1 00:00:00,500 --> 00:00:02,830 The following content is provided under a Creative 2 00:00:02,830 --> 00:00:04,370 Commons license. 3 00:00:04,370 --> 00:00:06,670 Your support will help MIT OpenCourseWare 4 00:00:06,670 --> 00:00:11,030 continue to offer high-quality educational resources for free. 5 00:00:11,030 --> 00:00:13,660 To make a donation or view additional materials 6 00:00:13,660 --> 00:00:17,610 from hundreds of MIT courses, visit Mit OpenCourseWare 7 00:00:17,610 --> 00:00:18,520 at ocw.mit.edu. 8 00:00:25,980 --> 00:00:28,410 ELIZABETH NOLAN: So where we left off last time, 9 00:00:28,410 --> 00:00:30,660 we were talking about using antibiotics 10 00:00:30,660 --> 00:00:33,720 as tools to study the ribosome. 11 00:00:33,720 --> 00:00:37,770 And recall that antibiotics have many different structures, 12 00:00:37,770 --> 00:00:40,440 can bind to the ribosome at different places. 13 00:00:40,440 --> 00:00:46,560 And we closed with talking about this antibiotic, puromycin, 14 00:00:46,560 --> 00:00:49,740 that can bind to the A-site and cause chain termination, 15 00:00:49,740 --> 00:00:51,870 and also molecules that are derivatives 16 00:00:51,870 --> 00:00:57,270 of puromycin, such as that more elaborate one with a C75 there. 17 00:00:57,270 --> 00:01:00,540 And so the example of a system where 18 00:01:00,540 --> 00:01:03,000 puromycin has been employed, and this 19 00:01:03,000 --> 00:01:05,459 is just one of many, many examples, 20 00:01:05,459 --> 00:01:08,830 but also gives us a little new information about players 21 00:01:08,830 --> 00:01:13,900 in translation, involves studies of elongation factor peak, so 22 00:01:13,900 --> 00:01:15,610 EFP. 23 00:01:15,610 --> 00:01:18,630 And if you recall, where I closed last time 24 00:01:18,630 --> 00:01:23,130 was with the comment that this EFP over the years 25 00:01:23,130 --> 00:01:26,880 was implicated in a variety of cellular processes. 26 00:01:26,880 --> 00:01:29,980 But its precise function remained unclear. 27 00:01:29,980 --> 00:01:33,720 And so Rodnina and co-workers conducted 28 00:01:33,720 --> 00:01:36,120 a series of experiments to ask, what 29 00:01:36,120 --> 00:01:41,100 is the effect of EFP on peptide bond formation 30 00:01:41,100 --> 00:01:46,170 when different dipeptides are in the P-site? 31 00:01:46,170 --> 00:01:46,860 OK? 32 00:01:46,860 --> 00:01:49,800 And their experiments were motivated by the fact 33 00:01:49,800 --> 00:01:53,640 that there was some preliminary work out there suggesting 34 00:01:53,640 --> 00:01:56,760 that EFP accelerates peptide bond formation, 35 00:01:56,760 --> 00:01:59,500 but really, the details were unclear. 36 00:01:59,500 --> 00:02:01,450 So we're going to look at the experiment, 37 00:02:01,450 --> 00:02:03,450 their initial experiment they did, 38 00:02:03,450 --> 00:02:06,660 which led to some new understanding about how 39 00:02:06,660 --> 00:02:11,039 EFP affects the translation process. 40 00:02:11,039 --> 00:02:14,510 So what is it that they want to do in this experiment 41 00:02:14,510 --> 00:02:17,280 effectively? 42 00:02:17,280 --> 00:02:26,480 Imagine we have our ribosome, and we have our three sites, 43 00:02:26,480 --> 00:02:27,630 OK? 44 00:02:27,630 --> 00:02:30,000 And so what they do in this experiment is 45 00:02:30,000 --> 00:02:39,380 they have a dipeptide loaded in the P-site, 46 00:02:39,380 --> 00:02:41,565 OK, where x is some amino acid. 47 00:02:47,960 --> 00:02:49,990 OK, and then what they want to do 48 00:02:49,990 --> 00:02:59,860 is have puromycin in the A-site and then effectively monitor 49 00:02:59,860 --> 00:03:10,310 for peptide bond formation with