Rachel Green (Johns Hopkins U., HHMI) 1: Protein synthesis: a high fidelity molecular event

By: IBiology

Hi. My name is Rachel Green. I'm at the Johns Hopkins University School of Medicine and in the Howard Hughes Medical Institute, and what I'm going to tell you about today is protein synthesis, a high fidelity molecular event. So, protein synthesis is also known as translation and, as many of you are familiar with, this is the final step in the central dogma. The central dogma, of course, is the process by which genetic information is transformed into the material - the proteins in the cell - that perform most of the functions of the cell. The genetic information is found in the form of DNA, that double-stranded molecule built of nucleotide building blocks, and that double-stranded DNA molecule has to be transcribed into a different form that we refer to as RNA, built of similar building blocks, called nucleotides. The RNA ultimately then needs to be translated into a different form, really a very, very different polymer, known as protein, and protein is composed of amino acids.

So, we see that the process of translation really refers to the fact that nucleic acid, which is the genetic information, is built of nucleotide building blocks known as A, C, G, and T in DNA, or A, C, G, and U in RNA, and this information has to be transformed, or translated, into the content of amino acids, or a polymer of amino acids that we refer to as proteins. And we see here that the DNA, in fact, is this rather linear molecule with two strands of nucleic acid, and this information encodes the great diversity of proteins found in all cells. So, we can think about translation as really a processive march along a linear template.

The template provides the information in the form of nucleotides, and along this linear template we're going to have adapter molecules that recognize the information in that linear template, and these adapter molecules are going to be attached to the amino acid building blocks that are actually going to be iteratively put together to make the peptide, or the protein. We see, here, the protein chain getting longer and longer and, ultimately, we reach the end of the protein's coding region, and we have a full-length protein that's been made. So, this is an iterative, linear march of an adapter molecule along a template, and I think that's the best way to begin thinking about what translation is, because this must be how translation began in the beginning.

We must have had simple adapter molecules that took the amino acid building blocks, which must have been chemically activated in some way, and marched along and ligated them together sequentially. And around this simple process, presumably in a primordial world, grew the process of translation, which is now a very sophisticated and highly regulated event performed by the ribosome, which is a machine that's stunning, sort of in its simplicity, in the fact that this is the simple event that it catalyzes, and stunning in the complexity with which... the way in which it catalyzes those events. So, what I'm going to do today is focus on two general areas. We're going to start by outlining the different players that are involved in the process, including the messenger RNAs, the tRNAs, the proteins, the ribosomes... all the players. And then we're going to talk sequentially about the process of translation, which really we can break down into four basic steps: the process of initiation, which you'll see is going to be called finding the AUG, or the start site; elongation, which is the process of iteratively adding an amino acid to the growing chain, the growing protein chain, and we're going to talk about elements of speed and accuracy and how they're relevant; then we'll get to the termination step, which is when, at the end of a coding region, the ribosome and the machinery has to stop protein synthesis; and finally, the protein synthetic machinery has to be recycled in order to begin again, and we'll take a walk through each of these steps together. So, the players..

Rachel Green (Johns Hopkins U., HHMI) 1: Protein synthesis: a high fidelity molecular event

what are we gonna focus on first? The players. We're going to start talking about the genetic code. We're going to talk about the adapters, which are known as aminoacyl-tRNAs.

We're going to talk about the messenger RNA template, which is the copy of the double-stranded DNA that encodes the genetic information. And we're going to talk about those both in the bacterial domain of life and the eukaryotic domain of life. That's where we've learned the most and there are some significant differences that we'd like to discuss. Then we're going to talk about the catalyst of this event, the ribosome, and finally about some of the many factors that are involved in facilitating this event. So, to start with the genetic code... the genetic code really is at the heart of translation. It's the set of rules that tells us how we get from one alphabet to another - how we get from nucleotides to amino acids. So, as I mentioned, there are 20 different amino acids, and there are four different building blocks, and so any code that would comprehensively cover those complementary amino acids probably needs to be bigger than a one-letter code or a two-letter code.

In fact, three-letter code can cover the 20 amino acid complexity, and the three-letter code, if you take 4 and raise it to the 3rd power, there are 64 different letter groups that could specify the 20 different amino acids, and it turns out that's what nature has settled on as the code... is three nucleotide letters correspond to a single amino acid. So, that means there are 64 codons that could cover, easily, the 20 amino acids, and we can see that if we look at the genetic code, the rules are outlined here for us. We see that at the first position of the codon, we can have a U, C, A, or G, at the second position, we can have a U, C, A, or G, and at the third position we can have a U, C, A, or G. These are the nucleotide letters in the RNA.

