字幕列表 影片播放 列印英文字幕 The following content is provided under a Creative Commons license. Your support will help MIT OpenCourseWare continue to offer high-quality educational resources for free. To make a donation or view additional materials from hundreds of MIT courses, visit MIT OpenCourseWare at ocw.mit.edu. ELIZABETH NOLAN: So last time, we were talking about these aminoacyl tRNA synthetases that are responsible for attaching amino acid monomers to the three prime end of tRNAs. And we were looking at the isoleucyl aminoacyl tRNA synthetase as an example, looking at experiments that were done to study mechanisms. So recall, we left off having discussed a two-step model, where there's an intermediate, an amino adenylate formed. And then, in the second step, there's transfer of that amino acid to the tRNA by the aaRS. And so we looked at some data from steady-state kinetic experiments. Recall that a C14 radiolabel was used to watch transfer, and then we closed discussing an ATP-PPi exchange assay which gave evidence for formation of that amino adenylate intermediate. Right? And then, lastly, we talked about use of a stopped-flow to do experiments that allow you to look at early points within a reaction. And so what we're going to do is to close these discussions of experiments and this aaRS mechanism is just look at one more experiment that was done to further probe the rate-determining step of this reaction using the stopped-flow. OK? And so this experiment pertains more to reaction kinetics, and the question is, let's monitor transfer of the amino acid to the tRNA by another method here. These experiments were set up in two different ways depending on what components were mixed. And if you just rewind to Monday and recall the ATP-PPi exchange assay and the steps in that assay, in that we showed that the amino adenylate intermediate remained bound to the enzyme there. Recall then only PPi was released in that assay. And so in these experiments, the fact that the amino adenylate can remain bound was taken advantage of. And the researchers were actually able to have a preformed complex there, so basically starting after step two. So in experiment one, how I'm going to show these is by drawing the two syringes and listing the components of each syringe. And this is a good way for setting up problems within the problem sets, thinking about stopped-flow experiments. So the question is what are we going to mix? So we have syringe one and syringe two, and recall that these go to some mixer. So the two solutions can be rapidly mixed, and that's where the chemistry is going to happen. So in experiment one, in syringe one, what we have is the purified complex. OK? So we have C-14 labeled isoleucine-AMP bound to the aminoacyl tRNA synthetase of a purified complex, here. And then in this other syringe two, what we have is the tRNA. OK? So imagine these are rapidly mixed. There'll be transfer of the radiolabeled isoleucine to the tRNA, and so formation of that aminoacyl tRNA can be monitored. OK? In the second experiment, we have just theme in variation, and if you're interested in more details, the reference is provided in the slides. So again, in syringe two, we have the tRNA, and in syringe one, what will be combined are the components here. OK? So then, the question is, in each case, what do we see? And those data are presented here from the paper, and there's some additional details about the experimental setup. So effectively, what we're looking at on the y-axis is the amount of tRNA that's been modified. So tRNA acylation measured by transfer of the radiolabel versus time. And in the black circles, we have the data from experiment one, shown here, and in the open circles, we have the data from experiment two. So what is the conclusion from these data? And this value here is not similar to something we've seen before in this system. Both experimental setups are giving the same result. Right? Effectively, these data are superimposable, and they can be fit the same. So what does that tell us about the rate-determining step? AUDIENCE: [INAUDIBLE] versus forming the intermediate. ELIZABETH NOLAN: Yeah. Right. Aminoacylation of tRNA is the rate-determining step. So some of you suggested that in class on Monday. Right? So that's the case here. OK? So formation of the intermediate is much more rapid than acylation of the tRNA here. So we've examined now the mechanism in terms of getting the amino acid onto the tRNA. What do we need to think about next here? So what we need to think about is fidelity. OK, and we've looked at the overall rate of error in protein biosynthesis, how often errors occur on the order of 10 to the 3. So how is the correct amino acid loaded onto the correct tRNA? Each tRNA has an anticodon that is a cognate pair with a codon. And so different tRNAs need to have different amino acids attached. OK, and what does that mean? That means, in general, there's a dedicated aminoacyl tRNA synthetase for each amino acid, in general here. So how are amino acids with similar side chains differentiated by these enzymes? And is it possible for an incorrect amino acid to get loaded onto a tRNA? And