or without EFP 50 00:03:10,310 --> 00:03:19,910 added such that the product is effectively 51 00:03:19,910 --> 00:03:24,000 a tripeptide, where we have fMat, the amino acid 52 00:03:24,000 --> 00:03:25,940 and puromycin, OK? 53 00:03:25,940 --> 00:03:28,400 And keep in mind, if this is what's being monitored, 54 00:03:28,400 --> 00:03:30,350 there needs to be a step to hydrolyze 55 00:03:30,350 --> 00:03:35,310 this tripeptide off the tRNA that's in the P-site, OK? 56 00:03:35,310 --> 00:03:37,290 And throughout this work, how they monitored 57 00:03:37,290 --> 00:03:44,940 this is that they have a radio label on the formal methionine. 58 00:03:44,940 --> 00:03:48,390 So you can imagine that you can somehow separate and see 59 00:03:48,390 --> 00:03:53,670 the dipeptide as well as this tripeptide-like molecule 60 00:03:53,670 --> 00:03:56,790 with the puromycin attached. 61 00:03:56,790 --> 00:04:02,080 So how to set up an experiment to test this? 62 00:04:02,080 --> 00:04:04,950 So they do a stop-flow experiment, 63 00:04:04,950 --> 00:04:08,220 so you heard some more about that method in recitation 64 00:04:08,220 --> 00:04:09,960 last week. 65 00:04:09,960 --> 00:04:11,730 And so in thinking about this, we 66 00:04:11,730 --> 00:04:15,640 need to think about what will be mixed. 67 00:04:15,640 --> 00:04:18,450 So what are the components of each syringe? 68 00:04:18,450 --> 00:04:22,180 How will this reaction be quenched? 69 00:04:22,180 --> 00:04:25,050 And so beginning to think about that, 70 00:04:25,050 --> 00:04:27,990 the question is, how do we even get the ribosome 71 00:04:27,990 --> 00:04:30,640 we need to start with in order to see the reaction? 72 00:04:30,640 --> 00:04:31,140 Right? 73 00:04:31,140 --> 00:04:34,350 So imagine that the goal is to have 74 00:04:34,350 --> 00:04:39,540 a pre-translocation ribosome, so effectively that dipeptide 75 00:04:39,540 --> 00:04:42,780 is in the P-site, and the A-site's empty. 76 00:04:42,780 --> 00:04:48,090 And then that assembled post-translocation ribosome 77 00:04:48,090 --> 00:04:50,970 needs to be mixed with puromycin such 78 00:04:50,970 --> 00:04:53,820 that puromycin can enter the A-site and peptide bond 79 00:04:53,820 --> 00:04:55,630 formation can occur. 80 00:04:55,630 --> 00:04:56,130 OK? 81 00:04:56,130 --> 00:04:57,588 So there's quite a bit of work that 82 00:04:57,588 --> 00:05:00,150 needs to happen to even get this experiment set up, 83 00:05:00,150 --> 00:05:03,240 because somehow that post-translocational ribosome 84 00:05:03,240 --> 00:05:04,740 needs to be made. 85 00:05:04,740 --> 00:05:06,960 OK, so if we think about this from the standpoint 86 00:05:06,960 --> 00:05:11,940 of the experiment and using the stop-flow to rapidly mix, 87 00:05:11,940 --> 00:05:22,470 we have syringe 1, and we have syringe 2, 88 00:05:22,470 --> 00:05:24,850 and we have our mixer. 89 00:05:24,850 --> 00:05:27,100 OK, so what are we going to put in syringe 1? 90 00:05:34,170 --> 00:05:45,670 OK, so here, we're going to have the post-translocation 91 00:05:45,670 --> 00:05:46,360 ribosome. 92 00:05:57,560 --> 00:06:06,280 The A-site is empty, and the P-site 93 00:06:06,280 --> 00:06:10,260 holds the dipeptide attached to the tRNA. 94 00:06:20,270 --> 00:06:27,860 And then in syringe 2, we're going to have puromycin here. 95 00:06:27,860 --> 00:06:31,080 OK, so before we get to EFP, I'm thinking 96 00:06:31,080 --> 00:06:33,510 about how we're going to look at that in this reaction 97 00:06:33,510 --> 00:06:34,740 and what it does. 98 00:06:34,740 --> 00:06:37,020 How are we going to get here? 99 00:06:37,020 --> 00:06:40,380 So what needs to be done to get this post-translocational 100 00:06:40,380 --> 00:06:41,453 ribosome? 