Each of those three-letter codes corresponds to a single amino acid, and so we can see, for example, that, if we look here at the top left corner, that a UUU corresponds to a phenylalanine amino acid. So, built into the code also has to be how you begin protein synthesis and how you end protein synthesis, and we see that protein synthesis, or we've learned that protein synthesis begins with a methionine. The triplet codon that specifies methionine is AUG; we see it here on the left side of the codon table.

So, it turns out, in every open reading frame or every element that specifies the production of a given protein, typically begins with a methionine residue, an AUG. We see that the code also specifies stops, and we look in the upper-right corner of the codon table and we see there are three stops: UAA, UGA, and UAG. And so when a ribosome reads along to the end of a coding region, it waits for a stop and that's how it knows to stop making proteins.

We also see that some amino acids are specified by just one codon. For example, tryptophan has just one codon, this UGG in the middle of the stop codon box, and in fact there's one methionine codon, which is AUG. Whereas other codons, for example, leucine, have as many as six codons that specify their identity.

So, this is what we mean by a redundant code: a number of amino acids are specified by more than one codon. The other feature you notice is that all of the codons specify something, and that's what we mean by non-ambiguous. No matter what codon you reach, no matter what combination of three nucleotides you reach, they'll always specify an amino acid. So, those are some of the key features of the code. Another feature of the code that's worth knowing is that the code is built, or has been assembled, in a way that's conservative, such that if there's a mutation that happens in your coding region, it ends up typically having a relatively minor consequence, and we can see that here. If we compare lysine and arginine, which are two amino acids that are positively charged, a single nucleotide substitution might take you from a lysine to an arginine, and that's a fairly conservative substitution. Similarly, we can look here in the bottom and we see aspartate and glutamate. These are two negatively charged amino acids, where single nucleotide substitution has a relatively modest consequence.

So, these are nice features that are built into the codon table, or the genetic code. So, the next component that we're going to talk about is tRNA. This is the adaptor molecule that actually interprets the genetic information and is going to bring along with it an amino acid, so really the tRNA is at the heart of protein synthesis. This is the molecule that takes one alphabet and puts it into another alphabet.

So, the idea of an adaptor was actually postulated as early as the 1950s by Francis Crick, who realized there had to be such a molecule that would interpret the genetic information and carry an amino acid, and some number of years after that, not very many years, Hoagland and Zamecnik actually found an activity, an RNA activity in extracts, that actually could become attached to radioactively-labeled amino acids, and that those radioactively-labeled amino acids ended up in the protein fractions of the cells. They also found that this was GTP- and ATP-dependent process, and this all makes sense in the context of how we understand translation now. So, ultimately, this RNA activity was identified as a tRNA, and the basic 2-dimensional and 3-dimensional structure of that molecule is shown here. On the left we see the typical 2-dimensional structure of a tRNA. We refer to it as a cloverleaf. It's composed of four stems and three different loops, and these elements bring about all the interesting properties of a tRNA. We see at the bottom of the tRNA there's this element called the anticodon loop, and it's on the anticodon stem, and what this portion of the molecule does is it has a three nucleotide motif that we refer to as the anticodon that's going to interact with the codon that's going to be in the messenger RNA in an antiparallel fashion. The machinery is gonna read 5' to 3' and the anticodon is going to position itself in the opposite direction to read the code by forming Watson and Crick pairing interactions between the codon and the anticodon.

The other end of the molecule that's probably most interesting is this acceptor end of the tRNA. We call it the acceptor end. There's always a CCA - 3' terminal CCA tail, and it's on that 3' CCA tail that the amino acid is placed. There are two other stem-loop structures, the T-loop and the D-loop. They're referred to as the T-loop and the D-loop because they contain interesting posttranscriptionally modified nucleotides that have given these loops their names, but the most important thing to know about these loops is that they help, in three dimensions, bring the molecule together.

So, we see that a tRNA, in three dimensions, looks more like an L-shaped structure rather than a cloverleaf, and we see that the pink loop and the orange loop come together - the D-loop and the T-loop - they form interesting 3-dimensional interactions that stabilize the elbow and give it the dynamic properties that a tRNA has to have. We see the acceptor loop out here, where the amino acid is going to come in and is going to be brought into the ribosome, and then there's the anticodon stem that's going to read the codon in the messenger RNA template. So, an additional feature that needs to be discussed about how the genetic code is interpreted is, how many tRNAs are there typically in a cell in order to recognize these 64 codons that specify what's in a protein? As I mentioned, three of them are stop codons that are not recognized by tRNAs, but that leaves 61 codons. And what we've found is that there are never as many as 61 tRNAs in any given cell. There tend to be 30-40 tRNAs in a cell.

