins if I could not produce the next cyclin would I turn on the next CDK? So that example alone should tell us our previous conceptions that transcription Has stops is not accurate Does that make sense? Okay, so this is one of those tables I really recommend that you sort of fill in on your own How do we initiate each of these processes? How do we elongate under each of these processes, and how do we terminate and of course now? We have a fun little example where we can move past termination by suppressing termination To be able to make a new piece of material Questions on the last bits of transcription that I expected for you Okay, are we ready to regulate this? Got a bunch of objectives with regulation If it is super important to life, do you think I'm going to turn it off and on I? Super need it all the time. Am I going to waste time having to turn that on? No, some genes are Constituatively expressed especially for prokaryotes. They are just on all the time So their regulation is going to be about what turning them off Most of our genes are off. So how are we going to really regulate them? Turning them on this is our most common transcriptional regulation prokaryotes are the exception because Transcription and translation are in the same compartment and they happen together So because they are coordinated Both of those regulation types are essential Ours are separated. We regulate Predominantly at the level of transcription. Do we have translational regulation? Yes, and I am going to teach it to you Even though you've probably already seen it But predominantly it's for us. It's transcription so Terms you may be familiar with repressor co-repressor inducer activator Silencer enhancer When it comes to prokaryotic gene regulation Is it on and I need to turn it off or is it off and I need to turn it on? It's the best way to think about it Negative regulation Turns off genes that are on Positive regulation turns on things that are off make sense Because we are talking about transcription we are talking about access to promoters prokaryotes have additional sequences that we'll get into at the very first lecture of block four, but We are talking about making it so that When we previously had access we no longer do or when we Didn't have proper association. We suddenly can We have fun little Friend molecules called small effector molecules that control these larger regulatory proteins One of our Fun little small effector molecules is called an inducer Here's where it gets fun an inducer Induces expression So depending on which type of regulation we're talking about positive or negative Ultimately expression is going to be induced. So if it is a Inducer role we are either going to block repression or enable activation still an inducer So I'm either Preventing turning it off or I'm enabling turning it on I'm inducing activity at the transcription start site We can have more complicated complexes like co-replica co-repressors where we are actually using a Binding with another small effector molecule, which is the co-repressor to create a repressor complex we can have general inhibitors and inhibitors can Prevent association of Key transcriptional machinery to the DNA. Key transcriptional machinery to the DNA. I can't land my polymerase can't do my job And so if we think about the operon model, which is what will literally our first lecture block for the operon model Has a key sequence that we are going to bind to to block the promoter and So these repressor and inducer molecules play a critical role to regulation Why would we do this? Why would a system? Create a situation where we turn things on Because there's times when you just don't need certain things and it's energy wasteful, right? If I have No need there's no glucose around Well, that would know that's a bad example There's no lactose around is a better example. Do I need all of the genes that break down lactose? So why would I bother? To make something I don't need Why would I have something always on? Why I'd have something that I can turn off but only under certain conditions So if we're looking for a big answer to this, it's energy efficiency It is more efficient to not waste time and energy Turning things on that you need all the time It's more efficient To induce things you only need when specific Scenarios are present an Inducer is a concept of I'm going to turn this on because I now have met the minimum requirements for this thing And so inducers can take on a lot of different forms Think back to our bacteria friends Compared to us who's more sensitive to environmental changes? Why why are we technically more sensitive? Why why are we technically more sensitive? Yeah, we can go get a coat But why are we technically more sensitive in terms of cellular responses than our bacteria or friends? Because they are designed to be able to rapidly change certain pieces to be able to deal with that now that is of course a gross generalization, but What kind of an environmental change would necessitate a response for a bacteria What about food if I suddenly have a totally different food and I can't change to eat that food What's gonna happen to me as a bacteria? That's all it's a common answer isn't no more eating. That's all it's a common answer isn't no more eating. We'll just leave it at that. What does no more eating constitute well imagination What about us Remember that we are also collections of cells So we do have some of those stressors in our tissue where suddenly we are lacking something we're supposed to have So in our tissue we respond so that the whole organism is okay What's the job of the liver Maintain blood glucose deals with toxins What if it could not do that for the whole system? So just remember a lot of times we're using our prokaryotic friends to explain some of the cellular changes you have And it'll help hopefully with Biocome We will talk about the operon model in block four, but I wanted to introduce eukaryotic regulation in the more human sense So remember eukaryotes Separate compartments prokaryotes have collinear Polycystronic material We have split genes We have introns stuck in the middle of our material. We have multiple promoters Each individual gene has its own promoter, but it can have more than one I Need I have situations where I need this material a lot. I'm gonna use the stronger promoter And so I might have one cell type Where that stronger promoter is controlling this gene, but in another cell type where I don't need that gene as much the weaker promoter is going to be available to control that and Rearranging the genetic material can help control which promoter is accessible Epigenetics is rearing its head again because it controls the ability to transcribe What is a CPG island? What is a CPG island? I've mentioned it like very briefly, but it's really important No Regions of DNA that contains cytosines with guanines CPG islands are segments where we have that repetition lots of cytosines lots of guanines Typically even only a few cytosines with a bunch of guanines These get highly recognized by DNA methylation machinery DNA methyltransferase is C CPG islands and start methylating cytosines What does methyl cytosine do? It's really dependent, but it does represent a place where we can have additional replication error, but more importantly What would sticking a bunch of posts or trees in the middle of a landing pad do? prevent landing CPG islands within a promoter can significantly reduce transcription Regulatory elements are just as important to transcription as other components transcription factor transcription factor availability Confers the ability of that polymerase to associate so we sort of have two levels here Do we have the proteins to enhance the polymerase, but do we also have the functional sequences to enhance polymerase landing? I'm really hoping a lot of this is still very much review And