13_BL_20231103_WhisperAI_3.docx

So, these stages of translation each involve a number of factors, and we will slowly discuss each one of them, but I just want to use one slide to kind of summarize what they are, what they are called. So, what are translation factors? Again, don't be overwhelmed. Basically, the whole 45 minutes we'...

So, these stages of translation each involve a number of factors, and we will slowly discuss each one of them, but I just want to use one slide to kind of summarize what they are, what they are called. So, what are translation factors? Again, don't be overwhelmed. Basically, the whole 45 minutes we'll be talking about this, so you'll have a chance to understand it better. So, for ribosomes to translate the information in the messenger RNA, they cannot do it on their own. So, it's not enough to have the ribosomes that kind of control tRNA messenger RNA interactions. You have to have some energy help the process. So, for example, during the process of finding the start codon, there are factors that help the ribosome in the process. There are factors that will deliver the tRNAs to the ribosomes as the elongation stage is occurring on the messenger RNA. There are factors that will help terminate protein synthesis at the end. You will remember that three codons are stop codons, and they're not recognized by amino acids. They're recognized by proteins, and these are some of these release factors or termination factors. So, all of these factors here that I'm talking about are actually proteins. Some of them actually hydrolyze GTP, so they are in a way GTPases, but they're proteins. They are not RNA molecules. And so, in bacteria, it is relatively easy to remember most of them because they have simple names. So, it's easy to remember that the initiation stage, there are three stages, initiation, elongation, termination, and each of these three stages requires three factors. And initiation factors are referred to as initiation factor one, initiation factor two, and initiation factor three. Release factors that release the polypeptide chain once the stop codon is encountered are referred to as release factor one, release factor two, and release factor three. Again, relatively easy to remember. The elongation factors are maybe the most difficult to remember. They have their own names, but you should know them and also know, as you'll see soon, what they do. So, one of them is called elongation factor TU, another one is called elongation factor TS, and the third one is called elongation factor G. And I mentioned to you some of these are GTPases. So, for example, initiation factor two binds GTP and hydrolyzes GTP, it also binds tRNA, elongation factor TU binds GTP and tRNA, and one of these release factor also hydrolyzes GTP in the process. So, let's go carefully through each one of these three stages of translation, starting with initiation. And you will see that during initiation, you have to assemble the ribosome, such that the small subunit first binds the messenger RNA, and this is helped with factors, then the first tRNA binds, and then the large subunit joins, and then you can have an elongation. So, what's happening during this initiation stage? So, initiation is not an iterative process, so it happens once for a particular message that specifies a gene. For a particular protein to be synthesized, initiation takes place only once, at the beginning. So, it's not a cycle, it's not called initiation cycle, it's called initiation, but then when you have elongation, then we are talking about an elongation cycle, because you have to have successive addition of amino acids, one after the other. But during initiation, all you need to do is bring together small subunit, large subunit, tRNA, base paired with a start codon. So, initiation happens at a particular start codon, and it is AUG, in most cases AUG. So, what these factors here are doing is the following, and you should be able to identify where these factors would be if you were shown such a scheme without them labeled. So, what do the three factors do, initiation factors? First, to initiate you have to have small subunit, so small subunit binds to the messenger RNA, so you have small subunit, you have messenger RNA. So, the small subunit binds, then you have two factors that will literally help this small subunit stay associated with the messenger RNA, because small subunit is kind of open, so there is no large subunit to kind of grab onto the messenger RNA from both sides. So, small subunit is open so that the messenger RNA doesn't go away, two factors bind and help. So, it's like having a little factor come here that prevents the messenger RNA from floating away, and these two factors are initiation factor one, initiation factor three. And then finally, the third factor, initiation factor two, is the one that will bring specifically the tRNA. So, initiation factor two is responsible for tRNA delivery, and there is a special tRNA that is used in the process. It's a chart, it's charged with a methionine, but it is also formulated, so it's modified methionine that is attached to this initiator tRNA. So, this is not the same tRNA that is used for recognition of a codon for a methionine, this is a special initiator tRNA. That is modified, it's formulated, that's why there is this little F, formal methionine tRNA, tRNA F, or initiator tRNA. And that's it, that's pretty much everything that has to happen. So, the small subunit finds the start codon, the two factors, initiation factor one, initiation factor three, prevent the messenger RNA from dissociating. Initiation factor two, with GTP, will attach the tRNA at the right place, so that the codon and the codon interactions take place, and then the large subunit joins. When the large subunit joins, the initiation factors depart, and in the process initiation factor two, which is the GTPase, hydrolyzes GTP. So, as a product, you have GTP and phosphate, inorganic phosphate, initiation factor two departs, and you have assembled what is called 70S initiation complex. 