So, how does that work? That means certain tRNAs must recognize more than one codon, and we refer to an event known as wobble to explain how one tRNA can recognize multiple codons. So, as an example, here are two phenylalanine codons: UUC is a phenylalanine codon, and so in UUU. And what we know is that there is one tRNA that in fact recognizes both of these phenylalanine codons. The anticodon of this tRNA has an AAG, or GAA in the 5' to 3' direction, and we see that, in one case - on the UUC codon for phenylalanine - there are perfect Watson-Crick pairing interactions at all three positions. However, in the case where the same tRNA is recognizing the UUU codon, the third position is mismatched, and we refer to that as wobble. Normal Watson-Crick base pairs... we think of them like this, as forming a very specific set of interactions that makes the nucleotides face one another like this, and the wobble interaction just bumps out a little bit... there are still several hydrogen bonds, it's still a stable interaction, but not as stable.

And it turns out that the protein synthesis machinery permits this sort of wobble interaction, only at the third position, and this explains how 30-40 tRNAs can in fact cover the 61 codons in the genetic code. So, the next thing I want to tell you about is how is it that this key step happens, which is that the amino acids, the 20 amino acids, get coupled with these 30-40 tRNAs that I just described. And at the heart of that process is an enzyme known as the aminoacyl tRNA synthetase and, truthfully, this machine, or this enzyme, is a huge part of the magic of translation, because this is the enzyme that realizes or understands which amino acid goes on which tRNA, and that is the translation of the genetic code. So, it turns out there are about... there are exactly 20 aminoacyl-tRNA synthetases in most cells. There's one for each amino acid in the cell. And the fidelity of the process of aminoacylation is extremely high: they make a mistake about 1 in 10^5 times.

So, that's an incredible level of fidelity. Almost always, the right amino acid gets attached to the right tRNA. And the process that the enzyme uses is something known as proofreading. I'm going to give you a little bit of an understanding of that here. So, the enzyme is shown in blue, here, and it's bound to the tRNA shown in grey, and we see that it sort of recognizes the whole face of the tRNA.

What we've also done in this enzyme, or in this molecular view of the aminoacyl-tRNA synthetase, is we've outlined two different sites that are critically important for the function of this enzyme. The first one is the aminoacylation site, and that's in fact the site that's responsible for taking an amino acid from solution that it recognizes, activating it with an ATP molecule by forming a chemical linkage, and then ultimately transferring that activated amino acid onto the end of the specific tRNAs that it recognizes. And that process is outlined here, where we see the square amino acid, here, the orange amino acid, gets attached to this tRNA, ultimately, with the help of ATP, because the amino acid binds to the synthetase, is activated with ATP, and is transferred onto the tRNA. So, that's the key step in aminoacylation. The other step to understand, however, is that there's this second site, here, which we refer to as the editing site.

And the editing site is a site on the enzyme that has the opportunity to correct wrong linkages, because occasionally the enzyme does activate the wrong amino acid, and it puts it on the wrong tRNA, or maybe the right tRNA, and what this editing site is responsible for is removing an amino acid that might be incorrectly aminoacylated onto the tRNA, and that's how this process achieves such a high level of fidelity. And that's really at the heart of protein synthesis. So, now we have activated aminoacyl-tRNAs. They're ready for action. They're ready to do protein synthesis. Well, let me tell you a little bit about the messenger RNAs.

These are the elements that are made from the double-stranded DNA in the bacterial and eukaryotic cell, and I'm not going to tell you about the processing steps that result in their final outcome. What I'm gonna tell you is what they look like when they're ready for protein synthesis. If we look on the left we see the elements of a bacterial messenger RNA, and we several things that are of interest.

First, we notice that there appear to be multiple coding regions on the bacterial messenger RNA. We refer to these are cistrons and we refer to bacterial messenger RNAs as typically polycistronic, meaning they typically encode for more than one protein, and so we see, for this particular messenger RNA, we're going to make protein A, B, and C. We see that each of those elements that encode a protein - we refer to that as an open reading frame - has an AUG associated with it, which is the start site, they have a stop codon associated with it, which is the ending site, and they have this interesting element upstream, which is referred to as Shine-Dalgarno region. And you see that each specific open reading frame has its own Shine-Dalgarno region.

Now, the Shine-Dalgarno region is simply a polypurine track that's found in the messenger RNA that's going to help facilitate the identification of appropriate start sites in bacterial messenger RNAs, and we'll describe that in a number of slides. We see that the eukaryotic messenger RNA is quite different. We see, first of all, that the messenger RNA we're showing here encodes a single protein, and we refer to this as monocistronic and this is typical of eukaryotic cells - most messenger RNA is expressed as a single open reading frame.

