WEBVTT 00:00:00.500 --> 00:00:02.820 The following content is provided under a Creative 00:00:02.820 --> 00:00:04.360 Commons license. 00:00:04.360 --> 00:00:06.660 Your support will help MIT OpenCourseWare 00:00:06.660 --> 00:00:11.020 continue to offer high-quality educational resources for free. 00:00:11.020 --> 00:00:13.650 To make a donation or view additional materials 00:00:13.650 --> 00:00:17.600 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.600 --> 00:00:18.540 at ocw.mit.edu. 00:00:25.745 --> 00:00:27.120 ELIZABETH NOLAN: So last time, we 00:00:27.120 --> 00:00:31.140 were talking about these aminoacyl tRNA synthetases that 00:00:31.140 --> 00:00:33.960 are responsible for attaching amino acid 00:00:33.960 --> 00:00:37.800 monomers to the three prime end of tRNAs. 00:00:37.800 --> 00:00:42.720 And we were looking at the isoleucyl aminoacyl tRNA 00:00:42.720 --> 00:00:47.160 synthetase as an example, looking at experiments that 00:00:47.160 --> 00:00:48.820 were done to study mechanisms. 00:00:48.820 --> 00:00:53.520 So recall, we left off having discussed a two-step model, 00:00:53.520 --> 00:00:56.730 where there's an intermediate, an amino adenylate formed. 00:00:56.730 --> 00:00:58.410 And then, in the second step, there's 00:00:58.410 --> 00:01:02.670 transfer of that amino acid to the tRNA by the aaRS. 00:01:02.670 --> 00:01:05.670 And so we looked at some data from steady-state kinetic 00:01:05.670 --> 00:01:06.950 experiments. 00:01:06.950 --> 00:01:12.050 Recall that a C14 radiolabel was used to watch transfer, 00:01:12.050 --> 00:01:16.020 and then we closed discussing an ATP-PPi exchange assay which 00:01:16.020 --> 00:01:19.140 gave evidence for formation of that amino adenylate 00:01:19.140 --> 00:01:20.350 intermediate. 00:01:20.350 --> 00:01:20.850 Right? 00:01:20.850 --> 00:01:25.050 And then, lastly, we talked about use of a stopped-flow 00:01:25.050 --> 00:01:28.800 to do experiments that allow you to look at early points 00:01:28.800 --> 00:01:30.490 within a reaction. 00:01:30.490 --> 00:01:33.960 And so what we're going to do is to close these discussions 00:01:33.960 --> 00:01:36.540 of experiments and this aaRS mechanism 00:01:36.540 --> 00:01:38.490 is just look at one more experiment that 00:01:38.490 --> 00:01:42.870 was done to further probe the rate-determining step 00:01:42.870 --> 00:01:46.020 of this reaction using the stopped-flow. 00:01:46.020 --> 00:01:47.010 OK? 00:01:47.010 --> 00:01:53.250 And so this experiment pertains more to reaction kinetics, 00:01:53.250 --> 00:01:55.500 and the question is, let's monitor 00:01:55.500 --> 00:02:00.240 transfer of the amino acid to the tRNA 00:02:00.240 --> 00:02:01.860 by another method here. 00:02:20.590 --> 00:02:25.450 These experiments were set up in two different ways 00:02:25.450 --> 00:02:28.000 depending on what components were mixed. 00:02:28.000 --> 00:02:33.670 And if you just rewind to Monday and recall the ATP-PPi exchange 00:02:33.670 --> 00:02:37.510 assay and the steps in that assay, in that we showed 00:02:37.510 --> 00:02:41.800 that the amino adenylate intermediate remained 00:02:41.800 --> 00:02:43.840 bound to the enzyme there. 00:02:43.840 --> 00:02:47.350 Recall then only PPi was released in that assay. 00:02:47.350 --> 00:02:50.170 And so in these experiments, the fact 00:02:50.170 --> 00:02:53.080 that the amino adenylate can remain bound 00:02:53.080 --> 00:02:54.310 was taken advantage of. 00:02:54.310 --> 00:02:55.720 And the researchers were actually 00:02:55.720 --> 00:03:00.580 able to have a preformed complex there, so basically 00:03:00.580 --> 00:03:02.410 starting after step two. 00:03:02.410 --> 00:03:08.760 So in experiment one, how I'm going to show 00:03:08.760 --> 00:03:10.980 these is by drawing the two syringes 00:03:10.980 --> 00:03:13.950 and listing the components of each syringe. 00:03:13.950 --> 00:03:16.380 And this is a good way for setting up problems 00:03:16.380 --> 00:03:17.880 within the problem sets, thinking 00:03:17.880 --> 00:03:20.290 about stopped-flow experiments. 00:03:20.290 --> 00:03:23.160 So the question is what are we going to mix? 00:03:23.160 --> 00:03:28.910 So we have syringe one and syringe two, 00:03:28.910 --> 00:03:32.820 and recall that these go to some mixer. 00:03:32.820 --> 00:03:35.430 So the two solutions can be rapidly mixed, 00:03:35.430 --> 00:03:39.640 and that's where the chemistry is going to happen. 00:03:39.640 --> 00:03:43.590 So in experiment one, in syringe one, 00:03:43.590 --> 00:03:46.540 what we have is the purified complex. 00:03:46.540 --> 00:03:47.040 OK? 00:03:47.040 --> 00:03:56.910 So we have C-14 labeled isoleucine-AMP 00:03:56.910 --> 00:04:02.370 bound to the aminoacyl tRNA synthetase 00:04:02.370 --> 00:04:10.130 of a purified complex, here. 00:04:10.130 --> 00:04:13.490 And then in this other syringe two, what we have is the tRNA. 00:04:17.453 --> 00:04:18.920 OK? 00:04:18.920 --> 00:04:21.089 So imagine these are rapidly mixed. 00:04:21.089 --> 00:04:24.260 There'll be transfer of the radiolabeled isoleucine 00:04:24.260 --> 00:04:28.790 to the tRNA, and so formation of that aminoacyl tRNA 00:04:28.790 --> 00:04:30.450 can be monitored. 00:04:30.450 --> 00:04:30.980 OK? 00:04:30.980 --> 00:04:38.830 In the second experiment, we have just theme in variation, 00:04:38.830 --> 00:04:41.440 and if you're interested in more details, 00:04:41.440 --> 00:04:43.940 the reference is provided in the slides. 00:04:47.970 --> 00:04:54.860 So again, in syringe two, we have the tRNA, 00:04:54.860 --> 00:05:02.900 and in syringe one, what will be combined 00:05:02.900 --> 00:05:06.650 are the components here. 00:05:06.650 --> 00:05:07.150 OK? 00:05:10.450 --> 00:05:16.420 So then, the question is, in each case, what do we see? 00:05:16.420 --> 00:05:20.182 And those data are presented here from the paper, 00:05:20.182 --> 00:05:21.640 and there's some additional details 00:05:21.640 --> 00:05:23.890 about the experimental setup. 00:05:23.890 --> 00:05:27.130 So effectively, what we're looking at on the y-axis 00:05:27.130 --> 00:05:30.400 is the amount of tRNA that's been modified. 00:05:30.400 --> 00:05:33.610 So tRNA acylation measured by transfer 00:05:33.610 --> 00:05:36.820 of the radiolabel versus time. 00:05:36.820 --> 00:05:39.010 And in the black circles, we have the data 00:05:39.010 --> 00:05:43.750 from experiment one, shown here, and in the open circles, 00:05:43.750 --> 00:05:48.290 we have the data from experiment two. 00:05:48.290 --> 00:05:53.520 So what is the conclusion from these data? 00:05:53.520 --> 00:05:57.060 And this value here is not similar to something we've 00:05:57.060 --> 00:05:59.865 seen before in this system. 00:06:11.190 --> 00:06:15.330 Both experimental setups are giving the same result. Right? 00:06:15.330 --> 00:06:18.210 Effectively, these data are superimposable, 00:06:18.210 --> 00:06:20.968 and they can be fit the same. 00:06:20.968 --> 00:06:23.385 So what does that tell us about the rate-determining step? 00:06:26.075 --> 00:06:29.293 AUDIENCE: [INAUDIBLE] versus forming the intermediate. 00:06:29.293 --> 00:06:30.210 ELIZABETH NOLAN: Yeah. 00:06:30.210 --> 00:06:30.710 Right. 