we're sort of thinking about this from the perspective of turn transcription off or on All genetic changes have the potential to do one of three things absolutely nothing no change downstream Increase activity or decrease activity in Terms of polymerase transcription factor enhancer changes promoter changes we can either increase transcription Decrease transcription or keep it exactly the same Those are our three choices the consequence of those changes is directly tied to What that material is doing? If I am a protein that drives cellular division Turning that on is going to promote division If I am material that drives cellular division turning that off is going to block division We cannot just pay attention to the regulation. We have to figure out how it's changing the outcome We essentially have to look at regulatory elements as binding sites Our general transcription factor is TF 2d TF 2d is going to bind to that tata box Its job is to recruit and orient Paul to It's pretty good. It is a general transcription factor Specific sequences require specific transcription factors when we say transcription factor, please take a look at all these bubbles That's a lot of components These are massive complexes if my enhancer is way over there and I'm the promoter right here To influence me there's got to be bending in three-dimensional space or a pretty massive complex to fill that space If I'm on a completely different Chromosome as an enhancer. I've got at least be in the general vicinity of that material Enhancers don't even have to be on the same chromosome. They can be anywhere so typically binding and recognition of our initial transcription factor is going to call in friends and their Entire job is to get what where? RNA polymerase onto the promoter Tire job So this is sort of showing you that bending that massive complex bringing in all these elements Every gene has multiple enhancers Which enhancers which transcription factors are being utilized controls the level of association of the polymerase The polymer the promoter itself can have variable binding affinity, but then so do these transcription factors so We are going to have enhancer elements our promoter and the whole job is to get that polymerase on there So if we look at here in terms of regulation We can have small effector molecules and playing a role here Getting us a better job of getting that transcription factor associated doing a worse job Of getting that transcription factor associated Ultimately, if I can't get my transcription factor to the right spot. I'm not going to get what where RNA polymerase to the promoter And so it doesn't matter how many different ways I show this ultimately polymerase doesn't land. What doesn't happen? We predominantly regulate at the level of transcription How your genetic material is organized? controls this methylation controls this We talked back in block two we talked about DNA versus histone methylation DNA methylation at a promoter the sudden presence of cytosines near guanines can literally prevent a polymerase from landing a single nucleotide polymorphism changing one base in a promoter has the potential to reduce or increase polymerase affinity if I literally do not have access to the promoter it is trapped against a nucleosome. Can I land a polymerase there? No, but couldn't we turn nucleosomes using chromatin remodeling complexes to give us accessibility? Big takeaway block any regulatory element you block transcription You have the potential to impact transcriptional levels If every gene has the potential to have more than one promoter you could be changing the regulation of that gene by taking away a more regulated promoter by giving it access to a more regulated promoter changes affect outcomes So I love this example because it really does tie that back to that CPG island I can have changes in cancer that give me multiple additional methylation sites within a promoter and It's literally like building a forest where your landing pad originally was you are creating a structural interference for the polymerase I literally cannot land where I should remember we talked about positional effects I Have a really great promoter its enhancers Allow my transcription factors to put me on over and over been very consistently. I am turning on this gene completely regulated in a nice positive way turning this gene on and I do this a lot I Suddenly move that material into heterochromatin What happens? That's not expressed somewhere like I don't have my promoter. It's heterochromatin my genes here Gene wants to do stuff, but now it can't I Have a Break right in the middle of a promoter We needed that tata box we needed that consensus sequence now, I'm split Can I land a polymerase? All of the same positional effects we were talking about was all about this affecting transcription Literally affecting the ability of our polymerase to land So we have to pay attention to this also from the perspective of transposition is supposed to happen There are times. We're moving genetic material around is Okay, and desirable We have two classes of transposons the retro and DNA retro transposons Use a reverse transcriptase and include RNA DNA transposons are a direct DNA to DNA transposition We have that nice tie back to what we did before because why in the world would we have a mechanism that allows for that? Kind of rearrangement. It's because there are times when it's necessary There are times when it's important and so this process in particular the idea of being able to move genetic material around Super important for your bacteria friends How do I get? resistance to an antibiotic By being able to grab resistance genes from friends Surviving and sharing that with my buddies Transposition is part of that Because this process exists in them turns out yeah exists for us, too If we do this wrong we can get something called insertional mutagenesis The genetic material enters into the wrong place disrupting or breaking Key material this is exactly like an insertion Like other structural rearrangements that we had talked about this should look familiar or will soon Here's our takeaway Here's our focus our takeaway Yes, you know, I should call back Our takeaway is how this works and the ultimate goal right you're doing that for all of your lovely antibodies Our takeaway is this slide Rag mediated recombination. So this is where we will pick up at the start of our Splicing because they actually go really well together. So remember on Monday. We have our activity It's a required session. Please be here. Make sure you notify us if you're not going to be and Then Wednesday will be our last like in-person content and you have every slide everything for the whole block already accessible to you questions or concerns I Don't know how to feel about the massive sighing that came with but questions So Monday is all about practicing these molecular processes. So if you come in sort of ready to talk about them We're going to replication transcription and translation although less on the translation front more of the splicing But I really want us to sort of come in ready to have that conversation and help each other We've got some sample questions. It's going to be all about make sure you know your objectives for those lectures. We've done before the more Successful we are on Monday the more questions and practice you will have for the exam Monday's goal is to write a bunch of questions other questions concerns I Promise we're going to try to make this less painful size indicated Hey, that makes my job easy You know every molecule I just get to go and here's how this works. And here's why we care This is back. Oh, this was about two. Yeah, I moved the blocks around in here to give us more time with us So immunology call back time. Yay Yay All right, any other last second questions because we went over again Have a good weekend