okay how do we go from a messenger RNA right that we have a five Prime cap and a poly tail on how do we go from that to an actual working protein in the cell okay this is a process called translation translation happens in the cytoplasm all proteins begin being translated in the cytoplasm okay they all start being translated they don't all finish there but they all start there okay so we're that's what we're going to kind of talk about today how does translation work and then how do cells that are staying in the cytoplasm what happens to them and then how what excuse me cells what happens to proteins that are staying in the cytoplasm what happens to proteins that H actually have to move to a different organel or have to be embedded in a membrane what happens or secreted from the cell how do they move uh after they start translation okay everybody remembers the central dogma which is what into protein so DNA to RNA to protein I can promise you I'm going to ask you that on the test okay all right so what do we need so this is translation right here right so we see the ribosomes in your book are always going to be the yellow little yellow things the protein the polypeptide chain that's being made is in green and the messenger RNA is in Red so what do we need for this process it's a pretty it's pretty elaborate actually okay well first of all we have to have a messenger RNA right and it has and this is a mature messenger RNA right so it has a five Prime cap and it has a polyat tail right we got to have a ribosome a ribosome as you can see there has two subunits it has a large subunit and a small one okay it also has three active sites inside that large subunit okay so that's where the polypeptide the peptide bond is going to be formed and the transfer rnas are going to be moving through there all right to bring in the amino acids we need another type of RNA we need a transfer RNA okay these guys and there is one Transfer RNA for every amino acid okay at least one okay the ribosome I just want to mention now has a ribosomal RNA and it's inside the large subunit and this is the enzyme that actually makes the peptide bond okay so it is a ribozyme that forms that peptide bond all right to begin um kind of you have to have something that attaches the amino acid to the transfer RNA okay that is another type of enzyme these guys right here called an amino ail TRNA synthetase it attacks that attaches that amino acid to the right Transfer RNA okay then when we start translation we have some proteins that help assemble the ribosome and get the messenger RNA in the correct positioning those are called initiation factors okay as the protein synthes as the peptide synthesis goes on we have other proteins that help do that they're called elongation factors and then when you reach that stop codon that we talked about right this the stop sign this is the end of the protein you now have to kind of break the whole thing apart so there are proteins called release factors that help disassemble the whole big complex okay and then it can all be recycled to start something else okay does that make sense so let's take a look at ribosomes okay so none of the pictures really show the ribosomal RNA it's but you cannot put the large and small subunit together without the ribosomal RNA okay and it is in this in the What's called the pite okay so these three there I told you there are three kind of active sites within this ribosome one is called the asite the amino acil site that's where where the transfer RNA comes in first the one in the middle is called the pepti the peptidal site this is where you form a peptide bond okay so this is like the start and then the last site is the exit site so this is going to be where once the the transfer RNA has given its amino acid to the new peptide it's released okay so it's a channel where the transfer RNA can leave all right so you have them moving down this kind of assembly line all right and everything is added one amino acid at a time you form one peptide bond at a time okay and the messenger RNA is is still going to go five Prime to three prime so it's going to be red in the ribosome five Prime to three prime okay so you can't just have it oriented in any direction it has to be in that direction so you have and that's one of the reasons you have that five Prime cap because the five Prime cap is where messenger RNA actually binds to the small subunit everything starts on the small subunit and gets organized and then the large comes in okay okay so I don't know if you can put something in the a peptide to make a polypeptide we're reading in sets of three right and that is the reading frame right okay yeah I'm going out tomorrow night my Budd what is the I don't want to worry about going to the backroom right and you don't remember how big the leg bags are do you okay and the code on is a u g Okay so frame call gab Max words okay so in this case if you look here we kind of a lot of nonsense until you get down here says the boy saw the man okay so in this good in tilt but like to drain it before I go out for the first codon and you would read through that okay it's very important that the reading frame is correct if you have different mutations that can take place so if you have a deletion that's going to throw it off one if you have an insertion that's going to throw it off one okay you can have you can even have insertions where you have multiple base pairs put in and deletions of the same flavor you can have multiple Bas pairs taken out that can be catastrophic because it can throw off the reading frame and now you have just this nonsense and while it might get trans while some of it might get translated probably