if that happens, what are the consequences? So we're going to examine fidelity some here. And as background, an observation made, say from studies like that ATP-PPi exchange assay, is that some aminoacyl tRNA synthetases can activate multiple amino acids, so not only the one they're supposed to activate but also others. So what does that mean? That means that the enzyme can bind and activate effectively the wrong amino acid, and if we think about fidelity, we can think about this as being a problem here. So what happens? What happens is that these enzymes have an editing function, and they're able to sense if a wrong amino acid is activated. And then they have a way to deal with it, and this is by hydrolysis. OK? And so let's consider an example, for instance, just similar side chains. So if we just consider, for instance, valine, isoleucine, and threonine, these will be the players for our discussion. OK? They're different, but they're not too different. Right? Oops, sorry about this. We're missing a methyl. Valine, an isoleucine, we have a difference of a methyl group. Threonine, we have this OH group. Right? And we can just ask the question, for instance, how is valine differentiated from isoleucine or threonine here? And so as an example, what's found is, if we consider our friend that we studied for the mechanism here, what we find is that this binds and activates isoleucine, as we saw, but it will also bind and activate valine here. And effectively, if this happens, we have a mismatch, because the end result will be isoleucine-RS with valine AMP bound here. OK? And what's found is that the catalytic efficiency or Kcat over Km, in this case, is about 150-fold less than the native substrate. So that doesn't account for the 10 to the 3 error rate here. So we need more specificity. So what's going on? So we're going to consider this editing function and a model that's often used to describe how these aaRS do editing is one of two sieves. These enzymes don't actually have a sieve. It's just a conceptual way to think about it. So this double-sieve editing model involves a first sieve which is considered to be a course one. So imagine if you have like a change sorter. It will let the quarters through as well as the and dimes and the pennies. There's some sort of discrimination of amino acids based on size, and then depending what gets through this first sieve or gate, there's a second sieve which is considered to be a fine one. And this one can differentiate perhaps on the basis of size or maybe on hydrophilicity or hydrophobic of the side chain. So effectively, if an incorrect amino acid passes through this first sieve-- so in other words, if it binds to the enzyme and becomes activated-- hydrolytic editing will occur. OK? So think about hydrolysis in terms of having breakdown of these species. So if the incorrect amino acid passes through and is adenylated, there'll be hydrolysis. So let's consider some examples so the first example here we can consider this guy and isoleucine and valine. So as I mentioned, this aaRS will activate both. So in this case, the first sieve can't differentiate isoleucine from valine. They have similar sizes according to this aaRS. But then what happens here in the second sieve, isoleucine is too big, and so there's no hydrolysis, and it moves on to form the desired charged tRNA. In contrast, valine's a bit smaller. It passes through the sieve, and it ends up being hydrolyzed. So these aaRS also have an editing domain, and this editing domain, as we'll see in a few slides in a structure, is responsible for this hydrolysis, so stated here. Right? Different sites, so there's an aminoacylation site and an editing site here. So valine can reach the editing site, but isoleucine cannot. So how do you predict? Just to keep in mind, every enzyme is different in terms of the model for discrimination and also when editing occurs. So you really need to look at the data when the data is presented to you to sort out how this works. Let's just look at another example with a cartoon depiction. So this is for the valine RS, and we're going to consider the three amino acids here-- valine, threonine, and isoleucine. So in green, we have the first sieve, and this is based on size. So what do we see in this cartoon? So threonine and valine make it through, but isoleucine does not. It's rejected right away, so it's never activated. So if threonine and valine pass through, what happens? We see each one is activated as the amino adenylate, and then what? Well, valine, we want to transfer the valine to the tRNA, so it can move on and help with protein synthesis. If threonine's activated, and here we see that threonine is transferred to the tRNA as well, this is hydrolyzed by the editing site, in this case. So the threonine is removed from the tRNA with the anticodon for valine. Right, so think about the ester bonds that we saw last time in terms of the three prime end of the tRNA being modified and the chemistry that will happen there to result in hydrolysis of and release of the amino acid here. So what that cartoon hints to is that the hydrolysis can occur at different steps.