101 00:06:47,740 --> 00:06:49,840 Is it in the sigma catalog? 102 00:06:49,840 --> 00:06:50,550 Bio rad? 103 00:06:56,340 --> 00:06:57,390 No way! 104 00:06:57,390 --> 00:06:59,580 And even if it were, you would be broke needing 105 00:06:59,580 --> 00:07:01,860 to purchase enough to do this experiment, right? 106 00:07:01,860 --> 00:07:04,470 You talked about needing high concentrations in recitation 107 00:07:04,470 --> 00:07:07,840 last week for these types of experiments. 108 00:07:07,840 --> 00:07:09,840 So where does this come from? 109 00:07:09,840 --> 00:07:12,990 What you need to do before even getting this into your syringe 110 00:07:12,990 --> 00:07:15,480 here, to do a rapid mixing experiment? 111 00:07:25,460 --> 00:07:28,090 AUDIENCE: You have to isolate it from cells? 112 00:07:28,090 --> 00:07:32,710 ELIZABETH NOLAN: OK, so what is the likelihood of isolating-- 113 00:07:32,710 --> 00:07:36,358 well, what's it, what do you need to isolate from cells? 114 00:07:36,358 --> 00:07:38,858 AUDIENCE: Well, you're going to need to modify it afterwards 115 00:07:38,858 --> 00:07:40,160 because there'll be all sorts of other things. 116 00:07:40,160 --> 00:07:41,743 ELIZABETH NOLAN: Right, but what's it? 117 00:07:41,743 --> 00:07:42,990 AUDIENCE: The ribosome. 118 00:07:42,990 --> 00:07:44,920 ELIZABETH NOLAN: OK, so we need a ribosome. 119 00:07:44,920 --> 00:07:45,590 Right? 120 00:07:45,590 --> 00:07:47,350 What else do we need? 121 00:07:47,350 --> 00:07:49,270 So we need the ribosome, and we need 122 00:07:49,270 --> 00:07:51,820 to get this into the P-site. 123 00:07:51,820 --> 00:07:56,470 So how are we going to get that dipeptidyl tRNA 124 00:07:56,470 --> 00:07:57,730 into the P-site? 125 00:07:57,730 --> 00:07:58,900 AUDIENCE: You need an mRNA. 126 00:07:58,900 --> 00:08:00,460 ELIZABETH NOLAN: We need an mRNA, 127 00:08:00,460 --> 00:08:05,140 and we're going to design that mRNA based on what amino acids 128 00:08:05,140 --> 00:08:06,860 we're interested in. 129 00:08:06,860 --> 00:08:09,370 So we need to come up with an mRNA. 130 00:08:09,370 --> 00:08:11,290 What else do we need? 131 00:08:11,290 --> 00:08:13,390 So think back to the whole cycle. 132 00:08:13,390 --> 00:08:16,060 AUDIENCE: You need EF-Tu, GTP. 133 00:08:16,060 --> 00:08:20,950 You need everything necessary to form the fMat to x-peptide. 134 00:08:20,950 --> 00:08:22,600 ELIZABETH NOLAN: Yeah. 135 00:08:22,600 --> 00:08:24,550 So what does that mean first? 136 00:08:24,550 --> 00:08:26,110 And when does that bond form? 137 00:08:26,110 --> 00:08:27,820 That's the next thing, right? 138 00:08:27,820 --> 00:08:32,230 So can we deliver this species to the P-site, 139 00:08:32,230 --> 00:08:34,630 based on what we understand about translation 140 00:08:34,630 --> 00:08:38,370 in the past four or five lectures? 141 00:08:38,370 --> 00:08:39,159 No. 142 00:08:39,159 --> 00:08:39,659 Right? 143 00:08:39,659 --> 00:08:41,970 So first, the initiation complex needs 144 00:08:41,970 --> 00:08:45,240 to be prepared in lab, which means 145 00:08:45,240 --> 00:08:50,010 you need initiation factors, a ribosome, mRNA, the initiator 146 00:08:50,010 --> 00:08:52,050 tRNA. 147 00:08:52,050 --> 00:08:56,220 And then that initiation complex needs to be purified, 148 00:08:56,220 --> 00:08:58,620 which is done by a type of sucrose gradient 149 00:08:58,620 --> 00:09:00,540 centrifugation. 