We see that the eukaryotic messenger RNAs, of course, have a start codon and a stop codon as well, but they have two key features that are quite different from what's seen in bacterial messenger RNAs. We see at the 5' end that the eukaryotic messenger RNA has what we refer to as a cap. It's an unusual nucleotide attached to the messenger RNA through an unusual 5'-5' linkage. And it is specifically recognized by key elements in the eukaryotic translation machinery. The eukaryotic messenger RNA also has an unusual feature at its 3' end that is referred to as the polyA tail. It can be anywhere from 20 or 50 nucleotides in length, to 100s of nucleotides in length, and it's particular to the messenger RNA, and to the cell cycle, and to the state of required gene expression. And those are key features that are going to be evaluated by the protein synthesis machinery to decide how efficiently to make these proteins. So, there are the messenger RNA building blocks, and finally we get to the ribosome.

The ribosome is the enzyme that catalyzes protein synthesis, and we're going to talk about a number of its interesting features. First of all, we show, here, the ribosome bound to a messenger RNA, and we see that the tRNA - the adaptor molecule - is extending from the messenger RNA up to the polypeptide chain. In fact, all ribosomes are composed of two subunits. There's one subunit that's principally responsible for interpreting the genetic code. It's where the mRNA and tRNA pairing interaction takes place. We refer to that as the small subunit. We see that there's a second subunit which is referred to as the large subunit, and that's where peptide bond formation takes place, and that's where the exit channel exists, where the polypeptide emerges out the backside of the ribosome.

So, all ribosomes from all organisms have two subunits. The other key feature to understand about a ribosome is that it's principally composed of RNA. Two-thirds of the mass of a ribosome is RNA and only one-third of the mass is proteins. There's a number of proteins, but generally a large RNA in the large subunit and a large RNA in the small subunit. And the RNA really is at the heart of the ribosome, the biosynthetic machinery for protein synthesis. You can see, however, that this looks a lot like that earlier slide that I showed, where translation is simply the iterative process of a linear march along a messenger RNA template. It's just that the ribosome is going to facilitate that march.

The other thing I'd like to point out is that the ribosome typically has three tRNA binding sites, and that allows for all of the different events that need to take place during protein synthesis. This tRNA here is bound in what we call the P site, the peptidyl site. It's where the growing polypeptide chain exists.

New tRNAs are going to come into this site of the ribosome. It's called the A site, for the aminoacyl site. And tRNAs leaving the ribosome will actually bind in this third site, here, known as the E site, the exit site. We can see this in the context of a crystal structure of the ribosome that is just actually focusing here on the RNA.

What we see on the top is the large subunit of the ribosome and on the bottom is the small subunit of the ribosome. We see tRNAs bound in their respective sites - the E site, the P site, and the A site. And you can see that the RNA component, which is very easy to see here, really forms the shape of the ribosome and facilitates interactions with the tRNAs in these three binding sites. I'm going to show another view on the next... this is another view of the ribosome - this is another crystal structure that came out - and it shows you, really, a picture of the ribosome from the side. So, now the tRNAs are bridging across the interface region, here, from the peptidyl transferase center, here, where amino acids are ligated together, to the decoding center where the genetic information is interpreted.

And I think this is a nice view of the ribosome because it makes it clear the extent to which translation has to be this incredibly coordinated event where the tRNAs and the messenger RNAs are being passaged through this tight interface, and that there's going to be a lot of movement and rearrangement that's going to need to facilitate these events. And that's what we're going to talk about next. So, I want to point out here that in the evolution of life, as we've gone from, likely, more simple organisms like bacteria to more complex organisms like ourselves, the ribosome has similarly gotten more complex and this gives you a feeling for this, here. At the top is the bacterial ribosome, which is really what most of the initial structures that were solved in the field were of, and so that grey outline is sort of what the bacterial ribosome looks like - the large and the small subunit. The next one that we see here is the archeal ribosome, which in fact you can see has some additional complexity. In fact, there's some additional proteins that came about in the archeal domain of life and add some mass and complexity to the ribosome. We then look down here - this is the structure of a yeast ribosome, and you can see that there's really even more complexity than there was in the archeal kingdom, or the archeal domain, and you can see that there's addition of protein and RNA components that make the ribosome larger and likely more able to do complex reactions or complex regulation. And finally, a higher eukaryote is shown here at the bottom.

And what we see is, in addition to the protein/RNA layer that's been added in the lower eukaryotes, we have an additional layer of RNA elements that extend from the surface of the ribosome. They kind of look crazy, and it's not clear what they do, but they likely are going to be recognized by various elements in the cell that help to impose more complex regulation. So, that's kind of an exciting way to think about ribosome function. So, now we're going to move on to the basic steps of translation and how all of these components that I've just described are going to come together to bring about the act of translating a specific protein.