00:06:30.710 --> 00:06:33.250 Aminoacylation of tRNA is the rate-determining step. 00:06:33.250 --> 00:06:37.850 So some of you suggested that in class on Monday. 00:06:37.850 --> 00:06:38.350 Right? 00:06:38.350 --> 00:06:40.090 So that's the case here. 00:06:40.090 --> 00:06:40.720 OK? 00:06:40.720 --> 00:06:43.630 So formation of the intermediate is much more rapid 00:06:43.630 --> 00:06:47.680 than acylation of the tRNA here. 00:06:47.680 --> 00:06:51.580 So we've examined now the mechanism 00:06:51.580 --> 00:06:53.830 in terms of getting the amino acid onto the tRNA. 00:06:56.350 --> 00:06:59.330 What do we need to think about next here? 00:06:59.330 --> 00:07:03.660 So what we need to think about is fidelity. 00:07:03.660 --> 00:07:06.790 OK, and we've looked at the overall rate of error 00:07:06.790 --> 00:07:09.970 in protein biosynthesis, how often errors occur 00:07:09.970 --> 00:07:12.220 on the order of 10 to the 3. 00:07:12.220 --> 00:07:17.740 So how is the correct amino acid loaded onto the correct tRNA? 00:07:17.740 --> 00:07:23.830 Each tRNA has an anticodon that is a cognate pair with a codon. 00:07:23.830 --> 00:07:26.530 And so different tRNAs need to have 00:07:26.530 --> 00:07:28.680 different amino acids attached. 00:07:28.680 --> 00:07:30.050 OK, and what does that mean? 00:07:30.050 --> 00:07:33.310 That means, in general, there's a dedicated aminoacyl tRNA 00:07:33.310 --> 00:07:37.690 synthetase for each amino acid, in general here. 00:07:37.690 --> 00:07:40.510 So how are amino acids with similar side chains 00:07:40.510 --> 00:07:43.150 differentiated by these enzymes? 00:07:43.150 --> 00:07:46.060 And is it possible for an incorrect amino acid 00:07:46.060 --> 00:07:48.310 to get loaded onto a tRNA? 00:07:48.310 --> 00:07:52.400 And if that happens, what are the consequences? 00:07:52.400 --> 00:07:56.290 So we're going to examine fidelity some here. 00:07:56.290 --> 00:08:01.090 And as background, an observation made, 00:08:01.090 --> 00:08:04.810 say from studies like that ATP-PPi exchange assay, 00:08:04.810 --> 00:08:09.220 is that some aminoacyl tRNA synthetases can activate 00:08:09.220 --> 00:08:12.160 multiple amino acids, so not only the one 00:08:12.160 --> 00:08:15.230 they're supposed to activate but also others. 00:08:15.230 --> 00:08:16.520 So what does that mean? 00:08:16.520 --> 00:08:18.820 That means that the enzyme can bind 00:08:18.820 --> 00:08:22.690 and activate effectively the wrong amino acid, 00:08:22.690 --> 00:08:24.940 and if we think about fidelity, we 00:08:24.940 --> 00:08:27.970 can think about this as being a problem here. 00:08:27.970 --> 00:08:30.530 So what happens? 00:08:30.530 --> 00:08:33.970 What happens is that these enzymes have an editing 00:08:33.970 --> 00:08:40.450 function, and they're able to sense if a wrong amino acid is 00:08:40.450 --> 00:08:41.470 activated. 00:08:41.470 --> 00:08:44.290 And then they have a way to deal with it, 00:08:44.290 --> 00:08:46.450 and this is by hydrolysis. 00:08:46.450 --> 00:08:47.440 OK? 00:08:47.440 --> 00:08:53.890 And so let's consider an example, for instance, just 00:08:53.890 --> 00:08:54.895 similar side chains. 00:09:07.350 --> 00:09:28.240 So if we just consider, for instance, valine, isoleucine, 00:09:28.240 --> 00:09:32.590 and threonine, these will be the players for our discussion. 00:09:40.526 --> 00:09:41.530 OK? 00:09:41.530 --> 00:09:43.930 They're different, but they're not too different. 00:09:43.930 --> 00:09:45.076 Right? 00:09:45.076 --> 00:09:46.270 Oops, sorry about this. 00:09:46.270 --> 00:09:48.520 We're missing a methyl. 00:09:48.520 --> 00:09:52.580 Valine, an isoleucine, we have a difference of a methyl group. 00:09:52.580 --> 00:09:55.630 Threonine, we have this OH group. 00:09:55.630 --> 00:09:56.230 Right? 00:09:56.230 --> 00:09:57.910 And we can just ask the question, 00:09:57.910 --> 00:10:02.045 for instance, how is valine differentiated from isoleucine 00:10:02.045 --> 00:10:05.280 or threonine here? 00:10:05.280 --> 00:10:07.720 And so as an example, what's found 00:10:07.720 --> 00:10:13.680 is, if we consider our friend that we studied 00:10:13.680 --> 00:10:17.130 for the mechanism here, what we find 00:10:17.130 --> 00:10:27.730 is that this binds and activates isoleucine, as we saw, 00:10:27.730 --> 00:10:35.470 but it will also bind and activate valine here. 00:10:35.470 --> 00:10:39.290 And effectively, if this happens, 00:10:39.290 --> 00:10:46.610 we have a mismatch, because the end result 00:10:46.610 --> 00:10:54.707 will be isoleucine-RS with valine AMP bound here. 00:10:54.707 --> 00:10:55.207 OK? 00:10:58.410 --> 00:11:02.560 And what's found is that the catalytic efficiency or Kcat 00:11:02.560 --> 00:11:07.400 over Km, in this case, is about 150-fold 00:11:07.400 --> 00:11:11.660 less than the native substrate. 00:11:11.660 --> 00:11:17.250 So that doesn't account for the 10 to the 3 error rate here. 00:11:17.250 --> 00:11:19.680 So we need more specificity. 00:11:19.680 --> 00:11:21.600 So what's going on? 00:11:21.600 --> 00:11:26.760 So we're going to consider this editing function and a model 00:11:26.760 --> 00:11:30.390 that's often used to describe how these aaRS do 00:11:30.390 --> 00:11:33.540 editing is one of two sieves. 00:11:33.540 --> 00:11:35.650 These enzymes don't actually have a sieve. 00:11:35.650 --> 00:11:39.430 It's just a conceptual way to think about it. 00:11:39.430 --> 00:11:41.670 So this double-sieve editing model 00:11:41.670 --> 00:11:46.380 involves a first sieve which is considered to be a course one. 00:11:46.380 --> 00:11:49.680 So imagine if you have like a change sorter. 00:11:49.680 --> 00:11:51.660 It will let the quarters through as well as 00:11:51.660 --> 00:11:54.150 the and dimes and the pennies. 00:11:54.150 --> 00:11:56.160 There's some sort of discrimination 00:11:56.160 --> 00:12:00.030 of amino acids based on size, and then 00:12:00.030 --> 00:12:03.870 depending what gets through this first sieve or gate, 00:12:03.870 --> 00:12:07.170 there's a second sieve which is considered to be a fine one. 00:12:07.170 --> 00:12:12.540 And this one can differentiate perhaps on the basis of size 00:12:12.540 --> 00:12:17.710 or maybe on hydrophilicity or hydrophobic of the side chain. 00:12:17.710 --> 00:12:22.560 So effectively, if an incorrect amino acid passes through this 00:12:22.560 --> 00:12:25.860 first sieve-- so in other words, if it binds to the enzyme 00:12:25.860 --> 00:12:27.690 and becomes activated-- 00:12:27.690 --> 00:12:29.320 hydrolytic editing will occur. 00:12:29.320 --> 00:12:29.820 OK? 00:12:29.820 --> 00:12:32.640 So think about hydrolysis in terms of having 00:12:32.640 --> 00:12:35.440 breakdown of these species. 00:12:35.440 --> 00:12:38.370 So if the incorrect amino acid passes through 00:12:38.370 --> 00:12:41.610 and is adenylated, there'll be hydrolysis. 00:12:41.610 --> 00:12:46.470 So let's consider some examples so the first example here we 00:12:46.470 --> 00:12:51.870 can consider this guy and isoleucine and valine. 