150 00:09:00,540 --> 00:09:02,160 OK, and then what? 151 00:09:02,160 --> 00:09:04,050 Once that initiation complex is formed, 152 00:09:04,050 --> 00:09:07,590 there needs to be a round of elongation, where 153 00:09:07,590 --> 00:09:10,200 the ternary complex of EF-Tu. 154 00:09:10,200 --> 00:09:15,570 The amino acid and GTP comes in to deliver that x-tRNA x 155 00:09:15,570 --> 00:09:20,070 to this A-site, and then have peptide bond formation occur. 156 00:09:20,070 --> 00:09:21,420 OK? 157 00:09:21,420 --> 00:09:24,480 And then, we also need the help of EFG 158 00:09:24,480 --> 00:09:27,420 to move that to the P-site, right? 159 00:09:27,420 --> 00:09:29,670 So that whole cycle we've talked about 160 00:09:29,670 --> 00:09:31,290 from a fundamental perspective needs 161 00:09:31,290 --> 00:09:35,268 to be done at the bench in order to get here. 162 00:09:35,268 --> 00:09:36,810 So there's a lot of factors that need 163 00:09:36,810 --> 00:09:39,270 to be purified and obtained, quite 164 00:09:39,270 --> 00:09:42,795 a bit of effort to just even set this experiment up. 165 00:09:42,795 --> 00:09:44,100 OK? 166 00:09:44,100 --> 00:09:48,600 So always think about where these things come from. 167 00:09:48,600 --> 00:09:50,040 So we have this. 168 00:09:50,040 --> 00:09:52,800 We have puromycin, right? 169 00:09:52,800 --> 00:09:58,800 And then we want to look at the effective EFP. 170 00:09:58,800 --> 00:10:02,760 So the idea is, are there differences 171 00:10:02,760 --> 00:10:04,530 in peptide bond formation? 172 00:10:04,530 --> 00:10:07,050 Is it accelerated in the presence of EFP, 173 00:10:07,050 --> 00:10:10,710 as how some of this preliminary data indicated? 174 00:10:10,710 --> 00:10:13,350 And if so, is that for all amino acids? 175 00:10:13,350 --> 00:10:17,100 Or is it specific for certain amino acids, right? 176 00:10:17,100 --> 00:10:19,860 So we need to include EFP. 177 00:10:19,860 --> 00:10:23,573 And in these experiments, it was either omitted or included 178 00:10:23,573 --> 00:10:24,240 in each syringe. 179 00:10:35,660 --> 00:10:37,430 And something just to think about when 180 00:10:37,430 --> 00:10:39,530 thinking about these rapid mixing experiments 181 00:10:39,530 --> 00:10:41,840 is what happens in the mixer, right? 182 00:10:41,840 --> 00:10:44,030 If you're having the same volume, which 183 00:10:44,030 --> 00:10:46,790 is the case coming from syringe 1 and 2, 184 00:10:46,790 --> 00:10:48,710 you're going to have a dilution in here 185 00:10:48,710 --> 00:10:50,360 of all of the components. 186 00:10:50,360 --> 00:10:51,830 Right? 187 00:10:51,830 --> 00:10:55,160 So these are going to be rapidly mixed in the absence 188 00:10:55,160 --> 00:10:57,890 or presence of EFP. 189 00:10:57,890 --> 00:11:01,260 There'll be some time to allow for reaction to occur. 190 00:11:01,260 --> 00:11:02,930 And then, in this case, the reaction 191 00:11:02,930 --> 00:11:04,190 is going to be quenched. 192 00:11:04,190 --> 00:11:05,960 So it's the quench flow-type setup 193 00:11:05,960 --> 00:11:10,560 that came up in the recitation notes from last week. 194 00:11:10,560 --> 00:11:12,500 So in this case, we're going to have a syringe 195 00:11:12,500 --> 00:11:18,440 3 with a quencher. 196 00:11:18,440 --> 00:11:21,020 And in this particular work, they used base, 197 00:11:21,020 --> 00:11:22,340 so sometimes it's acid. 198 00:11:22,340 --> 00:11:24,680 Sometimes it's base. 199 00:11:24,680 --> 00:11:30,140 And this was a solution of KOH. 200 00:11:30,140 --> 00:11:38,420 OK, so then after some time, OK, we 201 00:11:38,420 --> 00:11:39,710 can have the reaction quench. 202 00:11:47,810 --> 00:11:53,690 OK, and then there'll be some sort of workup and product 203 00:11:53,690 --> 00:11:54,560 analysis. 