As I mentioned in the beginning, we're going to talk first about initiation, and that's really about the process by which the ribosome is going to find the start codon - the AUG start codon - and it's going to happen, actually, through the actions simply of the small subunit of the ribosome. It's going to find the AUG and the large subunit is going to join, and we'll talk about those details in bacteria and eukaryotes, because they're quite different. N ext, we'll talk about the elongation cycle, which is the iterative process of adding amino acids to the growing polypeptide chain, and that can be broken down into a number of steps. Then we'll talk about termination, which is the recognition of a stop codon by the machinery specifically responsible for stop codon recognition. And finally, we'll talk about ribosome recycling, which is the process by which the entire complex is broken apart to allow for the next round of translation.

So, I want to mention, before we get going, that in addition to the ribosome, the messenger RNA, the tRNAs, and the genetic code, there's one other set of things that's quite critically important in modern-day biology, and that's a number of translation factors. These are proteins that function in the process. They make protein synthesis faster, they make it more efficient, they make it higher fidelity..

these are key components in every round of protein synthesis. And what I'm just going to point out in this first view is that there are some core initiation factors, both in eukaryotes and in bacteria. You see that in bacteria we refer to the initiation factors just as IF1, IF2, and IF3, and in eukaryotes we refer to them as eIF1A, eIF5B, and eIF1. So, these are essentially equivalent factors in bacteria and eukaryotes that perform equivalent functions that I'll refer to as core functions, and we'll describe those.

You can see also that there are a number of eukaryotic-specific factors that are thought to be responsible for part of the complexity of higher-order gene regulation in eukaryotic systems, and we'll talk about a number of these factors and how they differently participate in specifying the details of translation initiation. These core factors are fundamentally responsible for helping get that tRNA - the initiator tRNA - bound to that AUG and translation started, and that first step of subunit joining, whereas these other eukaryotic-specific factors are more focused on the cap and the polyA tail and their different mechanism of finding the AUG start site. We'll also focus on some factors that are involved in elongation and termination and recycling, and you can see again that there are names that correspond to the bacterial factors, on the left, and the eukaryotic factors, on the right. And what I'll you in advance is that elongation is an extremely well-conserved process, where the same three factors really function in both systems. I'll tell you about termination, which it turns out is mechanistically reasonably similar, but the factors that have evolved are really quite distinct from one another, and they've discovered similar ways of doing things, but different as well, and we'll talk about that.

And finally, I'll say that, in the end, recycling is really very different between bacteria and eukaryotes, and that ends up presenting some unique challenges for understanding translation in these systems. So, back to the steps of translation... we're gonna now start with initiation and follow how it is that ribosomes identify the initiating AUG in the two different systems.

As you're thinking about all these steps, however, what I want you to think about is that translation, in fact, happens in a very continuous process in the cell, and so you can imagine that you have a messenger RNA template in the cell onto which ribosomes are loading, and you recognize your AUG, and you translate along and reach the end, and the polypeptide chain is released. But the cell doesn't wait for the release of a given polypeptide chain before starting again. And so the way you should think about translation happening in the cell is in the context of what we call a polysome, which is multiple ribosomes loaded onto a single messenger RNA, like this, continually producing peptides, and we can see how this might look in a cell when we look at this EM (electron microscopy) image of translation in a cell taken a number of years ago.

We see a messenger RNA template, here, spread out along this grid, and we see the ribosomes accumulating along it, forming the polysomes as the messenger RNA is being translated. So... initiation, that's the process of finding the AUG and it's going to be a very different task in bacteria and eukaryotes. And in bacteria and eukaryotes it's going to depend on the specific features of the messenger RNA that I defined. In bacteria, we talked about the Shine-Dalgarno motif, and that's really the key to finding the AUG in bacteria. Whereas, in eukaryotes, we're going to talk about a process known as scanning, and it takes advantage of the cap and the polyA tail that are specific to the eukaryotic messenger RNAs. But the end goal in each case is the formation of an initiation complex that looks like this, with an initiator tRNA bound in the P site of the ribosome, recognizing the initiator AUG in the template. So, what do the factors..

what do the initiation factors do to help the ribosome to find the AUG? Well, I can tell you that the set of core factors in the eukaryotic and bacterial system really does about the same thing in both systems, which is that they bind to the ribosome in places where they don't want tRNAs to bind. So, we see that IF3, in bacteria, binds to the small subunit of the bacterial ribosome in this position, here. We see that another initiation factor binds to the ribosome, here, on the other side. And the place where we want the initiator tRNA to bind and find its AUG is in between those two positions where the proteins are effectively blocking the interaction of the tRNA from binding to the wrong tRNA binding site.

And we that schematically here in cartoon form. So, that's the role of these two simple initiation factors, here, IF1 and IF3, in bacteria. So, there it is.