00:12:51.870 --> 00:12:57.150 So as I mentioned, this aaRS will activate both. 00:12:57.150 --> 00:13:01.530 So in this case, the first sieve can't differentiate isoleucine 00:13:01.530 --> 00:13:02.440 from valine. 00:13:02.440 --> 00:13:05.970 They have similar sizes according to this aaRS. 00:13:05.970 --> 00:13:09.180 But then what happens here in the second sieve, 00:13:09.180 --> 00:13:13.560 isoleucine is too big, and so there's no hydrolysis, 00:13:13.560 --> 00:13:20.100 and it moves on to form the desired charged tRNA. 00:13:20.100 --> 00:13:23.280 In contrast, valine's a bit smaller. 00:13:23.280 --> 00:13:29.610 It passes through the sieve, and it ends up being hydrolyzed. 00:13:29.610 --> 00:13:34.260 So these aaRS also have an editing domain, 00:13:34.260 --> 00:13:35.640 and this editing domain, as we'll 00:13:35.640 --> 00:13:37.830 see in a few slides in a structure, 00:13:37.830 --> 00:13:43.150 is responsible for this hydrolysis, so stated here. 00:13:43.150 --> 00:13:43.660 Right? 00:13:43.660 --> 00:13:46.090 Different sites, so there's an aminoacylation site 00:13:46.090 --> 00:13:48.580 and an editing site here. 00:13:48.580 --> 00:13:53.890 So valine can reach the editing site, but isoleucine cannot. 00:13:53.890 --> 00:13:55.120 So how do you predict? 00:13:57.790 --> 00:13:59.620 Just to keep in mind, every enzyme 00:13:59.620 --> 00:14:01.810 is different in terms of the model 00:14:01.810 --> 00:14:05.360 for discrimination and also when editing occurs. 00:14:05.360 --> 00:14:07.150 So you really need to look at the data 00:14:07.150 --> 00:14:13.130 when the data is presented to you to sort out how this works. 00:14:13.130 --> 00:14:14.630 Let's just look at another example 00:14:14.630 --> 00:14:16.500 with a cartoon depiction. 00:14:16.500 --> 00:14:19.010 So this is for the valine RS, and we're 00:14:19.010 --> 00:14:22.100 going to consider the three amino acids here-- 00:14:22.100 --> 00:14:24.890 valine, threonine, and isoleucine. 00:14:24.890 --> 00:14:29.180 So in green, we have the first sieve, 00:14:29.180 --> 00:14:31.110 and this is based on size. 00:14:31.110 --> 00:14:33.680 So what do we see in this cartoon? 00:14:33.680 --> 00:14:36.500 So threonine and valine make it through, 00:14:36.500 --> 00:14:38.030 but isoleucine does not. 00:14:38.030 --> 00:14:42.080 It's rejected right away, so it's never activated. 00:14:42.080 --> 00:14:46.130 So if threonine and valine pass through, what happens? 00:14:46.130 --> 00:14:50.810 We see each one is activated as the amino adenylate, and then 00:14:50.810 --> 00:14:52.110 what? 00:14:52.110 --> 00:14:54.050 Well, valine, we want to transfer the valine 00:14:54.050 --> 00:14:56.210 to the tRNA, so it can move on and help 00:14:56.210 --> 00:14:58.640 with protein synthesis. 00:14:58.640 --> 00:15:01.130 If threonine's activated, and here we 00:15:01.130 --> 00:15:03.860 see that threonine is transferred to the tRNA 00:15:03.860 --> 00:15:09.340 as well, this is hydrolyzed by the editing site, in this case. 00:15:09.340 --> 00:15:12.470 So the threonine is removed from the tRNA 00:15:12.470 --> 00:15:14.750 with the anticodon for valine. 00:15:14.750 --> 00:15:17.540 Right, so think about the ester bonds 00:15:17.540 --> 00:15:20.030 that we saw last time in terms of the three prime end 00:15:20.030 --> 00:15:22.850 of the tRNA being modified and the chemistry that 00:15:22.850 --> 00:15:27.410 will happen there to result in hydrolysis of and release 00:15:27.410 --> 00:15:28.700 of the amino acid here. 00:15:31.560 --> 00:15:37.400 So what that cartoon hints to is that the hydrolysis can 00:15:37.400 --> 00:15:39.690 occur at different steps. 00:15:39.690 --> 00:15:44.540 So we can have hydrolysis that is pre-transfer, 00:15:44.540 --> 00:15:51.050 which means the editing occurs before the tRNA is modified. 00:15:51.050 --> 00:15:53.063 Or we can have post-transfer editing 00:15:53.063 --> 00:15:54.980 which is what we saw in the prior slide, where 00:15:54.980 --> 00:15:58.040 the editing and hydrolysis occurs after the amino acid 00:15:58.040 --> 00:16:00.900 monomer is transferred to the tRNA. 00:16:00.900 --> 00:16:01.400 OK? 00:16:01.400 --> 00:16:06.090 And this schematic here depicts that, so what do we have? 00:16:06.090 --> 00:16:13.280 We have the aaRS responsible for modifying tRNA for isoleucine, 00:16:13.280 --> 00:16:17.780 and we combine that with valine, the wrong amino acid, and ATP. 00:16:17.780 --> 00:16:18.460 What happens? 00:16:18.460 --> 00:16:19.880 So E is for enzyme. 00:16:19.880 --> 00:16:24.200 We have formulation of the amino adenylate intermediate. 00:16:24.200 --> 00:16:28.550 Here's the tRNA with the anticodon for isoleucine. 00:16:28.550 --> 00:16:29.450 What happens? 00:16:29.450 --> 00:16:32.990 So we have this complex form in this depiction. 00:16:32.990 --> 00:16:35.750 Pre-transfer editing would occur at this stage, 00:16:35.750 --> 00:16:39.680 before the valine is transferred to the tRNA, and so 00:16:39.680 --> 00:16:41.100 what do we see? 00:16:41.100 --> 00:16:44.480 We see breakdown and these species. 00:16:44.480 --> 00:16:47.690 If the valine is transferred to the tRNA, 00:16:47.690 --> 00:16:49.280 we don't want this, because that would 00:16:49.280 --> 00:16:52.310 result in this reading of the genetic code. 00:16:52.310 --> 00:16:57.140 Post-transfer editing, this species here is hydrolyzed. 00:16:57.140 --> 00:17:00.530 So whether pre or post-transfer editing occurs 00:17:00.530 --> 00:17:04.579 is going to depend on the aminoacyl tRNA synthetase, 00:17:04.579 --> 00:17:07.430 and some can use both mechanisms. 00:17:07.430 --> 00:17:09.520 That's what we're seeing here. 00:17:09.520 --> 00:17:10.160 OK? 00:17:10.160 --> 00:17:13.190 Some only use one, for instance, the valine RS 00:17:13.190 --> 00:17:16.740 only uses a post-transfer editing mechanism. 00:17:16.740 --> 00:17:19.460 So when presented with the data, look at the data 00:17:19.460 --> 00:17:23.089 and see what species is being hydrolyzed. 00:17:23.089 --> 00:17:26.000 And if both are, how did the steady-state kinetics, 00:17:26.000 --> 00:17:28.760 for instance, compare? 00:17:28.760 --> 00:17:32.840 Just to take a look in the context of a structure of one 00:17:32.840 --> 00:17:34.580 of these aaRS. 00:17:34.580 --> 00:17:38.420 So the sites where aminoacylation 00:17:38.420 --> 00:17:41.300 occur and editing occur are separated 00:17:41.300 --> 00:17:43.820 by about 30 Angstroms, and that's 00:17:43.820 --> 00:17:47.750 shown here, where we have the aminoacylation site, 00:17:47.750 --> 00:17:50.000 and here we have the editing site. 00:17:50.000 --> 00:17:54.960 That's responsible for pre and/or post-transfer editing. 00:17:54.960 --> 00:17:58.700 So in thinking about this and thinking 00:17:58.700 --> 00:18:02.780 about how one could leverage this 30 Angstrom 00:18:02.780 --> 00:18:05.540 separation and these two distinct sites 00:18:05.540 --> 00:18:12.260 in terms of experiments, what does that allow one to do? 00:18:12.260 --> 00:18:14.450 So imagine if you want to ask, what 00:18:14.450 --> 00:18:19.730 are the consequences of having aaRS that have faulty editing 00:18:19.730 --> 00:18:21.090 function? 