204 00:12:00,050 --> 00:12:01,070 OK? 205 00:12:01,070 --> 00:12:08,223 So in this case, they chose to hydrolyse the peptidyl tRNA's 206 00:12:08,223 --> 00:12:09,640 and look at the peptide fragments. 207 00:12:09,640 --> 00:12:12,050 So you can imagine you need a method that's 208 00:12:12,050 --> 00:12:15,420 going to separate fMet x, whatever amino acid x 209 00:12:15,420 --> 00:12:18,550 is, from that product there. 210 00:12:18,550 --> 00:12:20,420 And then the radio label on the fMat 211 00:12:20,420 --> 00:12:21,860 is used for quantification. 212 00:12:25,450 --> 00:12:28,555 So what happens here? 213 00:12:31,230 --> 00:12:33,610 And I'll just give a summary, and then we'll 214 00:12:33,610 --> 00:12:35,090 look at it in more detail. 215 00:12:35,090 --> 00:12:38,140 So what they did in these experiments-- 216 00:12:38,140 --> 00:12:40,780 and recall that JoAnne talked about in recitation 217 00:12:40,780 --> 00:12:42,850 last week, when doing these kinetic experiments, 218 00:12:42,850 --> 00:12:44,500 you have to tweak them quite a bit 219 00:12:44,500 --> 00:12:48,220 to get the exact good conditions to observe 220 00:12:48,220 --> 00:12:49,630 what you want to see. 221 00:12:49,630 --> 00:12:50,890 So imagine that happened. 222 00:12:50,890 --> 00:12:55,060 We have our k observed, and I'm going 223 00:12:55,060 --> 00:12:56,950 to show these on a log scale. 224 00:12:56,950 --> 00:13:04,120 So always keep in mind, paying attention to what type of scale 225 00:13:04,120 --> 00:13:06,860 the axes are in. 226 00:13:06,860 --> 00:13:09,310 And so what we're going to look at 227 00:13:09,310 --> 00:13:15,520 is the k observed for formation of this tripeptide, 228 00:13:15,520 --> 00:13:17,380 depending on amino acid. 229 00:13:17,380 --> 00:13:21,655 And I'm going to generalize a bunch of the data here, 230 00:13:21,655 --> 00:13:23,655 and then we'll look at all the individual cases. 231 00:13:27,140 --> 00:13:28,160 OK? 232 00:13:28,160 --> 00:13:34,790 So here, we have x does not equal proline OK, 233 00:13:34,790 --> 00:13:38,480 and here, not colored in, is no EFP. 234 00:13:43,180 --> 00:13:49,300 And shaded is k observed for the reactions conducted 235 00:13:49,300 --> 00:13:51,148 in the presence of EFP. 236 00:13:51,148 --> 00:13:51,648 OK? 237 00:14:05,220 --> 00:14:08,130 So what was observed in these studies, 238 00:14:08,130 --> 00:14:13,940 looking at having many different amino acids here? 239 00:14:16,480 --> 00:14:20,650 With that, many of these amino acids 240 00:14:20,650 --> 00:14:22,810 showed negligible difference, whether or not 241 00:14:22,810 --> 00:14:25,940 EFP was included in the reaction. 242 00:14:25,940 --> 00:14:27,070 OK? 243 00:14:27,070 --> 00:14:29,590 And we can look at that data in more detail 244 00:14:29,590 --> 00:14:31,780 from the paper on the slide. 245 00:14:31,780 --> 00:14:34,570 What was very striking about these initial experiments 246 00:14:34,570 --> 00:14:39,140 was what happened in this case, when x equals proline here. 247 00:14:39,140 --> 00:14:43,630 So effectively, what they observed in this case 248 00:14:43,630 --> 00:14:47,470 was about 90-fold rate acceleration. 249 00:14:53,790 --> 00:14:59,130 Effectively, if we compare the k observed for peptide bond 250 00:14:59,130 --> 00:15:03,510 formation in the absence of EFP, we 251 00:15:03,510 --> 00:15:05,970 see it's significantly diminished 252 00:15:05,970 --> 00:15:09,510 for proline if EFP isn't there. 253 00:15:09,510 --> 00:15:12,600 And along those lines, it was known 254 00:15:12,600 --> 00:15:19,230 before that proline attached to its tRNA 255 00:15:19,230 --> 00:15:21,360 is a poorly reactive tRNA. 256 00:15:21,360 --> 00:15:24,330 So different aminoacyl tRNA's react differently 257 00:15:24,330 --> 00:15:26,130 in the ribosome. 