the cell is just going to recognize it as garbage and destroy it okay so now you're going to be Mi you could potentially be missing a very important protein okay so in uh if you take biology of cancer uh this one of the things we're talking about I guess in in this lecture coming up you know there are lots of mutations that occur in cancers that are these insertions and deletions uh where you'll have uh you know different base pairs put in or you'll have mutations happen so that these Master regulator proteins don't work and so that's how the cells kind of get out of control okay and there are lots of different mechanisms behind that we'll talk about mutations later on in the course okay does that seem okay everybody's good okay so the three sites the a site is the amino AEL site it accepts the transfer RNA so you'll have your transfer RNA with an amino acid and it's going to come here okay the pep the P site is where the peptide bond forms the E site is literally a channel and that's how the transfer RNA actually is released okay all of those are in the large subunit okay the small subunit does not have any um active sites okay and that's because that ribosy the ribosy is actually that that ribosomal RNA is sitting in the P site okay where it can catalyze that reaction to make the peptide bond all right so let's talk a little bit about transfer rnas and the things that are associated with them okay so this is a transfer RNA remember we talked about RNA is single stranded and it has this kind of unique ability to fold on itself and it makes very elaborate shapes the transfer RNA is a great example um this is uh kind of more realistic so this is really more realistic of the three-dimensional shape of a transfer RNA okay just like any other nucleic acid it has a thre Prime end which is right here and it has a five Prime end which is right here okay I'm actually going to erase that the three prime it's a little bit hard to see all right at the three prime end that is where the amino acid attaches okay so if we had let's say we had methionine it would attach here right kind of Midway through the through the the transfer RNA is another part that has what's called the anti-codon okay the anti-codon is going to be complementary to the messenger RNA okay so you're going to have your messenger RNA in the ribosome the transfer RNA is going to come down what's going to recog that threel codon or three nucleotide codon is the anti-codon so it's going to bind there okay so it's the anti-codon that actually interacts with the messenger RNA all right so you have two kind of two ends of this and you can see that um RNA has these Loops so these places is where that that single strand is not complementary that's how you get a you get a loop when the base pairs are not able to pair okay so you get these Loops those Loops that you see in these more elaborate rnas are often sites where they're interacting with something or you have a catalytic site in the case of a ribosome okay so this is the transfer RNA we have a pool of transfer rnas in the cytoplasm we have a pool of amino acids in the cytoplasm so this is very efficient all right so you have to attach that amino acid to the transfer RNA it doesn't just happen spontaneously okay uh this is a very specific type of reaction right we want the right amino acid attached to the right Transfer RNA so to do that we need an enzyme right and those enzymes are these guys um the TRNA synthetases they actually you can see this is a nice a really nice figure because it shows you that Amino ACL TRNA synthetase in blue and you can see how the transfer RNA in purple and the amino acid in green are bound in that active site and you can see that the way it binds this is the three prime end of the transfer RNA this is the amino acid isoline you can see in that active site that three prime end where you need the amino acid added and the amino acid are right on top of each other they're very close okay we'll talk about enzymes I think in the next chapter but this is how this is one of the ways enzymes catalyze reaction they arrange everything so that the reaction takes place immediately okay so they they actually put the two things together so that the functional groups that are supposed to form the bond are right together and the bond can form immediately okay and this is nice because it also shows you that you have different um Amino ail transferases per amino acid okay now when you have an amino acid like these guys uh excuse me when you have a transfer RNA that is just free it doesn't have an amino acid added to it that is called an uncharged TRNA okay when you have one that has that amino acid added that is a charged TRNA okay yes yes they would because so they they have to have a different enzy that's a very good question because remember when we looked at the amino acids those R groups they're all different so they all have a different shape so to get this reaction to happen you got to have um a TRNA synthetase that has the right shape for every one of the amino acids so yes you have one for each one is that is that what you were asking this question over here no okay and you can see also that at the five at the not the five Prime at the um anti-codon Loop that also makes interactions with the TRNA synthetase okay so that actually helps Orient that TRNA synthetase in the active site okay and we get these active sites by protein folding which we talked about last time all right so this is kind of showing how you get the codon which is in the messenger RNA and the anticodon which is on the transfer RNA to come together okay so this is our messenger RNA here which we made in the nucleus it's been transported