00:18:21.090 --> 00:18:24.050 And effectively, mischarged tRNAs 00:18:24.050 --> 00:18:27.200 or put the wrong amino acid on a tRNA. 00:18:27.200 --> 00:18:30.770 What does that mean for a cell? 00:18:30.770 --> 00:18:33.410 There's an opportunity to do that here. 00:18:33.410 --> 00:18:36.950 So you could imagine mutating residues 00:18:36.950 --> 00:18:40.370 that are critical for editing function in the editing site. 00:18:40.370 --> 00:18:46.100 Such that you have an aaRS variant that can activate amino 00:18:46.100 --> 00:18:50.570 acids and transfer them to the tRNA but cannot edit when 00:18:50.570 --> 00:18:52.230 a mistake happens. 00:18:52.230 --> 00:18:52.730 Right? 00:18:52.730 --> 00:18:56.390 So you can imagine a site-directed mutagenesis, 00:18:56.390 --> 00:19:00.770 purifying the enzyme and doing some in vitro characterization 00:19:00.770 --> 00:19:02.730 to see how it behaves. 00:19:02.730 --> 00:19:04.940 And then you could also imagine translating this 00:19:04.940 --> 00:19:09.380 into a cellular context and asking say in cell culture what 00:19:09.380 --> 00:19:10.980 happens here? 00:19:10.980 --> 00:19:16.330 So basically, what are the consequences of faulty editing? 00:19:16.330 --> 00:19:18.940 And these types of studies have been done. 00:19:18.940 --> 00:19:21.540 We're not going to look at them in detail. 00:19:21.540 --> 00:19:26.910 But just as an overview and some concepts that will come up 00:19:26.910 --> 00:19:30.660 within our folding section, what's been shown 00:19:30.660 --> 00:19:35.520 is that a single point mutation in an editing domain of one 00:19:35.520 --> 00:19:38.700 of these aminoacyl tRNA synthetases 00:19:38.700 --> 00:19:41.550 may have deleterious consequences. 00:19:41.550 --> 00:19:45.960 And we can imagine that these consequences could result 00:19:45.960 --> 00:19:50.640 from proteins or enzymes that gain a new function 00:19:50.640 --> 00:19:53.830 or don't do their correct function. 00:19:53.830 --> 00:19:54.330 Right? 00:19:54.330 --> 00:19:59.880 So just imagine that some mischarged tRNAs, where 00:19:59.880 --> 00:20:03.690 mischarged means the wrong amino acid is attached, 00:20:03.690 --> 00:20:07.980 are around because of some mutant aaRS. 00:20:07.980 --> 00:20:10.440 And these tRNA that are mischarged 00:20:10.440 --> 00:20:12.810 can be delivered to the ribosome, which 00:20:12.810 --> 00:20:17.490 means that point mutations form within synthesized polypeptide 00:20:17.490 --> 00:20:18.000 chains. 00:20:18.000 --> 00:20:21.150 So there's some mixture where some of these proteins 00:20:21.150 --> 00:20:24.450 are native, and others are mutant, 00:20:24.450 --> 00:20:28.050 and what might happen here in terms of consequences? 00:20:28.050 --> 00:20:31.510 So native protein will go on and do its job. 00:20:31.510 --> 00:20:35.040 Imagine there's some mutant protein here 00:20:35.040 --> 00:20:39.020 that's altered in some way, and these are just some examples 00:20:39.020 --> 00:20:41.560 of possible outcomes. 00:20:41.560 --> 00:20:44.580 So maybe there's a breakdown of some essential cellular 00:20:44.580 --> 00:20:47.130 process. 00:20:47.130 --> 00:20:50.820 Here, we have triggering of autoimmune-like responses, 00:20:50.820 --> 00:20:53.280 things that are not good. 00:20:53.280 --> 00:20:55.560 What if these mutant proteins misfold? 00:20:55.560 --> 00:21:00.660 So they can't form their correct fold, 00:21:00.660 --> 00:21:02.850 and fold is important for function. 00:21:02.850 --> 00:21:04.800 Maybe there's aggregation. 00:21:04.800 --> 00:21:10.650 Maybe there's stress on the proteasome, ER response, 00:21:10.650 --> 00:21:13.690 unfolded protein response, cell death. 00:21:13.690 --> 00:21:16.410 So fidelity's important. 00:21:19.420 --> 00:21:23.960 And just some things to think about as we close this section. 00:21:23.960 --> 00:21:27.700 We can consider error rates of various biological 00:21:27.700 --> 00:21:32.270 polymerizations, whether that be DNA replication, transcription, 00:21:32.270 --> 00:21:39.500 or translation, and they vary quite a bit here from this. 00:21:39.500 --> 00:21:45.950 And what the take-home can be by comparing these error 00:21:45.950 --> 00:21:51.320 rates is infrequent mistakes in decoding the mRNA 00:21:51.320 --> 00:21:54.620 are accepted as a source of infidelity. 00:21:54.620 --> 00:21:57.110 So they do occur, and they occur more frequently 00:21:57.110 --> 00:22:00.080 than, say, an error in replicating the DNA, 00:22:00.080 --> 00:22:01.110 and that makes sense. 00:22:01.110 --> 00:22:01.610 Right? 00:22:01.610 --> 00:22:03.770 If an error occurs in DNA replication, 00:22:03.770 --> 00:22:06.650 there's a huge problem likely compared 00:22:06.650 --> 00:22:09.020 to an error in translation. 00:22:09.020 --> 00:22:12.740 So some questions just to think about, 00:22:12.740 --> 00:22:14.930 answers aren't going to come up within the context 00:22:14.930 --> 00:22:16.190 of this course. 00:22:16.190 --> 00:22:18.950 But higher accuracy is important, but actually 00:22:18.950 --> 00:22:21.080 how much accuracy is enough? 00:22:21.080 --> 00:22:26.810 And there is a cost in terms of cellular energy for accuracy, 00:22:26.810 --> 00:22:31.550 and is it that the cell tunes its accuracy to some point that 00:22:31.550 --> 00:22:34.610 could be considered optimal, and are 00:22:34.610 --> 00:22:37.310 there benefits to translational infidelity? 00:22:37.310 --> 00:22:37.810 Right? 00:22:37.810 --> 00:22:41.600 So the prior slide showed negative consequences, 00:22:41.600 --> 00:22:44.870 but are there benefits? 00:22:44.870 --> 00:22:51.110 So that discussion, we'll close considering 00:22:51.110 --> 00:22:55.670 how the amino acids get attached to tRNAs, 00:22:55.670 --> 00:22:59.688 and so where we're moving to now is the elongation cycle. 00:22:59.688 --> 00:23:02.556 AUDIENCE: So is there a specific part of the cytoplasm 00:23:02.556 --> 00:23:04.912 where the tRNAs and the amino acids 00:23:04.912 --> 00:23:08.110 come together, or does this happen everywhere? 00:23:08.110 --> 00:23:11.860 ELIZABETH NOLAN: So I actually don't know, 00:23:11.860 --> 00:23:13.540 but I think of them as being everywhere 00:23:13.540 --> 00:23:15.040 in terms of the tRNAs. 00:23:15.040 --> 00:23:17.820 Because as we'll see in a few slides, 00:23:17.820 --> 00:23:21.490 EF-Tu, which is required for delivering 00:23:21.490 --> 00:23:24.190 the tRNAs to the ribosome, is highly abundant. 00:23:24.190 --> 00:23:26.065 At least, that's my thinking for prokaryotes. 00:23:26.065 --> 00:23:28.010 Do you have anything to say? 00:23:28.010 --> 00:23:29.950 The question was effectively are there 00:23:29.950 --> 00:23:35.320 certain regions of the cell where tRNAs get modified more 00:23:35.320 --> 00:23:36.850 than other regions? 00:23:36.850 --> 00:23:38.017 JOANNE STUBBE: I don't know. 00:23:38.017 --> 00:23:40.650 In mammalian cells, they have weirdo complexes 00:23:40.650 --> 00:23:43.507 with tRNA synthases that they've been around forever. 00:23:43.507 --> 00:23:45.340 and I still think we don't really understand 00:23:45.340 --> 00:23:46.280 what the function is. 00:23:49.184 --> 00:23:52.572 AUDIENCE: [INAUDIBLE] 00:23:57.798 --> 00:23:59.840 JOANNE STUBBE: Can you speak a little bit louder? 