There's our bacterial messenger RNA. How do we find that Shine-Dalgarno? We have two initiation factors that seem to block the wrong sites on the ribosome, but how do we get the tRNA to bind to the AUG? And at the heart of that process is this Shine-Dalgarno motif that's in front of every open reading frame in bacteria. The way the Shine-Dalgarno motif works is it's a polypurine track that's found just upstream of the AUG start site, and it turns out that the bacterial ribosome, the small subunit of the ribosome, actually has something that we call the anti-Shine-Dalgarno motif in it. I mentioned that the small subunit of the ribosome, and the large subunit, are built principally of ribosomal RNA, and there's a motif within that ribosomal RNA that actually is directly complementary to the Shine-Dalgarno motif, and it actually binds directly to it and effectively tethers the messenger RNA to the small subunit of the ribosome, and in doing so the AUG start site is then effectively positioned in the P site of the ribosome where the tRNA can easily find it. And that is the magic of how bacterial ribosomes identify the appropriate AUGs in strings of messenger RNA. That's how they find the frame... they find the right one to get started on, is they use the Shine-Dalgarno to directly tether to an anti-Shine-Dalgarno motif in the ribosome. So, in eukaryotes, the process of finding an AUG is quite different because we don't have Shine-Dalgarno motifs, and what we've observed..

what researchers have observed as they were studying eukaryotic initiation was that in most messages - 95% of messenger RNAs - the first AUG encountered from the 5' end of the message is the one that's utilized to start an open reading frame. And so that led them to think about the idea of scanning, and in fact the scanning model is the predominant way that we understand how eukaryotic takes place, which is that somehow the ribosome starts at the 5' end of a messenger RNA and scans until it finds the first AUG, and when it finds that AUG, that's where it starts translating. This scanning takes place in the context of many of those initiation factors that were outlined several slides ago.

We see that certain initiation factors bind to the cap. Other initiation factors bind to the polyA tail. They form a circular complex; we think the messenger RNA tends to exist like this in the cytoplasm of eukaryotic cells. What happens next is a ribosome complex, that's actually bound to those factors that look like the bacterial factors IF1 and IF3, bind to the initiator tRNA in the context of an initiation factor known as eIF2, and it scans along this circularized messenger RNA until it hits the AUG..

boom, AUG recognition takes place, GTP hydrolysis takes place on eIF2, this key initiation factor, and now the complex is ready to go. It's found the AUG and it's ready to go. The next step in bacteria and eukaryotes is quite similar, which is that we need subunit joining. We need the large subunit to come in. I n bacteria, that's facilitated by this factor known as IF2, and IF2 is a GTPase that helps to facilitate this subunit joining step where the small subunit is joined by the large subunit and all of the initiation factors leave. Similar events take place in the eukaryotic system, where, once that AUG is recognized, eIF5B, which is a homologue of IF2, facilitates the same reaction where the large subunit joins and the many, many initiation factors leave. So, that's subunit joining.

That's AUG recognition. The ribosome is ready to go. Next, what we're going to do is talk about elongation. Elongation is the process by which each amino acid is added, and that can be broken down into three nice steps. The first step is the selection of the appropriate tRNA, the appropriate aminoacyl-tRNA.

The next is peptide bond formation, where the amino acids are ligated to one another. And then there's a step we refer to as translocation, where the whole complex needs to be moved along the messenger RNA template to open up the A site for the next incoming aminoacyl-tRNA. This is a highly accurate process and we'll talk about a number of steps where accuracy is imposed, and it's a relatively fast process, and these different requirements always need to be balanced. If you go too fast, you'll make more mistakes, and vice versa. So, at the heart of the elongation reaction is a protein known as EFTu. The 'u' referred to unstable in the earliest days of molecular biology, and it's probably the most abundant protein in biology. 5% of the mass of bacterial proteins is this particular protein, and its job..

the protein here is in blue... its job is to bind to the aminoacylated tRNA as soon as it comes off the synthetase. It protects the labile bond; this activated amino acid... it's a labile bond and this protein protects it. And this protein helps to load tRNAs rapidly, and with high fidelity, into the active site of the ribosome, or into the decoding center. That process is outlined here, and I'll just mention a few features of how that process is so accurate. The first thing to know is that EFTu - this protein that loads the tRNA into the ribosome - is a GTPase, and it uses the energy of GTP hydrolysis to help evaluate the quality of the codon-anticodon interaction that we discussed in the beginning.

You can see here... here's the ribosome. EFTu is going to load the aminoacyl-tRNA into the ribosome. You can see that there's an equilibrium that's going to take place here, where the ribosome gets to decide how well that tRNA that's being loaded in interacts with the codon that's presented in the A site, in the decoding center.

