1 00:00:00,500 --> 00:00:02,820 The following content is provided under a Creative 2 00:00:02,820 --> 00:00:04,360 Commons license. 3 00:00:04,360 --> 00:00:06,660 Your support will help MIT OpenCourseWare 4 00:00:06,660 --> 00:00:11,020 continue to offer high-quality educational resources for free. 5 00:00:11,020 --> 00:00:13,650 To make a donation or view additional materials 6 00:00:13,650 --> 00:00:17,600 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,600 --> 00:00:18,540 at ocw.mit.edu. 8 00:00:25,745 --> 00:00:27,120 ELIZABETH NOLAN: So last time, we 9 00:00:27,120 --> 00:00:31,140 were talking about these aminoacyl tRNA synthetases that 10 00:00:31,140 --> 00:00:33,960 are responsible for attaching amino acid 11 00:00:33,960 --> 00:00:37,800 monomers to the three prime end of tRNAs. 12 00:00:37,800 --> 00:00:42,720 And we were looking at the isoleucyl aminoacyl tRNA 13 00:00:42,720 --> 00:00:47,160 synthetase as an example, looking at experiments that 14 00:00:47,160 --> 00:00:48,820 were done to study mechanisms. 15 00:00:48,820 --> 00:00:53,520 So recall, we left off having discussed a two-step model, 16 00:00:53,520 --> 00:00:56,730 where there's an intermediate, an amino adenylate formed. 17 00:00:56,730 --> 00:00:58,410 And then, in the second step, there's 18 00:00:58,410 --> 00:01:02,670 transfer of that amino acid to the tRNA by the aaRS. 19 00:01:02,670 --> 00:01:05,670 And so we looked at some data from steady-state kinetic 20 00:01:05,670 --> 00:01:06,950 experiments. 21 00:01:06,950 --> 00:01:12,050 Recall that a C14 radiolabel was used to watch transfer, 22 00:01:12,050 --> 00:01:16,020 and then we closed discussing an ATP-PPi exchange assay which 23 00:01:16,020 --> 00:01:19,140 gave evidence for formation of that amino adenylate 24 00:01:19,140 --> 00:01:20,350 intermediate. 25 00:01:20,350 --> 00:01:20,850 Right? 26 00:01:20,850 --> 00:01:25,050 And then, lastly, we talked about use of a stopped-flow 27 00:01:25,050 --> 00:01:28,800 to do experiments that allow you to look at early points 28 00:01:28,800 --> 00:01:30,490 within a reaction. 29 00:01:30,490 --> 00:01:33,960 And so what we're going to do is to close these discussions 30 00:01:33,960 --> 00:01:36,540 of experiments and this aaRS mechanism 31 00:01:36,540 --> 00:01:38,490 is just look at one more experiment that 32 00:01:38,490 --> 00:01:42,870 was done to further probe the rate-determining step 33 00:01:42,870 --> 00:01:46,020 of this reaction using the stopped-flow. 34 00:01:46,020 --> 00:01:47,010 OK? 35 00:01:47,010 --> 00:01:53,250 And so this experiment pertains more to reaction kinetics, 36 00:01:53,250 --> 00:01:55,500 and the question is, let's monitor 37 00:01:55,500 --> 00:02:00,240 transfer of the amino acid to the tRNA 38 00:02:00,240 --> 00:02:01,860 by another method here. 39 00:02:20,590 --> 00:02:25,450 These experiments were set up in two different ways 40 00:02:25,450 --> 00:02:28,000 depending on what components were mixed. 41 00:02:28,000 --> 00:02:33,670 And if you just rewind to Monday and recall the ATP-PPi exchange 42 00:02:33,670 --> 00:02:37,510 assay and the steps in that assay, in that we showed 43 00:02:37,510 --> 00:02:41,800 that the amino adenylate intermediate remained 44 00:02:41,800 --> 00:02:43,840 bound to the enzyme there. 45 00:02:43,840 --> 00:02:47,350 Recall then only PPi was released in that assay. 46 00:02:47,350 --> 00:02:50,170 And so in these experiments, the fact 47 00:02:50,170 --> 00:02:53,080 that the amino adenylate can remain bound 48 00:02:53,080 --> 00:02:54,310 was taken advantage of. 49 00:02:54,310 --> 00:02:55,720 And the researchers were actually 50 00:02:55,720 --> 00:03:00,580 able to have a preformed complex there, so basically 51 00:03:00,580 --> 00:03:02,410 starting after step two. 52 00:03:02,410 --> 00:03:08,760 So in experiment one, how I'm going to show 53 00:03:08,760 --> 00:03:10,980 these is by drawing the two syringes 54 00:03:10,980 --> 00:03:13,950 and listing the components of each syringe. 55 00:03:13,950 --> 00:03:16,380 And this is a good way for setting up problems 56 00:03:16,380 --> 00:03:17,880 within the problem sets, thinking 57 00:03:17,880 --> 00:03:20,290 about stopped-flow experiments. 58 00:03:20,290 --> 00:03:23,160 So the question is what are we going to mix? 59 00:03:23,160 --> 00:03:28,910 So we have syringe one and syringe two, 60 00:03:28,910 --> 00:03:32,820 and recall that these go to some mixer. 61 00:03:32,820 --> 00:03:35,430 So the two solutions can be rapidly mixed, 62 00:03:35,430 --> 00:03:39,640 and that's where the chemistry is going to happen. 63 00:03:39,640 --> 00:03:43,590 So in experiment one, in syringe one, 64 00:03:43,590 --> 00:03:46,540 what we have is the purified complex. 65 00:03:46,540 --> 00:03:47,040 OK? 66 00:03:47,040 --> 00:03:56,910 So we have C-14 labeled isoleucine-AMP 67 00:03:56,910 --> 00:04:02,370 bound to the aminoacyl tRNA synthetase 68 00:04:02,370 --> 00:04:10,130 of a purified complex, here. 69 00:04:10,130 --> 00:04:13,490 And then in this other syringe two, what we have is the tRNA. 70 00:04:17,453 --> 00:04:18,920 OK? 71 00:04:18,920 --> 00:04:21,089 So imagine these are rapidly mixed. 72 00:04:21,089 --> 00:04:24,260 There'll be transfer of the radiolabeled isoleucine 73 00:04:24,260 --> 00:04:28,790 to the tRNA, and so formation of that aminoacyl tRNA 74 00:04:28,790 --> 00:04:30,450 can be monitored. 75 00:04:30,450 --> 00:04:30,980 OK? 76 00:04:30,980 --> 00:04:38,830 In the second experiment, we have just theme in variation, 77 00:04:38,830 --> 00:04:41,440 and if you're interested in more details, 78 00:04:41,440 --> 00:04:43,940 the reference is provided in the slides. 79 00:04:47,970 --> 00:04:54,860 So again, in syringe two, we have the tRNA, 80 00:04:54,860 --> 00:05:02,900 and in syringe one, what will be combined 81 00:05:02,900 --> 00:05:06,650 are the components here. 82 00:05:06,650 --> 00:05:07,150 OK? 83 00:05:10,450 --> 00:05:16,420 So then, the question is, in each case, what do we see? 84 00:05:16,420 --> 00:05:20,182 And those data are presented here from the paper, 85 00:05:20,182 --> 00:05:21,640 and there's some additional details 86 00:05:21,640 --> 00:05:23,890 about the experimental setup. 87 00:05:23,890 --> 00:05:27,130 So effectively, what we're looking at on the y-axis 88 00:05:27,130 --> 00:05:30,400 is the amount of tRNA that's been modified. 89 00:05:30,400 --> 00:05:33,610 So tRNA acylation measured by transfer 90 00:05:33,610 --> 00:05:36,820 of the radiolabel versus time. 91 00:05:36,820 --> 00:05:39,010 And in the black circles, we have the data 92 00:05:39,010 --> 00:05:43,750 from experiment one, shown here, and in the open circles, 93 00:05:43,750 --> 00:05:48,290 we have the data from experiment two. 94 00:05:48,290 --> 00:05:53,520 So what is the conclusion from these data? 95 00:05:53,520 --> 00:05:57,060 And this value here is not similar to something we've 96 00:05:57,060 --> 00:05:59,865 seen before in this system. 97 00:06:11,190 --> 00:06:15,330 Both experimental setups are giving the same result. Right? 98 00:06:15,330 --> 00:06:18,210 Effectively, these data are superimposable, 99 00:06:18,210 --> 00:06:20,968 and they can be fit the same. 100 00:06:20,968 --> 00:06:23,385 So what does that tell us about the rate-determining step? 101 00:06:26,075 --> 00:06:29,293 AUDIENCE: [INAUDIBLE] versus forming the intermediate. 102 00:06:29,293 --> 00:06:30,210 ELIZABETH NOLAN: Yeah. 103 00:06:30,210 --> 00:06:30,710 Right. 