00:23:59.840 --> 00:24:00.650 ELIZABETH NOLAN: The question is, do we 00:24:00.650 --> 00:24:03.440 have information about say the distribution of tRNAs 00:24:03.440 --> 00:24:06.735 as being amino acid modified versus unmodified? 00:24:06.735 --> 00:24:11.780 AUDIENCE: I think maybe we could [INAUDIBLE] I don't know. 00:24:11.780 --> 00:24:14.000 ELIZABETH NOLAN: There's always a way, probably. 00:24:14.000 --> 00:24:14.960 Right? 00:24:14.960 --> 00:24:17.510 But I don't know what that distribution is either 00:24:17.510 --> 00:24:21.440 in terms of the percentage of tRNAs that are aminoacylated 00:24:21.440 --> 00:24:24.490 at any one given time. 00:24:24.490 --> 00:24:26.730 Yeah, just don't know. 00:24:26.730 --> 00:24:28.880 I think one key thing to think about 00:24:28.880 --> 00:24:33.440 as we come to the next part is that these tRNAs are 00:24:33.440 --> 00:24:35.810 bound by EF-Tu. 00:24:35.810 --> 00:24:39.020 So to think of them as in complex with a translation 00:24:39.020 --> 00:24:42.560 factor as opposed to tRNAs floating around 00:24:42.560 --> 00:24:44.410 in the cytoplasm, so I think that that's 00:24:44.410 --> 00:24:47.000 a key point of focus. 00:24:47.000 --> 00:24:55.940 So moving into elongation, what do we need to think about here? 00:24:55.940 --> 00:24:57.830 So we need to think about delivery 00:24:57.830 --> 00:25:00.200 of the amino acid tRNAs. 00:25:00.200 --> 00:25:04.580 How does the ribosome ensure that the correct aminoacyl tRNA 00:25:04.580 --> 00:25:05.420 is delivered? 00:25:05.420 --> 00:25:08.750 So we have the correct amino acid onto the tRNA, 00:25:08.750 --> 00:25:10.870 but we also have to get the correct amino acid 00:25:10.870 --> 00:25:13.010 to the ribosome. 00:25:13.010 --> 00:25:16.490 How is peptide bond formation catalyzed? 00:25:16.490 --> 00:25:20.000 What is the method by which polypeptides 00:25:20.000 --> 00:25:24.840 leave the ribosome, and how is translation terminated here? 00:25:24.840 --> 00:25:26.660 So effectively, these are all questions 00:25:26.660 --> 00:25:29.030 we need to address in terms of thinking about how 00:25:29.030 --> 00:25:31.650 the ribosome translates the genetic code 00:25:31.650 --> 00:25:33.560 and synthesizes the polypeptide. 00:25:33.560 --> 00:25:36.260 So within the notes posted on Stellar, 00:25:36.260 --> 00:25:40.610 there's a number of pages of definitions, so terminology 00:25:40.610 --> 00:25:43.790 that comes up within these discussions of the ribosome 00:25:43.790 --> 00:25:45.500 to refer to. 00:25:45.500 --> 00:25:48.650 And in terms of our translation overview slide, 00:25:48.650 --> 00:25:52.050 where we are now is here, in elongation. 00:25:52.050 --> 00:25:55.250 So we have the mRNA our 70S, and we're 00:25:55.250 --> 00:25:59.240 going to focus for the rest of today on thinking about EF-Tu, 00:25:59.240 --> 00:26:02.930 this elongation factor that's responsible for delivering 00:26:02.930 --> 00:26:07.970 the amino acid tRNAs to the ribosome here. 00:26:07.970 --> 00:26:13.850 So as an overview in terms of a cartoon, where are we going? 00:26:13.850 --> 00:26:18.140 Here, we have our ribosome, and in this depiction, 00:26:18.140 --> 00:26:19.530 it has been translating. 00:26:19.530 --> 00:26:23.300 So we have a nascent polypeptide emerging through the exit 00:26:23.300 --> 00:26:25.130 tunnel of the 50S. 00:26:25.130 --> 00:26:28.580 So we see this peptidyl tRNA in the P-site, 00:26:28.580 --> 00:26:32.120 and we have this deacylated tRNA in the E-site. 00:26:32.120 --> 00:26:33.320 So what happens? 00:26:33.320 --> 00:26:37.040 That A-site is empty, and for another round of elongation 00:26:37.040 --> 00:26:41.630 to occur, the aminoacyl tRNA needs to be delivered. 00:26:41.630 --> 00:26:44.780 And as we'll see today and in recitation this week, 00:26:44.780 --> 00:26:48.450 EF-Tu is responsible for that. 00:26:48.450 --> 00:26:54.050 So there's a ternary complex that forms between EF-Tu-GTP. 00:26:54.050 --> 00:26:59.060 So EF-Tu is a GTPase and the aminoacyl tRNA. 00:26:59.060 --> 00:27:03.230 And this ternary complex delivers the aminoacyl tRNA 00:27:03.230 --> 00:27:05.050 to the A-site. 00:27:05.050 --> 00:27:05.750 OK? 00:27:05.750 --> 00:27:07.880 This allows for peptide bond formation 00:27:07.880 --> 00:27:10.040 to occur in the catalytic center. 00:27:10.040 --> 00:27:13.160 And then there's a process called translocation, 00:27:13.160 --> 00:27:17.780 in which the elongation factor-G in complex with GTP 00:27:17.780 --> 00:27:20.900 comes in and helps to reset the ribosome such 00:27:20.900 --> 00:27:24.290 that another aminoacyl tRNA can come in. 00:27:24.290 --> 00:27:27.230 So where we're going to focus for the rest of today 00:27:27.230 --> 00:27:30.860 is on this process here, thinking about EF-Tu 00:27:30.860 --> 00:27:34.950 and how that delivers amino acid attached 00:27:34.950 --> 00:27:36.650 to tRNAs to the A-site. 00:27:41.450 --> 00:27:52.390 OK, so just in our cartoon, where we left off, 00:27:52.390 --> 00:28:12.180 with initiation process, so we have 00:28:12.180 --> 00:28:21.280 that initiator tRNA in the P-site, 00:28:21.280 --> 00:28:23.710 and the A-site is empty. 00:28:23.710 --> 00:28:24.310 OK? 00:28:24.310 --> 00:28:28.690 And one other thing I'll just show here, 00:28:28.690 --> 00:28:32.350 I mentioned when describing ribosome structure 00:28:32.350 --> 00:28:35.410 that some ribosomal proteins have additional jobs. 00:28:35.410 --> 00:28:37.420 So it's not just that these proteins 00:28:37.420 --> 00:28:40.870 help with the overall structural integrity of the ribosome. 00:28:40.870 --> 00:28:44.890 And there's two ribosomal proteins, L7 and L12, 00:28:44.890 --> 00:28:47.440 and these are involved in recruitment 00:28:47.440 --> 00:28:51.310 of that ternary complex between EF-Tu, the GTP, 00:28:51.310 --> 00:28:52.585 and the aminoacyl tRNA. 00:29:11.720 --> 00:29:26.000 So now, we need to get the aminoacyl tRNA to the A-site, 00:29:26.000 --> 00:29:27.140 and this requires EF-Tu. 00:29:32.770 --> 00:29:35.290 And when we think about this, we always 00:29:35.290 --> 00:29:39.970 need to think about this ternary complex which 00:29:39.970 --> 00:29:54.905 is EF-Tu bound to the aminoacyl tRNA bound to GTP. 00:30:00.030 --> 00:30:04.110 So a little bit about EF-Tu. 00:30:04.110 --> 00:30:09.600 So in E. coli, EF-Tu is the most abundant protein. 00:30:09.600 --> 00:30:12.140 So there's tons of EF-Tu. 00:30:12.140 --> 00:30:17.520 OK, approximately here, we have 100,000 copies per cell. 00:30:17.520 --> 00:30:21.360 So it's about 5% of total cellular protein. 00:30:21.360 --> 00:30:24.030 And so, as I just said in response 00:30:24.030 --> 00:30:27.090 to a question about these tRNAs in the cells, 00:30:27.090 --> 00:30:30.360 we can think about this entire tRNA 00:30:30.360 --> 00:30:33.300 pool, or aminoacylated tRNA pool, 00:30:33.300 --> 00:30:37.320 as being sequestered by EF-Tu. 00:30:37.320 --> 00:30:40.800 So EF-Tu binds the aminoacyl tRNA, 00:30:40.800 --> 00:30:44.970 and it binds GTP to form the ternary complex. 