now out into the cytoplasm it has its five Prime cap it also has it's not showing here but a polyat which I'll put over here okay what's going to happen is and it'll show this I think in the probably in the next slide or two you have to have the the messenger RNA is going to sit on that small subunit and it's going to scan down messenger RNA until it hits the AUG right because this is the codon for methine okay so that indicates this is the start of the protein this is where we should start reading three base pairs at a time right and so you can see that here is a this is a charged trans for RNA with methionine it can come into the a site and associate with that codon right and this is the anti-codon right so it's complementary to that sequence it's not the exact same as that sequence okay and so this is nice because showing you that pairing does that make sense so this is how the process starts if we look at the codon so this is a table that shows you the basically the genetic codes these are all the codons and the different amino acids that they code for uh there are 64 total codons 61 actually uh specify the 20 amino acids I just went into Woods and Water for the first one and the other three are the in going to try and stay away from that went to go okay so the way you would read this I went in with the single goal of getting a belt and almost spent like $500 you start on this side so much that I then you move to this side so we have a okay and we have a u in the second position so this is going to put us here and we need to look down over here to G here's methine okay so that's how you read that chart you'll notice that a lot of amines in fact the majority of them have more than one yeah I've been trying to save money I need to stay away from that place something called redundancy and it is not just spec we don't have just uh redundancy in the genetic code we have a lot of redundancy in the cell in the important mechanisms and this is because if a point mutation happens and mutations happen at a pretty frequent rate uh in the cell over the course of an organism's lifetime you still have there's a little flexibility es especially in that third position that third nucleotide that's something called wobble okay so it gives you some flexibility in case something happens okay which is not unusual over the course of somebody's life time uh the stop sequences have a couple of different names some people call them Terminator codons uh some people say they are call them nonsense codons so you'll hear all those I always call them stop codons all right everybody good about codons so translation has a couple of different steps they're pretty straightforward you have initiation you have elongation and you have termination okay so we're going to focus on how this happens in eukaryotic cells right that's really the f of our book what does it mean to repent so if we look at initiation this is where you start to build the complex with the ribosome right so the five Prime cap on the messenger RNA is going to bind to the small subunit of the ribosome okay and it's actually kind of held in there by the two proteins you see in kind of that pinkish purple those are initiation factors okay then those initiation factors then and the ribosome itself are kind of going to scan down that messenger RNA and find the start site so they're going to scan for aug okay so once you find the start now what happens is you have other the other parts of this complex recruited in so everything is set the messenger RNA is set it's correct you have you found that initial um start you found the start codeon and now the large subunit can come in and bind okay so now you have this uh complex together to start translation right the first Transfer RNA with methionine also comes in at the beginning okay so you have to have that there to kind of start what are you going to add the peptide bond to okay so that gets together all of this gets together ready to start in initiation once this is formed those initiation factors can be released and they'll go to start initi initiate translations somewhere else else okay the next step is now elongation read along this messenger RNA form a new polypeptide okay so this molecular machine is going to scan along there's a there's a channel actually that forms when you have the large and the small subunit together for the messenger RNA to kind of flow through okay and as it reads along it's kind of protruding out the back think about it that way all right so we have our me it scans down three nucleotid next one is Gua which codes for veine so now we can have that charged Transfer RNA come into the a site and bind okay and once you have that what happens is the the amino acid in the pite forms a peptide bond to that new amino acid the veiling okay so this begins and so you're constantly going to just tack them on like that okay and it's the ribosomal RNA that catalyzes the formation of this peptide bond within the ribosome okay and then once you have this peptide bond everything moves down one okay so our first amino acid this was the one that had methine attached is released through the exit Channel okay we have our growing polypeptide right here in the P site you can see in red that's our new peptide bond and the ribus the asite has moved down now we have a new codon okay that codes for this guy Arginine so now this Transfer RNA can come in it can the anti-codon and the codon bind and there will be a new peptide bond formed between the veine and the Arginine okay and then everything shifts again okay does that make sense it's pretty straightforward so again your the the figure the you know picture doesn't the drawing doesn't