258 00:15:26,130 --> 00:15:28,450 So there's that layer of complexity 259 00:15:28,450 --> 00:15:33,000 we haven't really talked about in this class yet here. 260 00:15:33,000 --> 00:15:38,720 So if we take a look at all these different examples, 261 00:15:38,720 --> 00:15:41,300 this one is the outlier. 262 00:15:41,300 --> 00:15:42,600 OK? 263 00:15:42,600 --> 00:15:49,380 So what these data indicated is that EFP has some special role 264 00:15:49,380 --> 00:15:54,420 in accelerating peptide bond formation for peptide bonds 265 00:15:54,420 --> 00:16:00,420 that contain a C-terminal proline residue here for that. 266 00:16:00,420 --> 00:16:03,390 And so these experiments were just 267 00:16:03,390 --> 00:16:06,720 a starting point for many additional experiments 268 00:16:06,720 --> 00:16:11,250 that ended up showing EFP is really critical for helping 269 00:16:11,250 --> 00:16:15,000 the ribosome translate sequences that have 270 00:16:15,000 --> 00:16:17,340 consecutive prolines in a row. 271 00:16:17,340 --> 00:16:22,260 So either three prolines or maybe a PPG sequence here. 272 00:16:22,260 --> 00:16:24,900 And in the absence of EFP, what can happen 273 00:16:24,900 --> 00:16:27,000 is that the ribosome stalls. 274 00:16:27,000 --> 00:16:30,090 So these aminoacyl tRNA's are not very reactive, 275 00:16:30,090 --> 00:16:32,280 and the ribosome just gets kind of stuck. 276 00:16:32,280 --> 00:16:36,810 And you can imagine that's not good for the cell. 277 00:16:36,810 --> 00:16:39,960 And then if we bring these observations back around 278 00:16:39,960 --> 00:16:42,900 to some of these early works that were suggesting 279 00:16:42,900 --> 00:16:47,720 EFP has a role in a diversity of different cellular processes, 280 00:16:47,720 --> 00:16:48,720 what might we ask? 281 00:16:48,720 --> 00:16:51,570 We might ask, well, where do the sequences 282 00:16:51,570 --> 00:16:53,730 of multiple prolines come up? 283 00:16:53,730 --> 00:16:57,720 So what types of proteins have three prolines 284 00:16:57,720 --> 00:16:59,430 in a row some place in their sequence? 285 00:16:59,430 --> 00:17:02,070 Or something like PPG. 286 00:17:02,070 --> 00:17:04,200 And so they took a look at that. 287 00:17:04,200 --> 00:17:06,150 And if we think about E. coli, there's 288 00:17:06,150 --> 00:17:09,390 about 4,000 different proteins, and there's 289 00:17:09,390 --> 00:17:13,800 a subset of around 270 that have these types of sequences 290 00:17:13,800 --> 00:17:14,730 in them. 291 00:17:14,730 --> 00:17:18,190 So not hugely common, but they exist. 292 00:17:18,190 --> 00:17:21,790 And so then ask, what do these proteins do? 293 00:17:21,790 --> 00:17:22,290 Right? 294 00:17:22,290 --> 00:17:24,240 Provided a function is known. 295 00:17:24,240 --> 00:17:29,190 And so what we see is within that subset of about 296 00:17:29,190 --> 00:17:32,400 270 proteins, there's examples of proteins 297 00:17:32,400 --> 00:17:36,200 that are involved in regulation, in metabolism, you know, 298 00:17:36,200 --> 00:17:37,890 important cellular processes. 299 00:17:37,890 --> 00:17:39,960 So you can begin to understand why 300 00:17:39,960 --> 00:17:41,370 it might be that this protein got 301 00:17:41,370 --> 00:17:44,980 implicated in all these different types of phenomena, 302 00:17:44,980 --> 00:17:45,480 right? 303 00:17:45,480 --> 00:17:47,460 But in terms of the details, it's 304 00:17:47,460 --> 00:17:52,320 really back here in terms of how this translation factor is 305 00:17:52,320 --> 00:17:56,740 helping the ribosome make a certain subset of peptide bonds 306 00:17:56,740 --> 00:17:57,960 there. 