104 00:06:30,710 --> 00:06:33,250 Aminoacylation of tRNA is the rate-determining step. 105 00:06:33,250 --> 00:06:37,850 So some of you suggested that in class on Monday. 106 00:06:37,850 --> 00:06:38,350 Right? 107 00:06:38,350 --> 00:06:40,090 So that's the case here. 108 00:06:40,090 --> 00:06:40,720 OK? 109 00:06:40,720 --> 00:06:43,630 So formation of the intermediate is much more rapid 110 00:06:43,630 --> 00:06:47,680 than acylation of the tRNA here. 111 00:06:47,680 --> 00:06:51,580 So we've examined now the mechanism 112 00:06:51,580 --> 00:06:53,830 in terms of getting the amino acid onto the tRNA. 113 00:06:56,350 --> 00:06:59,330 What do we need to think about next here? 114 00:06:59,330 --> 00:07:03,660 So what we need to think about is fidelity. 115 00:07:03,660 --> 00:07:06,790 OK, and we've looked at the overall rate of error 116 00:07:06,790 --> 00:07:09,970 in protein biosynthesis, how often errors occur 117 00:07:09,970 --> 00:07:12,220 on the order of 10 to the 3. 118 00:07:12,220 --> 00:07:17,740 So how is the correct amino acid loaded onto the correct tRNA? 119 00:07:17,740 --> 00:07:23,830 Each tRNA has an anticodon that is a cognate pair with a codon. 120 00:07:23,830 --> 00:07:26,530 And so different tRNAs need to have 121 00:07:26,530 --> 00:07:28,680 different amino acids attached. 122 00:07:28,680 --> 00:07:30,050 OK, and what does that mean? 123 00:07:30,050 --> 00:07:33,310 That means, in general, there's a dedicated aminoacyl tRNA 124 00:07:33,310 --> 00:07:37,690 synthetase for each amino acid, in general here. 125 00:07:37,690 --> 00:07:40,510 So how are amino acids with similar side chains 126 00:07:40,510 --> 00:07:43,150 differentiated by these enzymes? 127 00:07:43,150 --> 00:07:46,060 And is it possible for an incorrect amino acid 128 00:07:46,060 --> 00:07:48,310 to get loaded onto a tRNA? 129 00:07:48,310 --> 00:07:52,400 And if that happens, what are the consequences? 130 00:07:52,400 --> 00:07:56,290 So we're going to examine fidelity some here. 131 00:07:56,290 --> 00:08:01,090 And as background, an observation made, 132 00:08:01,090 --> 00:08:04,810 say from studies like that ATP-PPi exchange assay, 133 00:08:04,810 --> 00:08:09,220 is that some aminoacyl tRNA synthetases can activate 134 00:08:09,220 --> 00:08:12,160 multiple amino acids, so not only the one 135 00:08:12,160 --> 00:08:15,230 they're supposed to activate but also others. 136 00:08:15,230 --> 00:08:16,520 So what does that mean? 137 00:08:16,520 --> 00:08:18,820 That means that the enzyme can bind 138 00:08:18,820 --> 00:08:22,690 and activate effectively the wrong amino acid, 139 00:08:22,690 --> 00:08:24,940 and if we think about fidelity, we 140 00:08:24,940 --> 00:08:27,970 can think about this as being a problem here. 141 00:08:27,970 --> 00:08:30,530 So what happens? 142 00:08:30,530 --> 00:08:33,970 What happens is that these enzymes have an editing 143 00:08:33,970 --> 00:08:40,450 function, and they're able to sense if a wrong amino acid is 144 00:08:40,450 --> 00:08:41,470 activated. 145 00:08:41,470 --> 00:08:44,290 And then they have a way to deal with it, 146 00:08:44,290 --> 00:08:46,450 and this is by hydrolysis. 147 00:08:46,450 --> 00:08:47,440 OK? 148 00:08:47,440 --> 00:08:53,890 And so let's consider an example, for instance, just 149 00:08:53,890 --> 00:08:54,895 similar side chains. 150 00:09:07,350 --> 00:09:28,240 So if we just consider, for instance, valine, isoleucine, 151 00:09:28,240 --> 00:09:32,590 and threonine, these will be the players for our discussion. 152 00:09:40,526 --> 00:09:41,530 OK? 153 00:09:41,530 --> 00:09:43,930 They're different, but they're not too different. 154 00:09:43,930 --> 00:09:45,076 Right? 155 00:09:45,076 --> 00:09:46,270 Oops, sorry about this. 156 00:09:46,270 --> 00:09:48,520 We're missing a methyl. 157 00:09:48,520 --> 00:09:52,580 Valine, an isoleucine, we have a difference of a methyl group. 158 00:09:52,580 --> 00:09:55,630 Threonine, we have this OH group. 159 00:09:55,630 --> 00:09:56,230 Right? 160 00:09:56,230 --> 00:09:57,910 And we can just ask the question, 161 00:09:57,910 --> 00:10:02,045 for instance, how is valine differentiated from isoleucine 162 00:10:02,045 --> 00:10:05,280 or threonine here? 163 00:10:05,280 --> 00:10:07,720 And so as an example, what's found 164 00:10:07,720 --> 00:10:13,680 is, if we consider our friend that we studied 165 00:10:13,680 --> 00:10:17,130 for the mechanism here, what we find 166 00:10:17,130 --> 00:10:27,730 is that this binds and activates isoleucine, as we saw, 167 00:10:27,730 --> 00:10:35,470 but it will also bind and activate valine here. 168 00:10:35,470 --> 00:10:39,290 And effectively, if this happens, 169 00:10:39,290 --> 00:10:46,610 we have a mismatch, because the end result 170 00:10:46,610 --> 00:10:54,707 will be isoleucine-RS with valine AMP bound here. 171 00:10:54,707 --> 00:10:55,207 OK? 172 00:10:58,410 --> 00:11:02,560 And what's found is that the catalytic efficiency or Kcat 173 00:11:02,560 --> 00:11:07,400 over Km, in this case, is about 150-fold 174 00:11:07,400 --> 00:11:11,660 less than the native substrate. 175 00:11:11,660 --> 00:11:17,250 So that doesn't account for the 10 to the 3 error rate here. 176 00:11:17,250 --> 00:11:19,680 So we need more specificity. 177 00:11:19,680 --> 00:11:21,600 So what's going on? 178 00:11:21,600 --> 00:11:26,760 So we're going to consider this editing function and a model 179 00:11:26,760 --> 00:11:30,390 that's often used to describe how these aaRS do 180 00:11:30,390 --> 00:11:33,540 editing is one of two sieves. 181 00:11:33,540 --> 00:11:35,650 These enzymes don't actually have a sieve. 182 00:11:35,650 --> 00:11:39,430 It's just a conceptual way to think about it. 183 00:11:39,430 --> 00:11:41,670 So this double-sieve editing model 184 00:11:41,670 --> 00:11:46,380 involves a first sieve which is considered to be a course one. 185 00:11:46,380 --> 00:11:49,680 So imagine if you have like a change sorter. 186 00:11:49,680 --> 00:11:51,660 It will let the quarters through as well as 187 00:11:51,660 --> 00:11:54,150 the and dimes and the pennies. 188 00:11:54,150 --> 00:11:56,160 There's some sort of discrimination 189 00:11:56,160 --> 00:12:00,030 of amino acids based on size, and then 190 00:12:00,030 --> 00:12:03,870 depending what gets through this first sieve or gate, 191 00:12:03,870 --> 00:12:07,170 there's a second sieve which is considered to be a fine one. 192 00:12:07,170 --> 00:12:12,540 And this one can differentiate perhaps on the basis of size 193 00:12:12,540 --> 00:12:17,710 or maybe on hydrophilicity or hydrophobic of the side chain. 194 00:12:17,710 --> 00:12:22,560 So effectively, if an incorrect amino acid passes through this 195 00:12:22,560 --> 00:12:25,860 first sieve-- so in other words, if it binds to the enzyme 196 00:12:25,860 --> 00:12:27,690 and becomes activated-- 197 00:12:27,690 --> 00:12:29,320 hydrolytic editing will occur. 198 00:12:29,320 --> 00:12:29,820 OK? 199 00:12:29,820 --> 00:12:32,640 So think about hydrolysis in terms of having 200 00:12:32,640 --> 00:12:35,440 breakdown of these species. 201 00:12:35,440 --> 00:12:38,370 So if the incorrect amino acid passes through 202 00:12:38,370 --> 00:12:41,610 and is adenylated, there'll be hydrolysis. 203 00:12:41,610 --> 00:12:46,470 So let's consider some examples so the first example here we 204 00:12:46,470 --> 00:12:51,870 can consider this guy and isoleucine and valine. 205 00:12:51,870 --> 00:12:57,150 So as I mentioned, this aaRS will activate both. 206 00:12:57,150 --> 00:13:01,530 So in this case, the first sieve can't differentiate isoleucine 207 00:13:01,530 --> 00:13:02,440 from valine. 