show this but there is also a channel that allows that growing peptide to just be extruded out okay so this is a this is a pretty big complex there a lot of channels for things to move in and out as because you could have you know if we have a peptide that has 34,000 amino acids that is a lot so we're going to be extruding something that's really large okay and so this is just showing you that movement um now we have the Arginine attached everything's going to shift over one we have now can have a new charged Transfer RNA come in with a new amino acid and it's going to move all the way until it reaches that stop codon okay and then when you get to the stock codon which you see here as UAA it's a little hard to see okay once that is in the as site what's actually recruited now is something called a release Factor so another protein comes in okay okay it's going to enter the asite and now this polypeptide whatever it is is going to be released okay so that polypeptide is released into the cytoplasm okay so all of this place in the cytoplasm everybody's good with that okay so once it's released in the cytoplasm a lot of proteins they work in the cytoplasm so they just fold it's a SP it is a spontaneous process um we don't know everything about it we're still really actively studying how protein folding happens um it does not require energy okay there's no energy required for a protein to fold into a three-dimensional structure all right so this this happens spontaneously and all the information needed for folding remember is in that primary amino acid all right so just to touch on procaryotes it's a lot simpler we don't have these different compartments everything's happening in the cytoplasm and it's not as you don't have as many protein players coming you know my weights my wrist weights make things happen basically the ribosomes and remind you they actually called a shine delaro sequence okay okay yeah and at that point they can start um protein translation I'll probably leave my room at like one so the the process is pretty similar going be a long day but it's it's a little bit this one compartment I know and I feel bad for so I have a life group which is my sequences so tic cells do not have a sh which is my like Bible study group that I've been going to complicated to stop going with sequen is that join Ducks Unlimited microbes right and it's the meetings are at Tuesday at 6:30 you can see from this table and thinking about how many how many I'm there a huge number of possible proteins and one of Reas eventually with fraternity Brothers the information stage molecule of the yeah I mean I've only been going you can make so there's so many possibilities and you know turn we talk about regulation in a minute we can also modify these Amino um so there's even more yeah I'm gonna start going on Mondays different amino acids different protein configurations so there you know there are a huge number of proteins compared to the roughly we have roughly 26,000 jeans but we make I don't even know hundreds of thousands of proteins so it's you know the gene is not reflective of the vast number of prots we have in the cell that are working to keep ourselves going no what any questions about translation roll got it all right so all these processes that we've talked about transcription uh processing of messenger R in and out of remind me toing my weights transation all of these are regulated very tightly okay and we've already talked about the fact that I don't have strength in cell they only in What's called the silent St it's not a duck hunting Duck Unlimited you know the license pl we have in the van that's duck uned pretty much we raise money oh you know I'm going to a meeting with um so Anna you know nurse Anna how her sister cut my hair yeah good so her sister was cutting another guy's hair who um is indu unlimited and was like I gotone that's perfect for that and uh an just gave him my number he just text me own kind of so that's that's one of the of that are being expressed and some that are just they're constantly just kind and they're actually to the edge of nucleus El micc I'm not a duck guy only birds are like shooting or andate process I know I'm going to go to the me thein isul remember my weights because we have all these transcription factors transcription factors you don't get gene expression we regul RNA processing because there are variants there are small rnas that can be used to made even the protein is made we can still modify it okay this modification takes place mostly in the we can add sugars we can all kind of you can add these modified amino acids I mean all kinds of things can go on okay so even after made not okay that makes sense yeah let me know that goes more can you hear me okay so how how does this work how do we get proteins from kind of this you know polypeptide in cytoplasm how do we get them folded and to the right place right how do we do that and that is a process called protein sorting so all proteins begin translation in the cytoplasm Okay so what you need wait what wait what do you mean now some some proteins are going to stay in the cytoplasm those are the easiest ones and some are going to go through the secretory system how do you know it's wrong what okay so how do you how can you tell the difference how do you tell the difference between Ming these two directions okay there is a special sequence added to a peptide that has to secretory system so it's called sequence oh it's because I have my notes going on in the background I'm recording okay so those proteins have a signal sequence these guys have no signal sequence just take another one tomorrow I see the faint line okay so that's the first