70S because it's the bacterial ribosome that has both subunits, 70S initiation complex. So, initiation of translation involves formation of the initiation complex. 70S initiation complex is the main product of initiation. What is next? Elongation. Here, you'll see we're talking about a cycle. So, elongation of stage of protein synthesis. You can start analyzing at any point, but let me just start at an arbitrary point here, okay? It is indicated here with number one. So, starting from some point, you have to realize that for the next amino acid to be added to the growing polypeptide chain, you have to have a possibility of binding simultaneously next to each other previous tRNA that has one particular amino acid, and the next one. So, this is the reason why the ribosomes have to be able to bind at least two different subunits. Two different tRNAs, and these two binding sites are called A and P. A is the place where amino acylated tRNA binds, and P is the site where tRNA that has a growing polypeptide chain binds. Okay, so how does that work? So, starting at some point during protein synthesis, the ribosome will have tRNA associated with the growing polypeptide chain. Successively, new amino acids will be added to it. The N-terminal amino acid was added first, that one is up here, and the next one is added one after the other. This tRNA that has a growing polypeptide chain covalently attached. So, during protein synthesis, this is another very important concept. The polypeptide chain is not released until it is fully synthesized. You do not want to separate and release partially synthesized polypeptides. Proteins will only be functional if they are fully synthesized. So, they are covalently attached to the tRNA that is bound to the ribosome until the protein is fully synthesized. So, you have tRNA interacting with its anti-coron, with a coron on the messenger RNA, triplet-coron, there is a growing polypeptide chain, then elongation factor TU that uses GTP will deliver the next tRNA to the ribosome. And then, the ribosome can catalyze peptide bond formation. That means that this amino acid is connected to this one, and this tRNA that was previously connected to the growing polypeptide chain now has nothing attached to it and can be released. So, as a result of this reaction, from two adjacent tRNAs, one with a single amino acid, one with a growing polypeptide, you end up with one tRNA that has a polypeptide chain that has been extended by one amino acid and the other tRNA that doesn't have anything attached to it. Then, elongation factor G translocates. So, this is the factor that hydrolyzes GTP and moves the ribosome one coron further on the messenger RNA. This is what's happening here. And now, the last site of the ribosome is introduced, the exit site. After this translocation, after the ribosome moves one step further, then this tRNA that has nothing attached to it is found in the so-called exit site. So, this is a short occupancy site that is occupied by this tRNA that is deaccelerated and then this tRNA dissociates and then the next tRNA can bind to the A site and the next amino acid is incorporated and this is what is referred to as the elongation cycle of protein synthesis. So, just to give you a sense of how this works in three dimensions rather than in this schematic, I would like to show you this. So, basically, it's the same concept. You have the large ribosomal subunit and the small one in front and they're positioned in the, what I already showed you before, this crown view of the ribosomes where these protuberances are sticking up. So, the messenger RNA will bind between the head and the body of the small subunit which is now here in front and it's made just as an outline in green. So, this is where the messenger RNA is. The two tRNAs, they are L-shaped molecules with the anticotons. They will point towards the messenger RNA on the small subunit. The small subunit is in front and the amino acids that are supposed to be connected, they will be here at the opposite end of the L-shaped tRNA right here in the active site and this is the active site. This active site is referred to as the peptidyl transferase center of the ribosome. The two amino acids will be connected. The polypeptide chain will be transferred to the A site bound tRNA, the red one. Then this elongation factor G will move the two tRNAs. Then the elongation factor G will hydrolyze GTP. It'll dissociate and then this ribosome is ready to accept the next tRNA in the A site which will be delivered by another GTPase called elongation factor TU. This one will bring it in. GTP will be hydrolyzed. The orange tRNA will be positioned just like the red one was before. The E site tRNA blue one will dissociate. Next polypeptide bond peptide bond will be synthesized and we keep going. This is a summary of the elongation cycle of protein synthesis. Now I would like to focus on this step here. What is happening in the active site of the ribosome in this catalytic center called peptidyl transferase center of the ribosome? Well, obviously there is a reaction that takes place which is referred to as the synthesis of the peptide bond. This reaction is one of the most fundamental reactions in biology and you should know and understand and be able to draw structurally components of this reaction. In a way it's a simple reaction and you can draw on your knowledge about how nucleotides look like and how amino acids look like and how amino acids are connected to the ribose to understand how this reaction takes