307 00:17:57,960 --> 00:18:02,460 So if you're curious about this, the paper's really wonderful. 308 00:18:02,460 --> 00:18:04,770 There's a number of additional interesting experiments 309 00:18:04,770 --> 00:18:07,080 that are done and additional methods to these kinetics 310 00:18:07,080 --> 00:18:07,580 there. 311 00:18:07,580 --> 00:18:12,190 I'm happy to point you in that direction. 312 00:18:12,190 --> 00:18:15,465 So yes? 313 00:18:15,465 --> 00:18:18,916 AUDIENCE: Does this rate of the reaction affect upon 314 00:18:18,916 --> 00:18:19,892 ribosome folding? 315 00:18:23,310 --> 00:18:24,910 ELIZABETH NOLAN: It could. 316 00:18:24,910 --> 00:18:26,370 I mean, basically, you're talking 317 00:18:26,370 --> 00:18:28,980 about what happens as the polypeptide extrudes 318 00:18:28,980 --> 00:18:30,570 from the ribosome, right? 319 00:18:30,570 --> 00:18:32,940 And if you're stalled and have some piece 320 00:18:32,940 --> 00:18:37,160 of this nascent polypeptide on the outside. 321 00:18:37,160 --> 00:18:39,150 Ribosomes stalling, yeah, what does 322 00:18:39,150 --> 00:18:41,700 that do in terms of how trigger factor, for instance, 323 00:18:41,700 --> 00:18:42,870 interacts. 324 00:18:42,870 --> 00:18:45,270 That's something we'll talk about in the next module, 325 00:18:45,270 --> 00:18:47,370 and we'll be getting there on Wednesday, 326 00:18:47,370 --> 00:18:51,090 I hope, if not Friday. 327 00:18:51,090 --> 00:18:55,230 So with that, we're going to close discussions of module 1 328 00:18:55,230 --> 00:18:59,730 in the ribosome with looking at some biotechnology 329 00:18:59,730 --> 00:19:01,860 and thinking about how we can use 330 00:19:01,860 --> 00:19:04,680 this fundamental understanding of the ribosome 331 00:19:04,680 --> 00:19:07,110 to do some new things. 332 00:19:07,110 --> 00:19:11,490 And so we're going to talk about re-engineering translation 333 00:19:11,490 --> 00:19:16,590 and ways to use this machinery to incorporate 334 00:19:16,590 --> 00:19:18,690 unnatural amino acids. 335 00:19:18,690 --> 00:19:21,600 And so to begin thinking about this, 336 00:19:21,600 --> 00:19:25,140 we can just consider some questions. 337 00:19:25,140 --> 00:19:28,680 And so many of us in this room are chemists or chemistry 338 00:19:28,680 --> 00:19:30,450 majors. 339 00:19:30,450 --> 00:19:33,280 We can think about organic chemistry, so 5.12, 340 00:19:33,280 --> 00:19:37,620 5.13, and all of the different organic transformations 341 00:19:37,620 --> 00:19:39,850 that are presented. 342 00:19:39,850 --> 00:19:42,720 So if we think about all these organic transformations 343 00:19:42,720 --> 00:19:47,580 and how they're available to synthetic chemists, 344 00:19:47,580 --> 00:19:49,860 we see a lot of versatility. 345 00:19:49,860 --> 00:19:53,230 And we can simply ask ourselves, can such versatility 346 00:19:53,230 --> 00:19:57,850 be achieved for protein modification? 347 00:19:57,850 --> 00:19:58,900 What is the toolkit? 348 00:19:58,900 --> 00:20:02,050 How can that toolkit be expanded? 349 00:20:02,050 --> 00:20:04,870 And then thinking about this further, 350 00:20:04,870 --> 00:20:07,790 can we use the translation machinery? 351 00:20:07,790 --> 00:20:12,460 So is it possible to modify the translation machinery 352 00:20:12,460 --> 00:20:15,820 to allow us to make peptides or proteins that 353 00:20:15,820 --> 00:20:17,630 have unnatural amino acids? 354 00:20:17,630 --> 00:20:22,150 So amino acids are moieties that are not the canonical ones. 355 00:20:22,150 --> 00:20:23,920 And can we do this in cells? 