00:30:44.970 --> 00:30:49.050 And this allows EF-Tu to deliver these amino acids attached 00:30:49.050 --> 00:30:53.550 to the tRNAs to the A-site, and it's a GTPase. 00:30:53.550 --> 00:30:57.180 And we need to think a lot about how this activity relates 00:30:57.180 --> 00:31:00.030 to its function and fidelity. 00:31:00.030 --> 00:31:06.780 So here is a depiction of the structure of a ternary complex. 00:31:06.780 --> 00:31:11.160 So what we see is that we have a tRNA here, 00:31:11.160 --> 00:31:15.210 and here we have EF-Tu bound to the tRNA. 00:31:15.210 --> 00:31:19.560 So here is the anticodon loop, and if we consider 00:31:19.560 --> 00:31:23.760 this structure of the ternary complex bound to mRNA, 00:31:23.760 --> 00:31:25.260 what do we see? 00:31:25.260 --> 00:31:28.700 So we have an mRNA in green. 00:31:28.700 --> 00:31:32.130 OK, here's the tRNA, and the anticodon end, 00:31:32.130 --> 00:31:34.860 and here's EF-Tu. 00:31:34.860 --> 00:31:38.070 And as I said, EF-Tu is a GTPase. 00:31:38.070 --> 00:31:40.500 Where is the GTPase center? 00:31:40.500 --> 00:31:42.720 That's up here. 00:31:42.720 --> 00:31:48.600 So this GTPase center of EF-Tu is quite far from the tRNA 00:31:48.600 --> 00:31:50.520 anticodon, down here. 00:31:55.330 --> 00:31:57.880 This distance is about 70 Angstroms. 00:32:02.050 --> 00:32:05.500 And so this is something quite incredible 00:32:05.500 --> 00:32:09.670 to think about, because as we'll see, 00:32:09.670 --> 00:32:12.070 when there's codon recognition-- 00:32:12.070 --> 00:32:14.440 meaning this codon-anticodon interaction, 00:32:14.440 --> 00:32:16.160 that's a cognate pair-- 00:32:16.160 --> 00:32:18.850 GTP hydrolysis is stimulated. 00:32:18.850 --> 00:32:22.360 So how is that communicated over 70 Angstroms? 00:32:22.360 --> 00:32:24.100 If there's a recognition of that here 00:32:24.100 --> 00:32:28.120 between the mRNA and the tRNA anticodon, 00:32:28.120 --> 00:32:31.090 and GTP hydrolysis happens up here, 00:32:31.090 --> 00:32:34.190 how is that signaled over 70 Angstroms? 00:32:34.190 --> 00:32:34.690 Right? 00:32:34.690 --> 00:32:37.540 So clearly, there's going to be some conformational changes 00:32:37.540 --> 00:32:41.920 that occur that allow this GTPase activity to turn on. 00:32:44.850 --> 00:32:48.810 Just another view, so here, again, we 00:32:48.810 --> 00:32:51.420 have the structure of the ternary complex bound 00:32:51.420 --> 00:32:57.000 to the mRNA, and here, we can look at the ternary complex 00:32:57.000 --> 00:32:59.880 bound to a 70S ribosome. 00:32:59.880 --> 00:33:03.100 So we have the ribosome in this orangey-gold color, 00:33:03.100 --> 00:33:05.190 the 50S the 30S. 00:33:05.190 --> 00:33:08.790 Here, we have the PTC and decoding site. 00:33:08.790 --> 00:33:14.670 The tRNA is in green, and EF-Tu is in this darker orange here, 00:33:14.670 --> 00:33:20.700 to place that in the perspective of the 70S ribosome here. 00:33:20.700 --> 00:33:22.800 So conformational change is required 00:33:22.800 --> 00:33:26.130 to signal code on recognition to the GTPase center, 00:33:26.130 --> 00:33:28.590 and this is something that will be 00:33:28.590 --> 00:33:34.590 spoken about in quite some detail this week in recitation. 00:33:34.590 --> 00:33:40.650 One other point of review before moving forward with delivery 00:33:40.650 --> 00:33:43.150 of the amino acid tRNA. 00:33:43.150 --> 00:33:48.450 We need to think about codon-anticodon interactions 00:33:48.450 --> 00:33:50.730 here for decoding. 00:34:14.580 --> 00:34:24.040 So we have cognate versus near-cognate 00:34:24.040 --> 00:34:33.699 versus non-cognate, and this is for the codon-anticodon 00:34:33.699 --> 00:34:34.570 interaction. 00:34:42.290 --> 00:34:47.940 OK, and so if we imagine we have some mRNA, 00:34:47.940 --> 00:34:50.719 and you need to think about the five prime and three prime ends 00:34:50.719 --> 00:34:51.980 with this. 00:34:51.980 --> 00:34:59.990 And then we have some tRNA, three prime, five prime, 00:34:59.990 --> 00:35:01.830 we need to ask how do these match? 00:35:01.830 --> 00:35:08.610 So for instance here, if we have AAG, 00:35:08.610 --> 00:35:11.180 and we have positions one, two, three, 00:35:11.180 --> 00:35:14.060 from left to right of the mRNA, right here 00:35:14.060 --> 00:35:15.940 we have a cognate match. 00:35:15.940 --> 00:35:17.060 OK? 00:35:17.060 --> 00:35:19.940 So we have the AU match in positions one and two, 00:35:19.940 --> 00:35:23.720 and then wobble's allowed in position three, this GU here. 00:35:23.720 --> 00:35:25.980 So no, no interaction. 00:35:25.980 --> 00:35:36.660 OK, just as another example here, 00:35:36.660 --> 00:35:44.970 imagine we have GAG, here. 00:35:44.970 --> 00:35:47.370 What we see is that there's only one 00:35:47.370 --> 00:35:52.600 match, meaning Watson-Crick base pairing, in position two. 00:35:52.600 --> 00:35:53.250 OK. 00:35:53.250 --> 00:35:58.050 Here, this GU, that's not a match 00:35:58.050 --> 00:36:01.600 based on Watson-Crick base pairing, and as a result, 00:36:01.600 --> 00:36:05.360 the ribosome is going to want to reject this tRNA, 00:36:05.360 --> 00:36:08.940 if this is what's happening in the A-site here. 00:36:08.940 --> 00:36:17.160 And then, we can just imagine some situation, 00:36:17.160 --> 00:36:26.790 where we have a tRNA and an mRNA where there's just no match. 00:36:31.460 --> 00:36:32.060 OK? 00:36:32.060 --> 00:36:33.870 No Watson-Crick base pairing here. 00:36:37.870 --> 00:36:43.900 So what we need to ask is, as EF-Tu is delivering 00:36:43.900 --> 00:36:47.350 these aminoacyl tRNAs, what happens 00:36:47.350 --> 00:36:51.190 if it's a cognate match versus a near-cognate 00:36:51.190 --> 00:36:53.650 versus a non-cognate? 00:36:53.650 --> 00:36:58.075 How does the ribosome deal with the wrong tRNA entering 00:36:58.075 --> 00:36:58.940 the A-site? 00:36:58.940 --> 00:36:59.440 Right? 00:36:59.440 --> 00:37:09.987 So again, this is something important for fidelity, 00:37:09.987 --> 00:37:11.445 and these both need to be rejected. 00:37:17.840 --> 00:37:19.990 So why are we reviewing this? 00:37:19.990 --> 00:37:21.850 We're reviewing this, because it's 00:37:21.850 --> 00:37:25.810 important in terms of what happens 00:37:25.810 --> 00:37:28.510 during initial binding of aminoacyl tRNAs 00:37:28.510 --> 00:37:29.890 to the ribosome. 00:37:29.890 --> 00:37:32.410 So we're going to go over some of this in words 00:37:32.410 --> 00:37:35.860 and then look at a cartoon that explains this process. 00:37:35.860 --> 00:37:40.390 And what we're focused on is delivery of the aminoacyl tRNA 00:37:40.390 --> 00:37:42.310 to the A-site. 00:37:42.310 --> 00:37:44.240 So what happens first? 00:37:44.240 --> 00:37:44.740 OK. 00:37:44.740 --> 00:37:46.870 First, there needs to be an initial binding 00:37:46.870 --> 00:37:51.520 event, where the ternary complex binds to the ribosome. 00:37:51.520 --> 00:37:55.220 So initial binding, it binds to the 70S, 00:37:55.220 --> 00:37:58.330 and these ribosomal proteins are involved in the recruitment 00:37:58.330 --> 00:38:00.910 of the ternary complex. 