place. So I told you that for this reaction for peptidyl transferase reaction to take place you have to have two tRNAs next to each other and then you know that the reaction takes place such that the polypeptide chain is transferred onto the next tRNA with a single amino acid. So how does that work? Well when the two tRNAs, so these are long tRNAs here, are next to each other then the one that has a single amino acid. This one is the P site that means peptidyl tRNA has potentially it can have a long polypeptide chain attached to it. So when this tRNA delivers the next amino acid there is a nucleophilic attack between the nitrogen atom of the amino group of the incoming tRNA, amino isolated tRNA onto the carbonyl carbon of the peptidyl tRNA and as a result you have this nucleophilic attack. As a result this bond is broken and there is a new covalent bond between the nitrogen and the carbon and so that's the end result of this reaction. So you have as a result of this reaction deacylated tRNA in the P site. There is nothing on this OH groups of the ribose on the three prime end of the tRNA. However this tRNA now has one extra amino acid added to the growing polypeptide chain. In the next cycle the third one will be added and so forth. So this is what is referred to as the peptidyl transferase reaction. And now of course finally the last stage of protein synthesis is termination. What happens during termination? During termination you have to encounter a special codon on the messenger RNA. So basically the ribosome that is translating message keeps encountering codons that can be recognized by tRNAs and tRNAs will deliver the next amino acid. But then suddenly when there is a codon present, one out of three, any one of these three, that cannot be recognized by any tRNA then suddenly you have to have, this indicates a stop and this codon then has to be recognized rather than by tRNA with a protein. So it's a protein that will recognize the codon and the protein is here shown in red. That's a release factor one. So release factors one and two will each recognize a pair of codons together. One of them, each one of them will recognize one unique codon and one that is the same between the two. So together they'll recognize three different codons. So this two binding side on the ribosome, this A side binding side of the ribosome called A will suddenly be empty. There'll be no tRNA and in that case a protein that in a way behaves a little bit like a tRNA, it recognizes the stop codon. So the protein has features that will recognize the nucleotides in the stop codon and that will recognize the nucleotides in the stop codon and then this protein will cause instead of addition of the next amino acid, which would happen with this nucleophilic attack of the amino group of the next amino acid to the carbonyl carbon of the growing polypeptide chain, here instead of an amino acid performing a nucleophilic attack, this release factor will make a water molecule hydrolyze away this polypeptide chain. So you have a released polypeptide chain, that's why these factors are called release factors and the ribosome will then disassemble. So this is the termination stage of protein synthesis. So with this I think we have covered some of the most complex topics in terms of the composition of machinery involved in both initiation, elongation and termination of protein synthesis and I would like to give you a bit more of a full picture of how protein synthesis takes place. So the ribosomes not only have several binding sites for tRNAs and they are going to continue synthesizing the protein. Ribosomes can also just like RNA polymerase transcribing a single region of DNA simultaneously with other RNA polymerases and they're just temporary like sequence-wise offset different RNA polymerases. In terms of protein synthesis the same thing can happen. So you can have one ribosome bind and find the start codon on the messenger RNA and keep translating and this one has already translated most of this messenger RNA, most of this protein is synthesized. But the next one that comes will just start and then it has a short polypeptide chain when it moves further it gets longer. So you can have simultaneously bound multiple ribosomes translating a single messenger RNA. What are those tandem or multiple ribosomes on the same messenger RNA called? They're called polysomes. So ribosome is a single one and poly means multiple ribosomes, polysomes bound to the single messenger RNA. How does that look? Well exactly like this. So you have a RNA polymerase that will read off the message on the DNA. RNA molecules will be synthesized. These are RNA molecules and these little dots, well not little, they're big compared to RNA polymerase, these ribosomes will read the message one after the other. So basically as the new message is synthesized and that is interesting because the RNA will be transcribed from 5 prime to 3 prime. So basically the first part of RNA that will be made is its 5 prime end which is exactly the place where the ribosomes will start translating the message. So basically this is the reason why you can have co-transcriptional translation. So basically these RNAs are getting longer and longer and as soon as they appear the messenger RNAs they can be recognized and translated by the ribosome. So this is a process of co-transcriptional translation that is happening in bacteria. In eukaryotes this is different. Okay so with this I will stop, take a break and after the break you'll see we will continue discussing some other features of translation and then I will try to put together the entire concept of central dogma to illustrate to you how you can actually use the knowledge for purposes of designing proteins and changing proteins and making proteins for certain needs.