208 00:13:02,440 --> 00:13:05,970 They have similar sizes according to this aaRS. 209 00:13:05,970 --> 00:13:09,180 But then what happens here in the second sieve, 210 00:13:09,180 --> 00:13:13,560 isoleucine is too big, and so there's no hydrolysis, 211 00:13:13,560 --> 00:13:20,100 and it moves on to form the desired charged tRNA. 212 00:13:20,100 --> 00:13:23,280 In contrast, valine's a bit smaller. 213 00:13:23,280 --> 00:13:29,610 It passes through the sieve, and it ends up being hydrolyzed. 214 00:13:29,610 --> 00:13:34,260 So these aaRS also have an editing domain, 215 00:13:34,260 --> 00:13:35,640 and this editing domain, as we'll 216 00:13:35,640 --> 00:13:37,830 see in a few slides in a structure, 217 00:13:37,830 --> 00:13:43,150 is responsible for this hydrolysis, so stated here. 218 00:13:43,150 --> 00:13:43,660 Right? 219 00:13:43,660 --> 00:13:46,090 Different sites, so there's an aminoacylation site 220 00:13:46,090 --> 00:13:48,580 and an editing site here. 221 00:13:48,580 --> 00:13:53,890 So valine can reach the editing site, but isoleucine cannot. 222 00:13:53,890 --> 00:13:55,120 So how do you predict? 223 00:13:57,790 --> 00:13:59,620 Just to keep in mind, every enzyme 224 00:13:59,620 --> 00:14:01,810 is different in terms of the model 225 00:14:01,810 --> 00:14:05,360 for discrimination and also when editing occurs. 226 00:14:05,360 --> 00:14:07,150 So you really need to look at the data 227 00:14:07,150 --> 00:14:13,130 when the data is presented to you to sort out how this works. 228 00:14:13,130 --> 00:14:14,630 Let's just look at another example 229 00:14:14,630 --> 00:14:16,500 with a cartoon depiction. 230 00:14:16,500 --> 00:14:19,010 So this is for the valine RS, and we're 231 00:14:19,010 --> 00:14:22,100 going to consider the three amino acids here-- 232 00:14:22,100 --> 00:14:24,890 valine, threonine, and isoleucine. 233 00:14:24,890 --> 00:14:29,180 So in green, we have the first sieve, 234 00:14:29,180 --> 00:14:31,110 and this is based on size. 235 00:14:31,110 --> 00:14:33,680 So what do we see in this cartoon? 236 00:14:33,680 --> 00:14:36,500 So threonine and valine make it through, 237 00:14:36,500 --> 00:14:38,030 but isoleucine does not. 238 00:14:38,030 --> 00:14:42,080 It's rejected right away, so it's never activated. 239 00:14:42,080 --> 00:14:46,130 So if threonine and valine pass through, what happens? 240 00:14:46,130 --> 00:14:50,810 We see each one is activated as the amino adenylate, and then 241 00:14:50,810 --> 00:14:52,110 what? 242 00:14:52,110 --> 00:14:54,050 Well, valine, we want to transfer the valine 243 00:14:54,050 --> 00:14:56,210 to the tRNA, so it can move on and help 244 00:14:56,210 --> 00:14:58,640 with protein synthesis. 245 00:14:58,640 --> 00:15:01,130 If threonine's activated, and here we 246 00:15:01,130 --> 00:15:03,860 see that threonine is transferred to the tRNA 247 00:15:03,860 --> 00:15:09,340 as well, this is hydrolyzed by the editing site, in this case. 248 00:15:09,340 --> 00:15:12,470 So the threonine is removed from the tRNA 249 00:15:12,470 --> 00:15:14,750 with the anticodon for valine. 250 00:15:14,750 --> 00:15:17,540 Right, so think about the ester bonds 251 00:15:17,540 --> 00:15:20,030 that we saw last time in terms of the three prime end 252 00:15:20,030 --> 00:15:22,850 of the tRNA being modified and the chemistry that 253 00:15:22,850 --> 00:15:27,410 will happen there to result in hydrolysis of and release 254 00:15:27,410 --> 00:15:28,700 of the amino acid here. 255 00:15:31,560 --> 00:15:37,400 So what that cartoon hints to is that the hydrolysis can 256 00:15:37,400 --> 00:15:39,690 occur at different steps. 257 00:15:39,690 --> 00:15:44,540 So we can have hydrolysis that is pre-transfer, 258 00:15:44,540 --> 00:15:51,050 which means the editing occurs before the tRNA is modified. 259 00:15:51,050 --> 00:15:53,063 Or we can have post-transfer editing 260 00:15:53,063 --> 00:15:54,980 which is what we saw in the prior slide, where 261 00:15:54,980 --> 00:15:58,040 the editing and hydrolysis occurs after the amino acid 262 00:15:58,040 --> 00:16:00,900 monomer is transferred to the tRNA. 263 00:16:00,900 --> 00:16:01,400 OK? 264 00:16:01,400 --> 00:16:06,090 And this schematic here depicts that, so what do we have? 265 00:16:06,090 --> 00:16:13,280 We have the aaRS responsible for modifying tRNA for isoleucine, 266 00:16:13,280 --> 00:16:17,780 and we combine that with valine, the wrong amino acid, and ATP. 267 00:16:17,780 --> 00:16:18,460 What happens? 268 00:16:18,460 --> 00:16:19,880 So E is for enzyme. 269 00:16:19,880 --> 00:16:24,200 We have formulation of the amino adenylate intermediate. 270 00:16:24,200 --> 00:16:28,550 Here's the tRNA with the anticodon for isoleucine. 271 00:16:28,550 --> 00:16:29,450 What happens? 272 00:16:29,450 --> 00:16:32,990 So we have this complex form in this depiction. 273 00:16:32,990 --> 00:16:35,750 Pre-transfer editing would occur at this stage, 274 00:16:35,750 --> 00:16:39,680 before the valine is transferred to the tRNA, and so 275 00:16:39,680 --> 00:16:41,100 what do we see? 276 00:16:41,100 --> 00:16:44,480 We see breakdown and these species. 277 00:16:44,480 --> 00:16:47,690 If the valine is transferred to the tRNA, 278 00:16:47,690 --> 00:16:49,280 we don't want this, because that would 279 00:16:49,280 --> 00:16:52,310 result in this reading of the genetic code. 280 00:16:52,310 --> 00:16:57,140 Post-transfer editing, this species here is hydrolyzed. 281 00:16:57,140 --> 00:17:00,530 So whether pre or post-transfer editing occurs 282 00:17:00,530 --> 00:17:04,579 is going to depend on the aminoacyl tRNA synthetase, 283 00:17:04,579 --> 00:17:07,430 and some can use both mechanisms. 284 00:17:07,430 --> 00:17:09,520 That's what we're seeing here. 285 00:17:09,520 --> 00:17:10,160 OK? 286 00:17:10,160 --> 00:17:13,190 Some only use one, for instance, the valine RS 287 00:17:13,190 --> 00:17:16,740 only uses a post-transfer editing mechanism. 288 00:17:16,740 --> 00:17:19,460 So when presented with the data, look at the data 289 00:17:19,460 --> 00:17:23,089 and see what species is being hydrolyzed. 290 00:17:23,089 --> 00:17:26,000 And if both are, how did the steady-state kinetics, 291 00:17:26,000 --> 00:17:28,760 for instance, compare? 292 00:17:28,760 --> 00:17:32,840 Just to take a look in the context of a structure of one 293 00:17:32,840 --> 00:17:34,580 of these aaRS. 294 00:17:34,580 --> 00:17:38,420 So the sites where aminoacylation 295 00:17:38,420 --> 00:17:41,300 occur and editing occur are separated 296 00:17:41,300 --> 00:17:43,820 by about 30 Angstroms, and that's 297 00:17:43,820 --> 00:17:47,750 shown here, where we have the aminoacylation site, 298 00:17:47,750 --> 00:17:50,000 and here we have the editing site. 299 00:17:50,000 --> 00:17:54,960 That's responsible for pre and/or post-transfer editing. 300 00:17:54,960 --> 00:17:58,700 So in thinking about this and thinking 301 00:17:58,700 --> 00:18:02,780 about how one could leverage this 30 Angstrom 302 00:18:02,780 --> 00:18:05,540 separation and these two distinct sites 303 00:18:05,540 --> 00:18:12,260 in terms of experiments, what does that allow one to do? 304 00:18:12,260 --> 00:18:14,450 So imagine if you want to ask, what 305 00:18:14,450 --> 00:18:19,730 are the consequences of having aaRS that have faulty editing 306 00:18:19,730 --> 00:18:21,090 function? 