difference if you don't have a signal sequence you're yeah I know that and that cytoplasm into it threedimensional shape if it needs to be multiunit it will join with other uh tertiary structures and form a quinary three-dimensional shape and then it will do its job so pretty simple hello right if you have this terminal sequence here the signal sequence here you're going to go to you can go to the chloroplast but we're going to really track to the mitochondria we're animal cells okay there are some that have an internal signal sequence okay that you see here in C if you took two I think you're good now these guys are special because if a Fain line I don't think it means anything and then I don't know give it another week so the position of the signal sequence is important in where you're going to Target a protein hello the most common is that you have a signal sequence at that Amino terminal end right at the end and it's going to be targeted to the ER yes right yeah why would you tell so how would it be his so remember we talked I you said you never did it wrong membrane system in chapter two right so this is composed you did wrong with yeah that's what I was thinking we have well it's one of those two so we also have vles here you're aable person and the plasma membrane a great okay so if you remember when we talked about the ER and the GOI they're these you got tell comp so the has a of it i' tell l so the space right here is called the ER lumen yeah actually that's a good point just wait wait also luminal spaces just wait okay the gold if you remember has an added feature right it has two different faces yeah just wait it has the face no no you're good i' wait to tell him this is called the CIS just try facing the plasma membrane this is called the trans well take another one do you want to just get a plan B just in case okay so this ISS that have that at the end are going to be targeted to they're going to be targeted first to the rough plasmic reticulum right so I just wait a little bit once you have that signal sequence comes out of the polypeptide well I'm not do I'm waiting for my there's another it what happened that binds shorter than name signal that's hilarious kind of freezes so funny yeah good say to is me that's in the cell to the r endoplasmic reticulum okay and you can see here I don't know there is a receptor hopefully you don't have a kid in the R membrane it recognizes the I dropped my parle so these guys all right I'll be right can you hang up okay this you can see next picture this positions that ribosome over a protein channel it's actually a tunnel right you can see that and now what's going to happen happen is you're actually going to have this part of the peptide rele released from the signal recognition particle and it moves into this Channel and the ribosome then it then Associates with that protein Channel okay once that's set up translation starts again okay but now that peptide is being extruded into ER because it's going through that protein channel so this is the ER Lumen okay so it's going to go until you reach the stop codeon okay when it when it reaches the stop coaton you're going to have the polypeptide terminated and then it's going to be released into the ER Lumen the ribosome and everything is going to you know break apart from that channel and it start over again okay does that make sense so that's how you would get approaching to enter this whole system going through the ru R to the gold G and then finally packaged in a vesicle to either be transported secreted out of the cell or transported to another organel whatever its Target is okay does that make sense so this is just showing you this anchoring okay and the new polypeptide being synthesized and secreted into the ER Lumen the ER Lumin has a lot of enzymes and one of them actually cuts off that signal sequence okay because the signal sequence is not part of the mature protein its job is to get the the polypeptide that's being translated the whole complex to the ER it gets it there and translation starts again it's done its job so it's just really it's it's degraded okay does that make sense so we have these proteins in the ER Lumen and so let me go back here so the protein you see here that has been completely released into the ER Lumin it is it's going to go somewhere uh probably targeted to an organella or it maybe secreted out but it's not going to be embedded in a membrane okay A protein that has to be embedded in the membrane has a very has stretches of hydrophobic amino acids in it that actually going to be embedded in the membrane and they also have to enter the secretory system okay so if we have a transmembrane protein remember a transmembrane protein is a protein that goes through the entire plasma membrane it has a extracellular side and an intracellular side okay if a protein H is going to be in the membrane you can see this kind of purple here that is a stretch of hydrophobic amino acids you can't put those into the ER Lumen that would be a big flashing you know red light those actually what happens is this this channel is actually very elaborate it can open on the side and what it does is it actually moves those that hydrophobic stretch that's been translated into the membrane okay so what you end up with is a transmembrane protein right it has the hydrophobic part embedded in the hydrophobic part of the membrane it has a c terminal region facing out into the cytoplasm and it has an inter terminal region that is in the ER Lumen okay so in this case what's going to happen is as these are made you have these proteins that are sitting in the ER membrane they actually form vesicles and then they're transported out