00:38:00.910 --> 00:38:04.360 This initial binding event of the ternary complex 00:38:04.360 --> 00:38:06.670 to the ribosome is independent of the mRNA. 00:38:09.830 --> 00:38:14.270 What happens next is that there's codon recognition. 00:38:14.270 --> 00:38:19.460 So we need to think about that tRNA entering the A-site, 00:38:19.460 --> 00:38:20.960 and there's some sort of sampling 00:38:20.960 --> 00:38:23.620 that occurs in the decoding center, so 00:38:23.620 --> 00:38:28.130 sampling of codon-anticodon pairs in the A-site, 00:38:28.130 --> 00:38:29.390 and so what happens? 00:38:29.390 --> 00:38:32.840 What happens if there's a cognate event 00:38:32.840 --> 00:38:34.890 or a non-cognate event? 00:38:34.890 --> 00:38:40.190 So if a cognate anticodon recognition event occurs, 00:38:40.190 --> 00:38:45.300 there's a series of steps that then happen. 00:38:45.300 --> 00:38:49.040 So with a cognate codon-anticodon interaction, 00:38:49.040 --> 00:38:53.300 there will be a conformational change in EF-Tu, 00:38:53.300 --> 00:38:56.330 and this activates the GTPase center which 00:38:56.330 --> 00:38:59.060 allows for GTP hydrolysis. 00:38:59.060 --> 00:39:02.930 OK, and effectively this conformational change 00:39:02.930 --> 00:39:07.740 stabilizes the codon-anticodon interaction here, 00:39:07.740 --> 00:39:11.870 and that stabilization accelerates the GTP hydrolysis 00:39:11.870 --> 00:39:12.978 step. 00:39:12.978 --> 00:39:15.020 So this is all building towards a kinetic scheme. 00:39:17.660 --> 00:39:21.800 In terms of enhancements, what's found is that the rate of GTP 00:39:21.800 --> 00:39:26.390 hydrolysis by EF-Tu increases by about 5 times 10 00:39:26.390 --> 00:39:29.030 to the 4th with cognate anticodon 00:39:29.030 --> 00:39:31.580 recognition in the A-site. 00:39:31.580 --> 00:39:34.850 So we have GTP hydrolysis, and then there's 00:39:34.850 --> 00:39:37.280 another conformational change. 00:39:37.280 --> 00:39:41.690 So we have EF-Tu in its GDP-bound form, 00:39:41.690 --> 00:39:44.360 and effectively, EF-Tu will dissociate 00:39:44.360 --> 00:39:49.700 from the aminoacyl tRNA, and the aminoacyl tRNA will fully 00:39:49.700 --> 00:39:51.700 enter the A-site. 00:39:51.700 --> 00:39:54.710 OK so this process is called accommodation, 00:39:54.710 --> 00:39:58.560 and once that happens, peptide bond formation can occur. 00:39:58.560 --> 00:40:00.890 So this is the good scenario. 00:40:00.890 --> 00:40:03.650 The polypeptide can keep being made. 00:40:03.650 --> 00:40:05.180 What if it's not a cognate? 00:40:05.180 --> 00:40:09.890 So what if a near-cognate tRNA is delivered to that A-site 00:40:09.890 --> 00:40:11.870 during this initial binding event which 00:40:11.870 --> 00:40:14.060 is independent of the mRNA? 00:40:14.060 --> 00:40:15.920 That's why this can occur. 00:40:15.920 --> 00:40:20.745 If it's a near-cognate anticodon, what we observe-- 00:40:20.745 --> 00:40:22.370 and this is all from experiments you'll 00:40:22.370 --> 00:40:24.770 be learning about this week-- 00:40:24.770 --> 00:40:29.420 the ternary complex rapidly dissociates from the ribosome. 00:40:29.420 --> 00:40:31.850 And what's found from kinetic measurements 00:40:31.850 --> 00:40:35.120 is that the dissociation of the ternary complex, 00:40:35.120 --> 00:40:39.860 when it's a near-cognate situation, 00:40:39.860 --> 00:40:45.410 is about 350-fold faster than cognate. 00:40:45.410 --> 00:40:51.080 So let's look at this stepwise within a cartoon format. 00:40:51.080 --> 00:40:53.600 You'll see another depiction of this scheme 00:40:53.600 --> 00:40:57.170 in the recitation notes and in problem set two. 00:40:57.170 --> 00:41:03.440 So here, we have multiple steps in this overall process. 00:41:03.440 --> 00:41:05.450 All of these steps have some rate 00:41:05.450 --> 00:41:08.720 that's been measured by multiple types of methods, 00:41:08.720 --> 00:41:10.580 and Joanne will be presenting this week 00:41:10.580 --> 00:41:14.000 on a lot of pre-steady-state kinetic analysis that were done 00:41:14.000 --> 00:41:16.150 to measure these rates here. 00:41:20.270 --> 00:41:24.830 And basically, the key point to keep in mind, and that I'd 00:41:24.830 --> 00:41:27.980 like to stress from what was just said on the prior slide, 00:41:27.980 --> 00:41:30.110 is that what you'll see throughout this 00:41:30.110 --> 00:41:33.470 is that conformational changes are coupled 00:41:33.470 --> 00:41:35.640 to these rapid chemical steps. 00:41:35.640 --> 00:41:37.490 And the chemical steps are irreversible, 00:41:37.490 --> 00:41:40.010 this GTP hydrolysis. 00:41:40.010 --> 00:41:41.930 So what do we see? 00:41:41.930 --> 00:41:44.330 We begin with initial selection. 00:41:44.330 --> 00:41:46.880 Here, we have our ribosome, and there's a polypeptide 00:41:46.880 --> 00:41:48.550 being synthesized. 00:41:48.550 --> 00:41:50.790 Here's the ternary complex-- 00:41:50.790 --> 00:41:54.260 EF-Tu, GTP, and the aminoacyl tRNA. 00:41:54.260 --> 00:41:55.970 So there's an initial binding step 00:41:55.970 --> 00:41:59.480 that's governed by k1 in the forward direction and k minus 1 00:41:59.480 --> 00:42:02.480 in the back direction, and said before, this 00:42:02.480 --> 00:42:05.030 is independent of the mRNA. 00:42:05.030 --> 00:42:06.190 So what happens? 00:42:06.190 --> 00:42:09.140 The ternary complex binds the ribosome, 00:42:09.140 --> 00:42:13.400 there's sampling in the A-site of the anticodon, 00:42:13.400 --> 00:42:17.570 and then there is a step described as codon recognition 00:42:17.570 --> 00:42:19.950 with k2 and k minus 2. 00:42:19.950 --> 00:42:20.450 OK? 00:42:20.450 --> 00:42:23.690 In this scheme, if an arrow is colored, 00:42:23.690 --> 00:42:26.780 red arrow indicates the rate is greater 00:42:26.780 --> 00:42:29.310 for near-cognate than cognate. 00:42:29.310 --> 00:42:29.810 OK? 00:42:29.810 --> 00:42:35.120 Which means in the event here of a cognate pair, 00:42:35.120 --> 00:42:38.180 this is going to push forward in the forward direction. 00:42:38.180 --> 00:42:42.080 If it's near-cognate, this back step 00:42:42.080 --> 00:42:45.580 has a greater rate of about 350-fold. 00:42:45.580 --> 00:42:46.080 OK? 00:42:46.080 --> 00:42:48.950 So we're going to end up back here. 00:42:48.950 --> 00:42:50.930 With cognate recognition, next, we 00:42:50.930 --> 00:42:55.040 have GTPase activation, again, forward and reverse. 00:42:55.040 --> 00:42:58.760 Green indicates the rate is greater for a cognate match 00:42:58.760 --> 00:43:01.170 than near-cognate. 00:43:01.170 --> 00:43:03.680 So if it's the correct anticodon, 00:43:03.680 --> 00:43:05.870 it's going to plow through to here. 00:43:05.870 --> 00:43:08.340 We have GTPase activation. 00:43:08.340 --> 00:43:11.090 And then what happens down here? 00:43:11.090 --> 00:43:13.025 We have a GTP hydrolysis step. 00:43:15.840 --> 00:43:20.730 We have a conformational change in EF-Tu, and then what? 00:43:20.730 --> 00:43:26.640 We can have accommodation such that the tRNA was installed 00:43:26.640 --> 00:43:28.710 fully into the A-site and then rapid 00:43:28.710 --> 00:43:32.460 peptide bond formation or peptidyl transfer. 00:43:32.460 --> 00:43:36.390 The ribosome has one last chance to correct a mistake. 