307 00:18:21,090 --> 00:18:24,050 And effectively, mischarged tRNAs 308 00:18:24,050 --> 00:18:27,200 or put the wrong amino acid on a tRNA. 309 00:18:27,200 --> 00:18:30,770 What does that mean for a cell? 310 00:18:30,770 --> 00:18:33,410 There's an opportunity to do that here. 311 00:18:33,410 --> 00:18:36,950 So you could imagine mutating residues 312 00:18:36,950 --> 00:18:40,370 that are critical for editing function in the editing site. 313 00:18:40,370 --> 00:18:46,100 Such that you have an aaRS variant that can activate amino 314 00:18:46,100 --> 00:18:50,570 acids and transfer them to the tRNA but cannot edit when 315 00:18:50,570 --> 00:18:52,230 a mistake happens. 316 00:18:52,230 --> 00:18:52,730 Right? 317 00:18:52,730 --> 00:18:56,390 So you can imagine a site-directed mutagenesis, 318 00:18:56,390 --> 00:19:00,770 purifying the enzyme and doing some in vitro characterization 319 00:19:00,770 --> 00:19:02,730 to see how it behaves. 320 00:19:02,730 --> 00:19:04,940 And then you could also imagine translating this 321 00:19:04,940 --> 00:19:09,380 into a cellular context and asking say in cell culture what 322 00:19:09,380 --> 00:19:10,980 happens here? 323 00:19:10,980 --> 00:19:16,330 So basically, what are the consequences of faulty editing? 324 00:19:16,330 --> 00:19:18,940 And these types of studies have been done. 325 00:19:18,940 --> 00:19:21,540 We're not going to look at them in detail. 326 00:19:21,540 --> 00:19:26,910 But just as an overview and some concepts that will come up 327 00:19:26,910 --> 00:19:30,660 within our folding section, what's been shown 328 00:19:30,660 --> 00:19:35,520 is that a single point mutation in an editing domain of one 329 00:19:35,520 --> 00:19:38,700 of these aminoacyl tRNA synthetases 330 00:19:38,700 --> 00:19:41,550 may have deleterious consequences. 331 00:19:41,550 --> 00:19:45,960 And we can imagine that these consequences could result 332 00:19:45,960 --> 00:19:50,640 from proteins or enzymes that gain a new function 333 00:19:50,640 --> 00:19:53,830 or don't do their correct function. 334 00:19:53,830 --> 00:19:54,330 Right? 335 00:19:54,330 --> 00:19:59,880 So just imagine that some mischarged tRNAs, where 336 00:19:59,880 --> 00:20:03,690 mischarged means the wrong amino acid is attached, 337 00:20:03,690 --> 00:20:07,980 are around because of some mutant aaRS. 338 00:20:07,980 --> 00:20:10,440 And these tRNA that are mischarged 339 00:20:10,440 --> 00:20:12,810 can be delivered to the ribosome, which 340 00:20:12,810 --> 00:20:17,490 means that point mutations form within synthesized polypeptide 341 00:20:17,490 --> 00:20:18,000 chains. 342 00:20:18,000 --> 00:20:21,150 So there's some mixture where some of these proteins 343 00:20:21,150 --> 00:20:24,450 are native, and others are mutant, 344 00:20:24,450 --> 00:20:28,050 and what might happen here in terms of consequences? 345 00:20:28,050 --> 00:20:31,510 So native protein will go on and do its job. 346 00:20:31,510 --> 00:20:35,040 Imagine there's some mutant protein here 347 00:20:35,040 --> 00:20:39,020 that's altered in some way, and these are just some examples 348 00:20:39,020 --> 00:20:41,560 of possible outcomes. 349 00:20:41,560 --> 00:20:44,580 So maybe there's a breakdown of some essential cellular 350 00:20:44,580 --> 00:20:47,130 process. 351 00:20:47,130 --> 00:20:50,820 Here, we have triggering of autoimmune-like responses, 352 00:20:50,820 --> 00:20:53,280 things that are not good. 353 00:20:53,280 --> 00:20:55,560 What if these mutant proteins misfold? 354 00:20:55,560 --> 00:21:00,660 So they can't form their correct fold, 355 00:21:00,660 --> 00:21:02,850 and fold is important for function. 356 00:21:02,850 --> 00:21:04,800 Maybe there's aggregation. 357 00:21:04,800 --> 00:21:10,650 Maybe there's stress on the proteasome, ER response, 358 00:21:10,650 --> 00:21:13,690 unfolded protein response, cell death. 359 00:21:13,690 --> 00:21:16,410 So fidelity's important. 360 00:21:19,420 --> 00:21:23,960 And just some things to think about as we close this section. 361 00:21:23,960 --> 00:21:27,700 We can consider error rates of various biological 362 00:21:27,700 --> 00:21:32,270 polymerizations, whether that be DNA replication, transcription, 363 00:21:32,270 --> 00:21:39,500 or translation, and they vary quite a bit here from this. 364 00:21:39,500 --> 00:21:45,950 And what the take-home can be by comparing these error 365 00:21:45,950 --> 00:21:51,320 rates is infrequent mistakes in decoding the mRNA 366 00:21:51,320 --> 00:21:54,620 are accepted as a source of infidelity. 367 00:21:54,620 --> 00:21:57,110 So they do occur, and they occur more frequently 368 00:21:57,110 --> 00:22:00,080 than, say, an error in replicating the DNA, 369 00:22:00,080 --> 00:22:01,110 and that makes sense. 370 00:22:01,110 --> 00:22:01,610 Right? 371 00:22:01,610 --> 00:22:03,770 If an error occurs in DNA replication, 372 00:22:03,770 --> 00:22:06,650 there's a huge problem likely compared 373 00:22:06,650 --> 00:22:09,020 to an error in translation. 374 00:22:09,020 --> 00:22:12,740 So some questions just to think about, 375 00:22:12,740 --> 00:22:14,930 answers aren't going to come up within the context 376 00:22:14,930 --> 00:22:16,190 of this course. 377 00:22:16,190 --> 00:22:18,950 But higher accuracy is important, but actually 378 00:22:18,950 --> 00:22:21,080 how much accuracy is enough? 379 00:22:21,080 --> 00:22:26,810 And there is a cost in terms of cellular energy for accuracy, 380 00:22:26,810 --> 00:22:31,550 and is it that the cell tunes its accuracy to some point that 381 00:22:31,550 --> 00:22:34,610 could be considered optimal, and are 382 00:22:34,610 --> 00:22:37,310 there benefits to translational infidelity? 383 00:22:37,310 --> 00:22:37,810 Right? 384 00:22:37,810 --> 00:22:41,600 So the prior slide showed negative consequences, 385 00:22:41,600 --> 00:22:44,870 but are there benefits? 386 00:22:44,870 --> 00:22:51,110 So that discussion, we'll close considering 387 00:22:51,110 --> 00:22:55,670 how the amino acids get attached to tRNAs, 388 00:22:55,670 --> 00:22:59,688 and so where we're moving to now is the elongation cycle. 389 00:22:59,688 --> 00:23:02,556 AUDIENCE: So is there a specific part of the cytoplasm 390 00:23:02,556 --> 00:23:04,912 where the tRNAs and the amino acids 391 00:23:04,912 --> 00:23:08,110 come together, or does this happen everywhere? 392 00:23:08,110 --> 00:23:11,860 ELIZABETH NOLAN: So I actually don't know, 393 00:23:11,860 --> 00:23:13,540 but I think of them as being everywhere 394 00:23:13,540 --> 00:23:15,040 in terms of the tRNAs. 395 00:23:15,040 --> 00:23:17,820 Because as we'll see in a few slides, 396 00:23:17,820 --> 00:23:21,490 EF-Tu, which is required for delivering 397 00:23:21,490 --> 00:23:24,190 the tRNAs to the ribosome, is highly abundant. 398 00:23:24,190 --> 00:23:26,065 At least, that's my thinking for prokaryotes. 399 00:23:26,065 --> 00:23:28,010 Do you have anything to say? 400 00:23:28,010 --> 00:23:29,950 The question was effectively are there 401 00:23:29,950 --> 00:23:35,320 certain regions of the cell where tRNAs get modified more 402 00:23:35,320 --> 00:23:36,850 than other regions? 403 00:23:36,850 --> 00:23:38,017 JOANNE STUBBE: I don't know. 404 00:23:38,017 --> 00:23:40,650 In mammalian cells, they have weirdo complexes 405 00:23:40,650 --> 00:23:43,507 with tRNA synthases that they've been around forever. 406 00:23:43,507 --> 00:23:45,340 and I still think we don't really understand 407 00:23:45,340 --> 00:23:46,280 what the function is. 408 00:23:49,184 --> 00:23:52,572 AUDIENCE: [INAUDIBLE] 409 00:23:57,798 --> 00:23:59,840 JOANNE STUBBE: Can you speak a little bit louder? 