And if it's a nice tight interaction, that tRNA is going to sit longer and it's going to be more likely to go through the next step, which is a GTP hydrolysis step by EFTu. If it's a poor binder it'll fall off more readily and it'll be rejected before it's even past that GTP hydrolysis step. We also know that after the GTP hydrolysis step EFTu goes away and there's another point at which the ribosome evaluates, again, how well that tRNA binds and how well it is rejected..

or if it's going to be rejected. And that's again based on the codon-anticodon pairing. So, there are two opportunities at which the ribosome can reject the wrong tRNA, and those events are in fact facilitated by this protein EFTu. We also know that there's a very magical event that takes place, which is that when the ribosome recognizes the correct tRNA, conformational changes take place within the ribosome that accelerate some of the forward rate constants in this scheme - the green arrows - making these events go more quickly when the right tRNA is bound.

And we get a feeling for how this happens by looking at some of the details of the crystal structures. In particular, these structures came out of Venki Ramakrishnan's lab, and they tell us something important about how the ribosome recognizes that codon-anticodon interaction. What we see on the left are some universally conserved nucleotides that are in the ribosomal RNA in the small subunit in the decoding center, where the codon-anticodon interaction is being interpreted. And we see, for example, these adenosines here are in a configuration that actually places them within another helix, and that's where they are normally, in the absence of a tRNA ligand. But in the presence of a messenger RNA and a cognate tRNA, a tRNA that matches that messenger RNA, we see that these adenosines in fact come into a completely different conformation, and that movement of these adenosines is critical to the ribosome knowing that this is a good interaction, this is a good tRNA, and one they want to keep. So, these are the sorts of things we've come to understand from using crystallography and biochemistry to decipher ribosomal events or events in translation. And this is one beautiful example of universally conserved nucleotides playing an essential role in the function of the ribosome. This is an RNA machine.

So, next I'm going to talk a little bit about peptide bond formation. This is really the chemical reaction that joins two amino acids together, and arguably it's one of the more simple reactions that the ribosome catalyzes. What we see here is a basic outline of this event where the pink ball becomes attached to the blue ball and in fact the polypeptide chain is transferred to the pink tRNA, and that's how peptide bond formation takes place. It takes place in an RNA-rich active site in the large subunit of the ribosome that I'll show you in the next slide.

And we also show here that the tRNAs move into a funny position after peptide bond formation and that funny position is going to be resolved by subsequent factors - the translocating factors - that come in and take care of this. But this is a very simplified view of that intermediate state. As I mentioned, this catalysis takes place in an RNA-rich active site, and that's emphasized by this view, here, of the ribosome. This is a crystal structure that came originally out of Tom Steitz's lab. The blue protein components are shown and they're scattered sort of around the exterior of the ribosome.

This is the interface side of the ribosome that looks at the small subunit, and we see the active site here. There's an analog that's found in the active site right here where this red molecule is found - it's a transition state analog - and you see that the active stie for peptide bond formation is formed exclusively, really, of RNA components, which helps us to understand how this machine evolved in an RNA world, in a primordial world, and that the proteins were later adaptations. This truly is an RNA enzyme and that's where catalysis takes place.

How does catalysis take place? Well, it's a pretty simple reaction. It's the attack of a nucleophile on an electron-deficient bond. This is the peptidyl-tRNA, here.

There's that terminal adenosine, the CCA end of the tRNA. This is the growing polypeptide chain. This is the aminoacyl-tRNA that's been loaded. Again, the adenosine at the end of the tRNA, the amino acid that's charged on it, and that’s the attack of the nucleophile on the electron-deficient center.

This is simple chemistry, and we think the main thing that the ribosome does to facilitate this chemistry is it brings these two components together. It brings them together with universally conserved elements. There's an element in the large subunit called the A loop and one called the P loop, and both of these elements use Watson-Crick pairing interactions with the CC of that CCA end, and here with the C of the CCA end, to bring these two substrates together to perform a relatively simple chemical reaction, a nucleophilic displacement. So, again, universally conserved nucleotides in the ribosomal RNA, in the heart of this RNA machine, are facilitating the chemistry of protein synthesis. So, now we've formed a peptide bond. The next step is translocation. This is the process by which the mRNA:tRNA complex is moved within the context of the ribosome to open up the aminoacyl site, here, on the right, for the next incoming aminoacyl tRNA and the next round of elongation.

This is a process facilitated by a protein enzyme known as EFG. It's a GTPase and it's going to couple that energy of GTP hydrolysis to movement of the complex. You can imagine that a nice way to promote this reaction might be to insert something into the A site of the ribosome, and it turns out that's how we think translocation really does take place.