00:43:36.390 --> 00:43:40.740 So you can imagine that after GTP hydrolysis, 00:43:40.740 --> 00:43:45.540 after the conformational change in EF-Tu and its dissociation, 00:43:45.540 --> 00:43:49.140 there's a last chance at rejection here. 00:43:49.140 --> 00:43:54.380 Realize that step is occurring at the expense of GTP here. 00:43:59.850 --> 00:44:04.710 So in thinking about how to deconvolute this model 00:44:04.710 --> 00:44:11.860 or how to design experiments to test this model, 00:44:11.860 --> 00:44:13.450 there's a lot that needs to be done. 00:44:13.450 --> 00:44:13.950 Right? 00:44:13.950 --> 00:44:18.480 A lot of rates that need to be measured, 00:44:18.480 --> 00:44:21.700 a lot of different species along the way with the ribosome. 00:44:21.700 --> 00:44:22.200 Right? 00:44:22.200 --> 00:44:24.960 So how do you get a read out of each of these steps? 00:44:24.960 --> 00:44:28.440 That's what we'll be focused on in recitation this week 00:44:28.440 --> 00:44:29.370 and next here. 00:44:33.220 --> 00:44:39.160 So here are some more details on this initial binding process 00:44:39.160 --> 00:44:45.460 with some information related to the k1s and k minus 1s here. 00:44:45.460 --> 00:44:48.820 That's provided to help navigate the reading this week 00:44:48.820 --> 00:44:50.010 for recitation here. 00:44:54.680 --> 00:44:59.440 So what happens in the GTPase center of EF-Tu? 00:45:03.670 --> 00:45:05.625 What are some of these conformational changes? 00:45:08.440 --> 00:45:15.010 And effectively, there are conformational changes 00:45:15.010 --> 00:45:19.730 in the decoding center that are critical on one hand. 00:45:19.730 --> 00:45:21.280 So that's not at the GTPase center, 00:45:21.280 --> 00:45:24.340 but first asking what's happening when the mRNA 00:45:24.340 --> 00:45:26.860 and tRNA codon interact? 00:45:26.860 --> 00:45:31.090 And then what's happening in the GTPase center here? 00:45:31.090 --> 00:45:35.410 So just to note, not shown in the slide in terms 00:45:35.410 --> 00:45:44.860 of the decoding center. 00:46:09.660 --> 00:46:19.350 OK, what we need to be focusing on are changes in the 16S RNA, 00:46:19.350 --> 00:46:26.520 and effectively, I'll just point out three of the positions. 00:46:26.520 --> 00:46:46.730 So we have A1492, A1493, and G530 of the 16S, here. 00:46:46.730 --> 00:46:52.550 And what we find is that these bases effectively 00:46:52.550 --> 00:47:01.275 change conformation with a cognate match. 00:47:04.140 --> 00:47:07.140 And they effectively flip and interact 00:47:07.140 --> 00:47:09.990 with that cognate anticodon to help 00:47:09.990 --> 00:47:12.780 stabilize the codon-anticodon interaction. 00:47:35.480 --> 00:47:39.740 So this stabilizes the codon-anticodon interaction, 00:47:39.740 --> 00:47:43.730 and that stabilization accelerates the forward steps. 00:47:43.730 --> 00:47:48.200 So that results in this acceleration of GTP hydrolysis. 00:47:48.200 --> 00:47:50.750 So then the question is, what's happening 00:47:50.750 --> 00:47:54.380 in the GTPase center of EF-Tu? 00:47:54.380 --> 00:47:58.400 Because there has to be a change in conformation at that GTPase 00:47:58.400 --> 00:48:01.970 center 70 Angstroms away to allow for GTP 00:48:01.970 --> 00:48:05.060 hydrolysis to occur, and somehow, 00:48:05.060 --> 00:48:09.000 that all has to be signaled from here to there. 00:48:09.000 --> 00:48:16.070 So what we're looking at here is an excerpt 00:48:16.070 --> 00:48:20.600 of the structures looking at this GTPase center, 00:48:20.600 --> 00:48:23.120 and so what do we see? 00:48:23.120 --> 00:48:29.720 Effectively, two residues, so isoleucine-60 and valine-20 00:48:29.720 --> 00:48:34.670 have been described as a hydrophobic gate in the GTPase 00:48:34.670 --> 00:48:36.140 center. 00:48:36.140 --> 00:48:41.000 OK, and the idea is that if this gate is closed, 00:48:41.000 --> 00:48:44.840 it prevents a certain histidine residue, histidine-84, 00:48:44.840 --> 00:48:49.160 from activating a water molecule which then allows for the GTP 00:48:49.160 --> 00:48:51.140 to be hydrolyzed. 00:48:51.140 --> 00:48:54.320 OK, but if there's a change in conformation, 00:48:54.320 --> 00:48:58.950 and this gate opens, that chemistry can occur. 00:48:58.950 --> 00:49:04.370 So what are we looking at here in these structures? 00:49:04.370 --> 00:49:08.240 Effectively here, we have the two hydrophobic residues 00:49:08.240 --> 00:49:12.380 of the gate, so valine-20, isoleucine-60, 00:49:12.380 --> 00:49:16.250 and here's that histidine-84 I told you about, 00:49:16.250 --> 00:49:18.400 and what is this, GTPCP? 00:49:23.370 --> 00:49:28.240 So what we have there is a nonhydrolizable GTP analog. 00:49:28.240 --> 00:49:31.380 These types of molecules are very 00:49:31.380 --> 00:49:35.220 helpful in terms of getting structural information, 00:49:35.220 --> 00:49:38.770 in terms of doing certain types of biochemical experiments. 00:49:38.770 --> 00:49:39.270 OK? 00:49:39.270 --> 00:49:42.780 So effectively, we can have an analog bound that cannot 00:49:42.780 --> 00:49:45.210 hydrolyze. 00:49:45.210 --> 00:49:47.970 What are we looking at here? 00:49:47.970 --> 00:49:53.410 Here, we're looking at the, say, activated species, 00:49:53.410 --> 00:49:55.090 and what do we see? 00:49:55.090 --> 00:49:58.170 We see that this histidine has changed position. 00:49:58.170 --> 00:50:04.650 So here, it's flipped that way, here this way and here, 00:50:04.650 --> 00:50:11.760 what we see is a view with EF-Tu in the GTP-bound form. 00:50:11.760 --> 00:50:16.440 So the idea is that overall conformational changes that 00:50:16.440 --> 00:50:21.960 occur 70 Angstroms away, because of codon-anticodon recognition, 00:50:21.960 --> 00:50:24.300 effectively signal conformational changes 00:50:24.300 --> 00:50:28.860 in GTPase center that allow for GTP hydrolysis to occur 00:50:28.860 --> 00:50:33.360 and things to move in the forward direction there. 00:50:33.360 --> 00:50:36.150 So that's where we'll close for today. 00:50:36.150 --> 00:50:39.330 On Friday, we'll continue moving forward in this elongation 00:50:39.330 --> 00:50:42.060 cycle, and starting in recitation tomorrow, 00:50:42.060 --> 00:50:46.110 you'll look at experiments that allowed for this kinetic model 00:50:46.110 --> 00:50:50.450 to be analyzed and presented. 00:50:50.450 --> 00:50:52.570 You really need to come to recitation this week 00:50:52.570 --> 00:50:53.500 and read the paper. 00:50:53.500 --> 00:50:56.042 JOANNE STUBBE: And you need to read the paper more than once. 00:50:56.042 --> 00:50:57.283 It's a complicated paper. 00:50:57.283 --> 00:50:58.950 ELIZABETH NOLAN: That's on [INAUDIBLE].. 00:50:58.950 --> 00:51:01.380 It's a complicated paper which is why we have 00:51:01.380 --> 00:51:03.150 two weeks of recitation for it. 00:51:03.150 --> 00:51:06.090 There's a lot of methods, and I'll also point out 00:51:06.090 --> 00:51:10.230 that problem set three has very similar types of experiments, 00:51:10.230 --> 00:51:13.200 but it's looking at EFG instead of EF-Tu. 00:51:13.200 --> 00:51:17.070 So spending the time on this paper in the upcoming weeks 00:51:17.070 --> 00:51:19.310 is really important.