410 00:23:59,840 --> 00:24:00,650 ELIZABETH NOLAN: The question is, do we 411 00:24:00,650 --> 00:24:03,440 have information about say the distribution of tRNAs 412 00:24:03,440 --> 00:24:06,735 as being amino acid modified versus unmodified? 413 00:24:06,735 --> 00:24:11,780 AUDIENCE: I think maybe we could [INAUDIBLE] I don't know. 414 00:24:11,780 --> 00:24:14,000 ELIZABETH NOLAN: There's always a way, probably. 415 00:24:14,000 --> 00:24:14,960 Right? 416 00:24:14,960 --> 00:24:17,510 But I don't know what that distribution is either 417 00:24:17,510 --> 00:24:21,440 in terms of the percentage of tRNAs that are aminoacylated 418 00:24:21,440 --> 00:24:24,490 at any one given time. 419 00:24:24,490 --> 00:24:26,730 Yeah, just don't know. 420 00:24:26,730 --> 00:24:28,880 I think one key thing to think about 421 00:24:28,880 --> 00:24:33,440 as we come to the next part is that these tRNAs are 422 00:24:33,440 --> 00:24:35,810 bound by EF-Tu. 423 00:24:35,810 --> 00:24:39,020 So to think of them as in complex with a translation 424 00:24:39,020 --> 00:24:42,560 factor as opposed to tRNAs floating around 425 00:24:42,560 --> 00:24:44,410 in the cytoplasm, so I think that that's 426 00:24:44,410 --> 00:24:47,000 a key point of focus. 427 00:24:47,000 --> 00:24:55,940 So moving into elongation, what do we need to think about here? 428 00:24:55,940 --> 00:24:57,830 So we need to think about delivery 429 00:24:57,830 --> 00:25:00,200 of the amino acid tRNAs. 430 00:25:00,200 --> 00:25:04,580 How does the ribosome ensure that the correct aminoacyl tRNA 431 00:25:04,580 --> 00:25:05,420 is delivered? 432 00:25:05,420 --> 00:25:08,750 So we have the correct amino acid onto the tRNA, 433 00:25:08,750 --> 00:25:10,870 but we also have to get the correct amino acid 434 00:25:10,870 --> 00:25:13,010 to the ribosome. 435 00:25:13,010 --> 00:25:16,490 How is peptide bond formation catalyzed? 436 00:25:16,490 --> 00:25:20,000 What is the method by which polypeptides 437 00:25:20,000 --> 00:25:24,840 leave the ribosome, and how is translation terminated here? 438 00:25:24,840 --> 00:25:26,660 So effectively, these are all questions 439 00:25:26,660 --> 00:25:29,030 we need to address in terms of thinking about how 440 00:25:29,030 --> 00:25:31,650 the ribosome translates the genetic code 441 00:25:31,650 --> 00:25:33,560 and synthesizes the polypeptide. 442 00:25:33,560 --> 00:25:36,260 So within the notes posted on Stellar, 443 00:25:36,260 --> 00:25:40,610 there's a number of pages of definitions, so terminology 444 00:25:40,610 --> 00:25:43,790 that comes up within these discussions of the ribosome 445 00:25:43,790 --> 00:25:45,500 to refer to. 446 00:25:45,500 --> 00:25:48,650 And in terms of our translation overview slide, 447 00:25:48,650 --> 00:25:52,050 where we are now is here, in elongation. 448 00:25:52,050 --> 00:25:55,250 So we have the mRNA our 70S, and we're 449 00:25:55,250 --> 00:25:59,240 going to focus for the rest of today on thinking about EF-Tu, 450 00:25:59,240 --> 00:26:02,930 this elongation factor that's responsible for delivering 451 00:26:02,930 --> 00:26:07,970 the amino acid tRNAs to the ribosome here. 452 00:26:07,970 --> 00:26:13,850 So as an overview in terms of a cartoon, where are we going? 453 00:26:13,850 --> 00:26:18,140 Here, we have our ribosome, and in this depiction, 454 00:26:18,140 --> 00:26:19,530 it has been translating. 455 00:26:19,530 --> 00:26:23,300 So we have a nascent polypeptide emerging through the exit 456 00:26:23,300 --> 00:26:25,130 tunnel of the 50S. 457 00:26:25,130 --> 00:26:28,580 So we see this peptidyl tRNA in the P-site, 458 00:26:28,580 --> 00:26:32,120 and we have this deacylated tRNA in the E-site. 459 00:26:32,120 --> 00:26:33,320 So what happens? 460 00:26:33,320 --> 00:26:37,040 That A-site is empty, and for another round of elongation 461 00:26:37,040 --> 00:26:41,630 to occur, the aminoacyl tRNA needs to be delivered. 462 00:26:41,630 --> 00:26:44,780 And as we'll see today and in recitation this week, 463 00:26:44,780 --> 00:26:48,450 EF-Tu is responsible for that. 464 00:26:48,450 --> 00:26:54,050 So there's a ternary complex that forms between EF-Tu-GTP. 465 00:26:54,050 --> 00:26:59,060 So EF-Tu is a GTPase and the aminoacyl tRNA. 466 00:26:59,060 --> 00:27:03,230 And this ternary complex delivers the aminoacyl tRNA 467 00:27:03,230 --> 00:27:05,050 to the A-site. 468 00:27:05,050 --> 00:27:05,750 OK? 469 00:27:05,750 --> 00:27:07,880 This allows for peptide bond formation 470 00:27:07,880 --> 00:27:10,040 to occur in the catalytic center. 471 00:27:10,040 --> 00:27:13,160 And then there's a process called translocation, 472 00:27:13,160 --> 00:27:17,780 in which the elongation factor-G in complex with GTP 473 00:27:17,780 --> 00:27:20,900 comes in and helps to reset the ribosome such 474 00:27:20,900 --> 00:27:24,290 that another aminoacyl tRNA can come in. 475 00:27:24,290 --> 00:27:27,230 So where we're going to focus for the rest of today 476 00:27:27,230 --> 00:27:30,860 is on this process here, thinking about EF-Tu 477 00:27:30,860 --> 00:27:34,950 and how that delivers amino acid attached 478 00:27:34,950 --> 00:27:36,650 to tRNAs to the A-site. 479 00:27:41,450 --> 00:27:52,390 OK, so just in our cartoon, where we left off, 480 00:27:52,390 --> 00:28:12,180 with initiation process, so we have 481 00:28:12,180 --> 00:28:21,280 that initiator tRNA in the P-site, 482 00:28:21,280 --> 00:28:23,710 and the A-site is empty. 483 00:28:23,710 --> 00:28:24,310 OK? 484 00:28:24,310 --> 00:28:28,690 And one other thing I'll just show here, 485 00:28:28,690 --> 00:28:32,350 I mentioned when describing ribosome structure 486 00:28:32,350 --> 00:28:35,410 that some ribosomal proteins have additional jobs. 487 00:28:35,410 --> 00:28:37,420 So it's not just that these proteins 488 00:28:37,420 --> 00:28:40,870 help with the overall structural integrity of the ribosome. 489 00:28:40,870 --> 00:28:44,890 And there's two ribosomal proteins, L7 and L12, 490 00:28:44,890 --> 00:28:47,440 and these are involved in recruitment 491 00:28:47,440 --> 00:28:51,310 of that ternary complex between EF-Tu, the GTP, 492 00:28:51,310 --> 00:28:52,585 and the aminoacyl tRNA. 493 00:29:11,720 --> 00:29:26,000 So now, we need to get the aminoacyl tRNA to the A-site, 494 00:29:26,000 --> 00:29:27,140 and this requires EF-Tu. 495 00:29:32,770 --> 00:29:35,290 And when we think about this, we always 496 00:29:35,290 --> 00:29:39,970 need to think about this ternary complex which 497 00:29:39,970 --> 00:29:54,905 is EF-Tu bound to the aminoacyl tRNA bound to GTP. 498 00:30:00,030 --> 00:30:04,110 So a little bit about EF-Tu. 499 00:30:04,110 --> 00:30:09,600 So in E. coli, EF-Tu is the most abundant protein. 500 00:30:09,600 --> 00:30:12,140 So there's tons of EF-Tu. 501 00:30:12,140 --> 00:30:17,520 OK, approximately here, we have 100,000 copies per cell. 502 00:30:17,520 --> 00:30:21,360 So it's about 5% of total cellular protein. 503 00:30:21,360 --> 00:30:24,030 And so, as I just said in response 504 00:30:24,030 --> 00:30:27,090 to a question about these tRNAs in the cells, 505 00:30:27,090 --> 00:30:30,360 we can think about this entire tRNA 506 00:30:30,360 --> 00:30:33,300 pool, or aminoacylated tRNA pool, 507 00:30:33,300 --> 00:30:37,320 as being sequestered by EF-Tu. 508 00:30:37,320 --> 00:30:40,800 So EF-Tu binds the aminoacyl tRNA, 509 00:30:40,800 --> 00:30:44,970 and it binds GTP to form the ternary complex. 