When crystal structures were solved of EFG, and they were compared to crystal structures of EFTu - the tRNA loader - we see that there's a protein domain on EFG that seems to mimic, really, in shape and sort of structure, a tRNA bound to EFTu. And so, from that look at the crystal structure, it became clear that that might be a good way to facilitate translocation. And we know that's the case if we look actually look at cryo-EM structures of EFG bound to the ribosome. We see here a cryo-EM structure of the large subunit, on top, the small subunit on the bottom. This is the E site tRNA, the P site tRNA, and this is where the A site tRNA would be, and what we see is domain IV of EFG, that looked like the tRNA bound to EFTu, binds in the A site of the ribosome and likely functions much like a pawl in a motor to promote the forward movement of the mRNA:tRNA complex, and just biases forward movement by binding in that site and displacing the tRNA that was there before.

And that, sort of in a nutshell, is how we think translocation must take place. Okay, now for the final steps: termination - we need to recognize stop codons. We've started at an AUG, we've translated all the way through an open reading frame, and now the ribosome is looking for a stop codon to know when to stop. And, as we mentioned in the first slides, the genetic code specifies three different stop codons. Stop codons are not recognized by tRNAs, but instead by protein factors known an termination factors.

But we can think of them much like a tRNA in that they have to have two ends. They have to have an end that recognizes the codon - they have to recognize the information as 'STOP' - and they have to have an end, actually, that promotes chemistry. And the chemistry that they're going to promote is not peptide bond formation, but hydrolysis.

They're going to bring a water into the active site of the large subunit and promote the hydrolysis of the polypeptide chain so that it can leave and protein synthesis is done. So, it turns out we can see the similarities between termination factor or release factors and tRNAs, right here superimposed one on the other. In blue is the protein, a termination factor from bacteria. And it's superimposed on a tRNA structure. The CCA end is up here at the top and the anticodon at the bottom. And we see that the superimposition is really quite impressive and it again suggests that one of the ways that this protein functions is by binding in the same site as a tRNA, but with a protein end to promote catalysis, the hydrolytic reaction, and a protein motif that's going to recognize specifically the stop codons.

We see that's true when we look at the way they bind to the ribosome. Here's the termination factor bound, again, on that side of the ribosome in the A site of the ribosome, recognizing stop codons and promoting peptide release. So, that's a pretty simple view of how termination takes place. As an additional point, I'll mention that the bacterial and eukaryotic release factors, while they have similar names and do a really very similar function in that they have a motif that recognizes a codon and they have a motif that does chemistry, what we see is that these proteins are really quite different in bacteria and eukaryotes, suggesting that they evolved independently of one another after the lineages had split, very, very early in evolution. So, that's a pretty exciting idea is that there really wasn't termination before the split of these domains of life, and that these two factors evolved independently, and they kind of look the same and they do the same things, but they really are built with very different protein building blocks. A particular feature to notice, however, is that the way they solve chemistry is the same. This is an example of convergent evolution. They both perform the chemistry with a three amino acid motif, a GGQ motif that actually performs the chemistry of the reaction.

So finally, we're almost done. The ribosomes have released the growing polypeptide chain, they've recognized the stop codon in order to do that, but they still have things bound to the ribosome that need to be released, and their subunits need to come apart for another round of initiation. And this is a process known as recycling.

I'm really not going to give very many details on this beyond saying that in bacteria and eukaryotes very, very different solutions have come about. Much like the termination factors, they're completely different factors in this case that promote these events. But the process in each case... bacteria use a factor known as RRF and EFG again comes in... the eukaryotes use their termination factors and an independent factor known as ABCE1, and the result is that, in an energy-dependent reaction, the subunits are split, releasing the messenger RNAs, the tRNA, and the termination factors, and subunits for the next round of protein synthesis. So, that's how proteins are made. I hope you've learned a number of general points from this lecture.

First, the translation components are broadly conserved across biology. There are some differences in some of the specifics of it, but the events of translation are highly, highly conserved, suggesting that this was a process that evolved long before the divergence into the different domains of life. Protein synthesis is an RNA-driven process. At the heart of decoding, at the heart of peptidyl transfer, and as we would see if we looked at it in more detail, translocation... at the heart of all of these events there's RNA driving these steps, with the help of exogenous protein factors, of course.

The most different steps are going to be the process of initiation and termination and recycling, and the most similar step is the process of elongation, which is really very highly conserved. And in a number of points I mentioned the accuracy of the process. I mentioned it at the step of aminoacylation, where the synthetases rarely make mistakes in connecting amino acids to tRNAs.

I mentioned the decoding process, which has a multi-step process to make fidelity high. We know the fidelity of that process is also on the order of 10^-4. So, this is a very high fidelity, efficient process subject to much regulation, which could be the subject of a subsequent lecture. And with that I'll stop.

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