some of them might be going to the chloroplast the nucleus the mitochondria to be in those membranes or they could be headed for the plasma membrane to renew the plasma membrane okay so the plasma membrane because things are usually constantly budding off it loses a little bit all every time the vesicle buds off so you have to replenish that okay so as we said talked about in chapter three the membrane is a very Dynamic Place unlimited okay a joint Duck Unlimited see I joined Ducks Unlimited and the meetings are every proteins evolv just like everything else we have about protein families we group proteins really by their sequence and by different structural elements I love Jesus be so and we do this because if you see these common sequences that make these common you know larger structures when they're folded it gives you an idea about what the function is and so you have lots of so all the TRNA synthetases they're all different but they're all they all share a lot of similarities because they're doing the same job okay so we have lot of proteins that do the same job we kind of group them into these families and lots of people who are biochemists and biologists spend their whole lives studying you know one or two proteins within a group in my lab we study um a type of protein called E3 liases and these are the proteins that uh they target other proteins for Destruction so really important a lot of a lot of stress processes in our cell my lab studies inate immunity in plants so you know there's a lot of um study of these different proteins okay we've talked about we talked about protein folding we talked about Alpha Helix and beta pleed sheet so you can see here these are two examples uh this is this Protein that's on the left is Alpha helit and beta PL sheet and then the protein on the right is something called a beta Barrel which you probably are learning about in chemistry this is a shape that a lot of membrane channels have because you have the baa pleated sheets kind of embedded in the membrane and then there's a channel going through so you have you can have a lot of things moving in both directions actually and the loops that you see in GR that's where things can associate on both sides to be channeled back and forth okay so if we look at the number of folding domains there are a lot fewer of those right they're only about 2 two or three times um and that's you know that's just kind of how it is all right but I just joined you have one of the CL many different folding domains within the same protein a club that has meeting are very large so you have a lot of room for a lot of different the same to form and so we talked about um we haven't talked about dehydrogenases yet but we'll talk about those when production and respiration so dehydrogenases are huge proteins and they have three different enzymes inside of them each one with a totally different job and they channels that are moving things from place to place um things coming in things going out so each part of that protein has that overall protein the dehydrogen has many folding domains within it okay and we also kind of look at proteins uh these folding domains give us information find an protein we that has an unknown function you can look at the differents might haircut and's sister got me in touch with one of the guys um so a lot of evolution uh what you'll see as we kind of go on happens through mutation um and so some there's some mutation it can be large or small it could be a point mutation where only one nucleotides change it could be something really catastrophic where you have a whole piece of a chromosome that gets deleted that happens but sometimes when these things happen it's not bad it actually ends up creating something that gives that organism a survival advantage over the rest of the population and so in that case that's going to be conserved because in in most cases that individual of whatever population it is is going to going to be able to survive longer better produce more offspring so now this mutated or new version of whatever this protein is is going to be the majority of the population okay so things move forward like that um so if something is positive it has to have a survival advantage nothing is going to be conserved over evolutionary time in the genome if it doesn't provide an advantage to the organism if it's not providing an advantage that's a problem there are some things that can be neutral most of the time these are actually uh Gene duplications so if you look at our genome um over you know billions of years sometimes jeans just get copied multiple times and they're typically sitting right next to each other we don't really understand why that happens but it is a way that you can evolve The genome okay um negative if you have a negative mutation it typically takes away from a survival Advantage it causes the organism to be less fit we'll talk about populations later on and so that individual probably would not reach reproductive age or would not produce progyny so now this would just take it out of the je pool hello okay any questions about that so this chapter has two really nice um has I think four really nice um big figures looking at transcription overall RNA processing overall so this is going to be right in the nucleus so and then it also has transport out of the nucleus and translation in the cytoplasm so from I'm kind of I I'm a visual learner I learn a little better like that I like to look at pictures and uh this is a very nice tool I think you should take you know it's not for this exam but for next exam I think it'll be helpful okay all right please take advantage of Kel session tonight I will see you all on Tuesday