510 00:30:44,970 --> 00:30:49,050 And this allows EF-Tu to deliver these amino acids attached 511 00:30:49,050 --> 00:30:53,550 to the tRNAs to the A-site, and it's a GTPase. 512 00:30:53,550 --> 00:30:57,180 And we need to think a lot about how this activity relates 513 00:30:57,180 --> 00:31:00,030 to its function and fidelity. 514 00:31:00,030 --> 00:31:06,780 So here is a depiction of the structure of a ternary complex. 515 00:31:06,780 --> 00:31:11,160 So what we see is that we have a tRNA here, 516 00:31:11,160 --> 00:31:15,210 and here we have EF-Tu bound to the tRNA. 517 00:31:15,210 --> 00:31:19,560 So here is the anticodon loop, and if we consider 518 00:31:19,560 --> 00:31:23,760 this structure of the ternary complex bound to mRNA, 519 00:31:23,760 --> 00:31:25,260 what do we see? 520 00:31:25,260 --> 00:31:28,700 So we have an mRNA in green. 521 00:31:28,700 --> 00:31:32,130 OK, here's the tRNA, and the anticodon end, 522 00:31:32,130 --> 00:31:34,860 and here's EF-Tu. 523 00:31:34,860 --> 00:31:38,070 And as I said, EF-Tu is a GTPase. 524 00:31:38,070 --> 00:31:40,500 Where is the GTPase center? 525 00:31:40,500 --> 00:31:42,720 That's up here. 526 00:31:42,720 --> 00:31:48,600 So this GTPase center of EF-Tu is quite far from the tRNA 527 00:31:48,600 --> 00:31:50,520 anticodon, down here. 528 00:31:55,330 --> 00:31:57,880 This distance is about 70 Angstroms. 529 00:32:02,050 --> 00:32:05,500 And so this is something quite incredible 530 00:32:05,500 --> 00:32:09,670 to think about, because as we'll see, 531 00:32:09,670 --> 00:32:12,070 when there's codon recognition-- 532 00:32:12,070 --> 00:32:14,440 meaning this codon-anticodon interaction, 533 00:32:14,440 --> 00:32:16,160 that's a cognate pair-- 534 00:32:16,160 --> 00:32:18,850 GTP hydrolysis is stimulated. 535 00:32:18,850 --> 00:32:22,360 So how is that communicated over 70 Angstroms? 536 00:32:22,360 --> 00:32:24,100 If there's a recognition of that here 537 00:32:24,100 --> 00:32:28,120 between the mRNA and the tRNA anticodon, 538 00:32:28,120 --> 00:32:31,090 and GTP hydrolysis happens up here, 539 00:32:31,090 --> 00:32:34,190 how is that signaled over 70 Angstroms? 540 00:32:34,190 --> 00:32:34,690 Right? 541 00:32:34,690 --> 00:32:37,540 So clearly, there's going to be some conformational changes 542 00:32:37,540 --> 00:32:41,920 that occur that allow this GTPase activity to turn on. 543 00:32:44,850 --> 00:32:48,810 Just another view, so here, again, we 544 00:32:48,810 --> 00:32:51,420 have the structure of the ternary complex bound 545 00:32:51,420 --> 00:32:57,000 to the mRNA, and here, we can look at the ternary complex 546 00:32:57,000 --> 00:32:59,880 bound to a 70S ribosome. 547 00:32:59,880 --> 00:33:03,100 So we have the ribosome in this orangey-gold color, 548 00:33:03,100 --> 00:33:05,190 the 50S the 30S. 549 00:33:05,190 --> 00:33:08,790 Here, we have the PTC and decoding site. 550 00:33:08,790 --> 00:33:14,670 The tRNA is in green, and EF-Tu is in this darker orange here, 551 00:33:14,670 --> 00:33:20,700 to place that in the perspective of the 70S ribosome here. 552 00:33:20,700 --> 00:33:22,800 So conformational change is required 553 00:33:22,800 --> 00:33:26,130 to signal code on recognition to the GTPase center, 554 00:33:26,130 --> 00:33:28,590 and this is something that will be 555 00:33:28,590 --> 00:33:34,590 spoken about in quite some detail this week in recitation. 556 00:33:34,590 --> 00:33:40,650 One other point of review before moving forward with delivery 557 00:33:40,650 --> 00:33:43,150 of the amino acid tRNA. 558 00:33:43,150 --> 00:33:48,450 We need to think about codon-anticodon interactions 559 00:33:48,450 --> 00:33:50,730 here for decoding. 560 00:34:14,580 --> 00:34:24,040 So we have cognate versus near-cognate 561 00:34:24,040 --> 00:34:33,699 versus non-cognate, and this is for the codon-anticodon 562 00:34:33,699 --> 00:34:34,570 interaction. 563 00:34:42,290 --> 00:34:47,940 OK, and so if we imagine we have some mRNA, 564 00:34:47,940 --> 00:34:50,719 and you need to think about the five prime and three prime ends 565 00:34:50,719 --> 00:34:51,980 with this. 566 00:34:51,980 --> 00:34:59,990 And then we have some tRNA, three prime, five prime, 567 00:34:59,990 --> 00:35:01,830 we need to ask how do these match? 568 00:35:01,830 --> 00:35:08,610 So for instance here, if we have AAG, 569 00:35:08,610 --> 00:35:11,180 and we have positions one, two, three, 570 00:35:11,180 --> 00:35:14,060 from left to right of the mRNA, right here 571 00:35:14,060 --> 00:35:15,940 we have a cognate match. 572 00:35:15,940 --> 00:35:17,060 OK? 573 00:35:17,060 --> 00:35:19,940 So we have the AU match in positions one and two, 574 00:35:19,940 --> 00:35:23,720 and then wobble's allowed in position three, this GU here. 575 00:35:23,720 --> 00:35:25,980 So no, no interaction. 576 00:35:25,980 --> 00:35:36,660 OK, just as another example here, 577 00:35:36,660 --> 00:35:44,970 imagine we have GAG, here. 578 00:35:44,970 --> 00:35:47,370 What we see is that there's only one 579 00:35:47,370 --> 00:35:52,600 match, meaning Watson-Crick base pairing, in position two. 580 00:35:52,600 --> 00:35:53,250 OK. 581 00:35:53,250 --> 00:35:58,050 Here, this GU, that's not a match 582 00:35:58,050 --> 00:36:01,600 based on Watson-Crick base pairing, and as a result, 583 00:36:01,600 --> 00:36:05,360 the ribosome is going to want to reject this tRNA, 584 00:36:05,360 --> 00:36:08,940 if this is what's happening in the A-site here. 585 00:36:08,940 --> 00:36:17,160 And then, we can just imagine some situation, 586 00:36:17,160 --> 00:36:26,790 where we have a tRNA and an mRNA where there's just no match. 587 00:36:31,460 --> 00:36:32,060 OK? 588 00:36:32,060 --> 00:36:33,870 No Watson-Crick base pairing here. 589 00:36:37,870 --> 00:36:43,900 So what we need to ask is, as EF-Tu is delivering 590 00:36:43,900 --> 00:36:47,350 these aminoacyl tRNAs, what happens 591 00:36:47,350 --> 00:36:51,190 if it's a cognate match versus a near-cognate 592 00:36:51,190 --> 00:36:53,650 versus a non-cognate? 593 00:36:53,650 --> 00:36:58,075 How does the ribosome deal with the wrong tRNA entering 594 00:36:58,075 --> 00:36:58,940 the A-site? 595 00:36:58,940 --> 00:36:59,440 Right? 596 00:36:59,440 --> 00:37:09,987 So again, this is something important for fidelity, 597 00:37:09,987 --> 00:37:11,445 and these both need to be rejected. 598 00:37:17,840 --> 00:37:19,990 So why are we reviewing this? 599 00:37:19,990 --> 00:37:21,850 We're reviewing this, because it's 600 00:37:21,850 --> 00:37:25,810 important in terms of what happens 601 00:37:25,810 --> 00:37:28,510 during initial binding of aminoacyl tRNAs 602 00:37:28,510 --> 00:37:29,890 to the ribosome. 603 00:37:29,890 --> 00:37:32,410 So we're going to go over some of this in words 604 00:37:32,410 --> 00:37:35,860 and then look at a cartoon that explains this process. 605 00:37:35,860 --> 00:37:40,390 And what we're focused on is delivery of the aminoacyl tRNA 606 00:37:40,390 --> 00:37:42,310 to the A-site. 607 00:37:42,310 --> 00:37:44,240 So what happens first? 608 00:37:44,240 --> 00:37:44,740 OK. 609 00:37:44,740 --> 00:37:46,870 First, there needs to be an initial binding 610 00:37:46,870 --> 00:37:51,520 event, where the ternary complex binds to the ribosome. 611 00:37:51,520 --> 00:37:55,220 So initial binding, it binds to the 70S, 612 00:37:55,220 --> 00:37:58,330 and these ribosomal proteins are involved in the recruitment 613 00:37:58,330 --> 00:38:00,910 of the ternary complex. 614 00:38:00,910 --> 00:38:04,360 This initial binding event of the ternary complex 615 00:38:04,360 --> 00:38:06,670 to the ribosome is independent of the mRNA. 616 00:38:09,830 --> 00:38:14,270 What happens next is that there's codon recognition. 617 00:38:14,270 --> 00:38:19,460 So we need to think about that tRNA entering the A-site, 618 00:38:19,460 --> 00:38:20,960 and there's some sort of sampling 619 00:38:20,960 --> 00:38:23,620 that occurs in the decoding center, so 620 00:38:23,620 --> 00:38:28,130 sampling of codon-anticodon pairs in the A-site, 621 00:38:28,130 --> 00:38:29,390 and so what happens? 622 00:38:29,390 --> 00:38:32,840 What happens if there's a cognate event 623 00:38:32,840 --> 00:38:34,890 or a non-cognate event? 624 00:38:34,890 --> 00:38:40,190 So if a cognate anticodon recognition event occurs, 625 00:38:40,190 --> 00:38:45,300 there's a series of steps that then happen. 626 00:38:45,300 --> 00:38:49,040 So with a cognate codon-anticodon interaction, 627 00:38:49,040 --> 00:38:53,300 there will be a conformational change in EF-Tu, 628 00:38:53,300 --> 00:38:56,330 and this activates the GTPase center which 629 00:38:56,330 --> 00:38:59,060 allows for GTP hydrolysis. 630 00:38:59,060 --> 00:39:02,930 OK, and effectively this conformational change 631 00:39:02,930 --> 00:39:07,740 stabilizes the codon-anticodon interaction here, 632 00:39:07,740 --> 00:39:11,870 and that stabilization accelerates the GTP hydrolysis 633 00:39:11,870 --> 00:39:12,978 step. 634 00:39:12,978 --> 00:39:15,020 So this is all building towards a kinetic scheme. 635 00:39:17,660 --> 00:39:21,800 In terms of enhancements, what's found is that the rate of GTP 636 00:39:21,800 --> 00:39:26,390 hydrolysis by EF-Tu increases by about 5 times 10 637 00:39:26,390 --> 00:39:29,030 to the 4th with cognate anticodon 638 00:39:29,030 --> 00:39:31,580 recognition in the A-site. 639 00:39:31,580 --> 00:39:34,850 So we have GTP hydrolysis, and then there's 640 00:39:34,850 --> 00:39:37,280 another conformational change. 641 00:39:37,280 --> 00:39:41,690 So we have EF-Tu in its GDP-bound form, 642 00:39:41,690 --> 00:39:44,360 and effectively, EF-Tu will dissociate 643 00:39:44,360 --> 00:39:49,700 from the aminoacyl tRNA, and the aminoacyl tRNA will fully 644 00:39:49,700 --> 00:39:51,700 enter the A-site. 645 00:39:51,700 --> 00:39:54,710 OK so this process is called accommodation, 646 00:39:54,710 --> 00:39:58,560 and once that happens, peptide bond formation can occur. 647 00:39:58,560 --> 00:40:00,890 So this is the good scenario. 648 00:40:00,890 --> 00:40:03,650 The polypeptide can keep being made. 649 00:40:03,650 --> 00:40:05,180 What if it's not a cognate? 650 00:40:05,180 --> 00:40:09,890 So what if a near-cognate tRNA is delivered to that A-site 651 00:40:09,890 --> 00:40:11,870 during this initial binding event which 652 00:40:11,870 --> 00:40:14,060 is independent of the mRNA? 653 00:40:14,060 --> 00:40:15,920 That's why this can occur. 654 00:40:15,920 --> 00:40:20,745 If it's a near-cognate anticodon, what we observe-- 655 00:40:20,745 --> 00:40:22,370 and this is all from experiments you'll 656 00:40:22,370 --> 00:40:24,770 be learning about this week-- 657 00:40:24,770 --> 00:40:29,420 the ternary complex rapidly dissociates from the ribosome. 658 00:40:29,420 --> 00:40:31,850 And what's found from kinetic measurements 659 00:40:31,850 --> 00:40:35,120 is that the dissociation of the ternary complex, 660 00:40:35,120 --> 00:40:39,860 when it's a near-cognate situation, 661 00:40:39,860 --> 00:40:45,410 is about 350-fold faster than cognate. 662 00:40:45,410 --> 00:40:51,080 So let's look at this stepwise within a cartoon format. 663 00:40:51,080 --> 00:40:53,600 You'll see another depiction of this scheme 664 00:40:53,600 --> 00:40:57,170 in the recitation notes and in problem set two. 665 00:40:57,170 --> 00:41:03,440 So here, we have multiple steps in this overall process. 666 00:41:03,440 --> 00:41:05,450 All of these steps have some rate 667 00:41:05,450 --> 00:41:08,720 that's been measured by multiple types of methods, 668 00:41:08,720 --> 00:41:10,580 and Joanne will be presenting this week 669 00:41:10,580 --> 00:41:14,000 on a lot of pre-steady-state kinetic analysis that were done 670 00:41:14,000 --> 00:41:16,150 to measure these rates here. 671 00:41:20,270 --> 00:41:24,830 And basically, the key point to keep in mind, and that I'd 672 00:41:24,830 --> 00:41:27,980 like to stress from what was just said on the prior slide, 673 00:41:27,980 --> 00:41:30,110 is that what you'll see throughout this 674 00:41:30,110 --> 00:41:33,470 is that conformational changes are coupled 675 00:41:33,470 --> 00:41:35,640 to these rapid chemical steps. 676 00:41:35,640 --> 00:41:37,490 And the chemical steps are irreversible, 677 00:41:37,490 --> 00:41:40,010 this GTP hydrolysis. 678 00:41:40,010 --> 00:41:41,930 So what do we see? 679 00:41:41,930 --> 00:41:44,330 We begin with initial selection. 680 00:41:44,330 --> 00:41:46,880 Here, we have our ribosome, and there's a polypeptide 681 00:41:46,880 --> 00:41:48,550 being synthesized. 682 00:41:48,550 --> 00:41:50,790 Here's the ternary complex-- 683 00:41:50,790 --> 00:41:54,260 EF-Tu, GTP, and the aminoacyl tRNA. 684 00:41:54,260 --> 00:41:55,970 So there's an initial binding step 685 00:41:55,970 --> 00:41:59,480 that's governed by k1 in the forward direction and k minus 1 686 00:41:59,480 --> 00:42:02,480 in the back direction, and said before, this 687 00:42:02,480 --> 00:42:05,030 is independent of the mRNA. 688 00:42:05,030 --> 00:42:06,190 So what happens? 689 00:42:06,190 --> 00:42:09,140 The ternary complex binds the ribosome, 690 00:42:09,140 --> 00:42:13,400 there's sampling in the A-site of the anticodon, 691 00:42:13,400 --> 00:42:17,570 and then there is a step described as codon recognition 692 00:42:17,570 --> 00:42:19,950 with k2 and k minus 2. 693 00:42:19,950 --> 00:42:20,450 OK? 694 00:42:20,450 --> 00:42:23,690 In this scheme, if an arrow is colored, 695 00:42:23,690 --> 00:42:26,780 red arrow indicates the rate is greater 696 00:42:26,780 --> 00:42:29,310 for near-cognate than cognate. 697 00:42:29,310 --> 00:42:29,810 OK? 698 00:42:29,810 --> 00:42:35,120 Which means in the event here of a cognate pair, 699 00:42:35,120 --> 00:42:38,180 this is going to push forward in the forward direction. 700 00:42:38,180 --> 00:42:42,080 If it's near-cognate, this back step 701 00:42:42,080 --> 00:42:45,580 has a greater rate of about 350-fold. 702 00:42:45,580 --> 00:42:46,080 OK? 703 00:42:46,080 --> 00:42:48,950 So we're going to end up back here. 704 00:42:48,950 --> 00:42:50,930 With cognate recognition, next, we 705 00:42:50,930 --> 00:42:55,040 have GTPase activation, again, forward and reverse. 706 00:42:55,040 --> 00:42:58,760 Green indicates the rate is greater for a cognate match 707 00:42:58,760 --> 00:43:01,170 than near-cognate. 708 00:43:01,170 --> 00:43:03,680 So if it's the correct anticodon, 709 00:43:03,680 --> 00:43:05,870 it's going to plow through to here. 710 00:43:05,870 --> 00:43:08,340 We have GTPase activation. 711 00:43:08,340 --> 00:43:11,090 And then what happens down here? 712 00:43:11,090 --> 00:43:13,025 We have a GTP hydrolysis step. 713 00:43:15,840 --> 00:43:20,730 We have a conformational change in EF-Tu, and then what? 714 00:43:20,730 --> 00:43:26,640 We can have accommodation such that the tRNA was installed 715 00:43:26,640 --> 00:43:28,710 fully into the A-site and then rapid 716 00:43:28,710 --> 00:43:32,460 peptide bond formation or peptidyl transfer. 717 00:43:32,460 --> 00:43:36,390 The ribosome has one last chance to correct a mistake. 718 00:43:36,390 --> 00:43:40,740 So you can imagine that after GTP hydrolysis, 719 00:43:40,740 --> 00:43:45,540 after the conformational change in EF-Tu and its dissociation, 720 00:43:45,540 --> 00:43:49,140 there's a last chance at rejection here. 721 00:43:49,140 --> 00:43:54,380 Realize that step is occurring at the expense of GTP here. 722 00:43:59,850 --> 00:44:04,710 So in thinking about how to deconvolute this model 723 00:44:04,710 --> 00:44:11,860 or how to design experiments to test this model, 724 00:44:11,860 --> 00:44:13,450 there's a lot that needs to be done. 725 00:44:13,450 --> 00:44:13,950 Right? 726 00:44:13,950 --> 00:44:18,480 A lot of rates that need to be measured, 727 00:44:18,480 --> 00:44:21,700 a lot of different species along the way with the ribosome. 728 00:44:21,700 --> 00:44:22,200 Right? 729 00:44:22,200 --> 00:44:24,960 So how do you get a read out of each of these steps? 730 00:44:24,960 --> 00:44:28,440 That's what we'll be focused on in recitation this week 731 00:44:28,440 --> 00:44:29,370 and next here. 732 00:44:33,220 --> 00:44:39,160 So here are some more details on this initial binding process 733 00:44:39,160 --> 00:44:45,460 with some information related to the k1s and k minus 1s here. 734 00:44:45,460 --> 00:44:48,820 That's provided to help navigate the reading this week 735 00:44:48,820 --> 00:44:50,010 for recitation here. 736 00:44:54,680 --> 00:44:59,440 So what happens in the GTPase center of EF-Tu? 737 00:45:03,670 --> 00:45:05,625 What are some of these conformational changes? 738 00:45:08,440 --> 00:45:15,010 And effectively, there are conformational changes 739 00:45:15,010 --> 00:45:19,730 in the decoding center that are critical on one hand. 740 00:45:19,730 --> 00:45:21,280 So that's not at the GTPase center, 741 00:45:21,280 --> 00:45:24,340 but first asking what's happening when the mRNA 742 00:45:24,340 --> 00:45:26,860 and tRNA codon interact? 743 00:45:26,860 --> 00:45:31,090 And then what's happening in the GTPase center here? 744 00:45:31,090 --> 00:45:35,410 So just to note, not shown in the slide in terms 745 00:45:35,410 --> 00:45:44,860 of the decoding center. 746 00:46:09,660 --> 00:46:19,350 OK, what we need to be focusing on are changes in the 16S RNA, 747 00:46:19,350 --> 00:46:26,520 and effectively, I'll just point out three of the positions. 748 00:46:26,520 --> 00:46:46,730 So we have A1492, A1493, and G530 of the 16S, here. 749 00:46:46,730 --> 00:46:52,550 And what we find is that these bases effectively 750 00:46:52,550 --> 00:47:01,275 change conformation with a cognate match. 751 00:47:04,140 --> 00:47:07,140 And they effectively flip and interact 752 00:47:07,140 --> 00:47:09,990 with that cognate anticodon to help 753 00:47:09,990 --> 00:47:12,780 stabilize the codon-anticodon interaction. 754 00:47:35,480 --> 00:47:39,740 So this stabilizes the codon-anticodon interaction, 755 00:47:39,740 --> 00:47:43,730 and that stabilization accelerates the forward steps. 756 00:47:43,730 --> 00:47:48,200 So that results in this acceleration of GTP hydrolysis. 757 00:47:48,200 --> 00:47:50,750 So then the question is, what's happening 758 00:47:50,750 --> 00:47:54,380 in the GTPase center of EF-Tu? 759 00:47:54,380 --> 00:47:58,400 Because there has to be a change in conformation at that GTPase 760 00:47:58,400 --> 00:48:01,970 center 70 Angstroms away to allow for GTP 761 00:48:01,970 --> 00:48:05,060 hydrolysis to occur, and somehow, 762 00:48:05,060 --> 00:48:09,000 that all has to be signaled from here to there. 763 00:48:09,000 --> 00:48:16,070 So what we're looking at here is an excerpt 764 00:48:16,070 --> 00:48:20,600 of the structures looking at this GTPase center, 765 00:48:20,600 --> 00:48:23,120 and so what do we see? 766 00:48:23,120 --> 00:48:29,720 Effectively, two residues, so isoleucine-60 and valine-20 767 00:48:29,720 --> 00:48:34,670 have been described as a hydrophobic gate in the GTPase 768 00:48:34,670 --> 00:48:36,140 center. 769 00:48:36,140 --> 00:48:41,000 OK, and the idea is that if this gate is closed, 770 00:48:41,000 --> 00:48:44,840 it prevents a certain histidine residue, histidine-84, 771 00:48:44,840 --> 00:48:49,160 from activating a water molecule which then allows for the GTP 772 00:48:49,160 --> 00:48:51,140 to be hydrolyzed. 773 00:48:51,140 --> 00:48:54,320 OK, but if there's a change in conformation, 774 00:48:54,320 --> 00:48:58,950 and this gate opens, that chemistry can occur. 775 00:48:58,950 --> 00:49:04,370 So what are we looking at here in these structures? 776 00:49:04,370 --> 00:49:08,240 Effectively here, we have the two hydrophobic residues 777 00:49:08,240 --> 00:49:12,380 of the gate, so valine-20, isoleucine-60, 778 00:49:12,380 --> 00:49:16,250 and here's that histidine-84 I told you about, 779 00:49:16,250 --> 00:49:18,400 and what is this, GTPCP? 780 00:49:23,370 --> 00:49:28,240 So what we have there is a nonhydrolizable GTP analog. 781 00:49:28,240 --> 00:49:31,380 These types of molecules are very 782 00:49:31,380 --> 00:49:35,220 helpful in terms of getting structural information, 783 00:49:35,220 --> 00:49:38,770 in terms of doing certain types of biochemical experiments. 784 00:49:38,770 --> 00:49:39,270 OK? 785 00:49:39,270 --> 00:49:42,780 So effectively, we can have an analog bound that cannot 786 00:49:42,780 --> 00:49:45,210 hydrolyze. 787 00:49:45,210 --> 00:49:47,970 What are we looking at here? 788 00:49:47,970 --> 00:49:53,410 Here, we're looking at the, say, activated species, 789 00:49:53,410 --> 00:49:55,090 and what do we see? 790 00:49:55,090 --> 00:49:58,170 We see that this histidine has changed position. 791 00:49:58,170 --> 00:50:04,650 So here, it's flipped that way, here this way and here, 792 00:50:04,650 --> 00:50:11,760 what we see is a view with EF-Tu in the GTP-bound form. 793 00:50:11,760 --> 00:50:16,440 So the idea is that overall conformational changes that 794 00:50:16,440 --> 00:50:21,960 occur 70 Angstroms away, because of codon-anticodon recognition, 795 00:50:21,960 --> 00:50:24,300 effectively signal conformational changes 796 00:50:24,300 --> 00:50:28,860 in GTPase center that allow for GTP hydrolysis to occur 797 00:50:28,860 --> 00:50:33,360 and things to move in the forward direction there. 798 00:50:33,360 --> 00:50:36,150 So that's where we'll close for today. 799 00:50:36,150 --> 00:50:39,330 On Friday, we'll continue moving forward in this elongation 800 00:50:39,330 --> 00:50:42,060 cycle, and starting in recitation tomorrow, 801 00:50:42,060 --> 00:50:46,110 you'll look at experiments that allowed for this kinetic model 802 00:50:46,110 --> 00:50:50,450 to be analyzed and presented. 803 00:50:50,450 --> 00:50:52,570 You really need to come to recitation this week 804 00:50:52,570 --> 00:50:53,500 and read the paper. 805 00:50:53,500 --> 00:50:56,042 JOANNE STUBBE: And you need to read the paper more than once. 806 00:50:56,042 --> 00:50:57,283 It's a complicated paper. 807 00:50:57,283 --> 00:50:58,950 ELIZABETH NOLAN: That's on [INAUDIBLE].. 808 00:50:58,950 --> 00:51:01,380 It's a complicated paper which is why we have 809 00:51:01,380 --> 00:51:03,150 two weeks of recitation for it. 810 00:51:03,150 --> 00:51:06,090 There's a lot of methods, and I'll also point out 811 00:51:06,090 --> 00:51:10,230 that problem set three has very similar types of experiments, 812 00:51:10,230 --> 00:51:13,200 but it's looking at EFG instead of EF-Tu. 813 00:51:13,200 --> 00:51:17,070 So spending the time on this paper in the upcoming weeks 814 00:51:17,070 --> 00:51:19,310 is really important.