356 00:20:23,920 --> 00:20:25,600 Can we do this in a test tube? 357 00:20:25,600 --> 00:20:28,270 And if we can, what does that provide us 358 00:20:28,270 --> 00:20:32,450 with in terms of possibilities? 359 00:20:32,450 --> 00:20:35,200 So the answer is yes, and we're going 360 00:20:35,200 --> 00:20:37,740 to focus on the how and strengths 361 00:20:37,740 --> 00:20:41,470 and limitations in terms of our discussions of this machinery 362 00:20:41,470 --> 00:20:42,430 here. 363 00:20:42,430 --> 00:20:45,580 I also note-- I believe, JoAnne, this will come up. 364 00:20:45,580 --> 00:20:48,933 Will you be talking about this in the nucleotide parts, too? 365 00:20:48,933 --> 00:20:50,350 JOANNE STUBBE: If we get that far. 366 00:20:50,350 --> 00:20:51,850 ELIZABETH NOLAN: If we get that far. 367 00:20:51,850 --> 00:20:55,150 So in addition to here, this may come up again 368 00:20:55,150 --> 00:20:58,510 towards the end of the course, as a tool. 369 00:20:58,510 --> 00:21:03,520 So hopefully we'll get that far, because that's exciting. 370 00:21:03,520 --> 00:21:07,000 So let's think about re-engineering translation. 371 00:21:07,000 --> 00:21:08,680 And we can think about two things. 372 00:21:08,680 --> 00:21:11,780 We can think about the genetic code here, 373 00:21:11,780 --> 00:21:14,140 and we can think about the ribosome. 374 00:21:14,140 --> 00:21:18,070 And so I'll just present you with the questions. 375 00:21:18,070 --> 00:21:21,010 If we consider the genetic code, what 376 00:21:21,010 --> 00:21:23,200 can be done to this genetic code to change 377 00:21:23,200 --> 00:21:26,260 an amino acid in a protein? 378 00:21:26,260 --> 00:21:29,380 And if we think about the ribosome, what 379 00:21:29,380 --> 00:21:31,970 can be done to the ribosome to change 380 00:21:31,970 --> 00:21:34,450 an amino acid in a protein? 381 00:21:34,450 --> 00:21:38,260 And effectively, can we expand the genetic code 382 00:21:38,260 --> 00:21:41,320 to encode something other than what it's supposed to encode? 383 00:21:41,320 --> 00:21:47,320 So can this code allow us to encode an unnatural amino acid? 384 00:21:47,320 --> 00:21:50,200 And from the standpoint of the ribosome, 385 00:21:50,200 --> 00:21:53,380 is it possible to design new ribosomes? 386 00:21:53,380 --> 00:21:56,800 So can we make a new ribosome that 387 00:21:56,800 --> 00:22:00,830 can incorporate unnatural amino acids into proteins? 388 00:22:00,830 --> 00:22:03,710 So these are separate but related, 389 00:22:03,710 --> 00:22:09,130 and we're going to first discuss basically reassigning-- 390 00:22:09,130 --> 00:22:12,220 is it possible to reassign a codon? 391 00:22:12,220 --> 00:22:15,830 So why would we want to do this? 392 00:22:15,830 --> 00:22:19,740 And let's think about that for a minute. 393 00:22:24,010 --> 00:22:28,030 And what do I mean by expanding the genetic code? 394 00:22:28,030 --> 00:22:32,350 So if we think about the genetic code, 395 00:22:32,350 --> 00:22:35,800 we all know that it encodes these 20 amino acids building 396 00:22:35,800 --> 00:22:39,830 blocks, there's the start codons and the stop codon. 397 00:22:39,830 --> 00:22:42,880 And effectively, the codons are all used up, right? 398 00:22:42,880 --> 00:22:45,040 There aren't extra codons floating around 399 00:22:45,040 --> 00:22:50,150 that we could poach and assign to something else here. 400 00:22:50,150 --> 00:22:54,070 So can we overcome this? 401 00:22:54,070 --> 00:22:57,970 And why would we want to do that? 402 00:22:57,970 --> 00:23:01,930 Just broadly, if we think about being able to put something 403 00:23:01,930 --> 00:23:06,610 other than a natural amino acid in a protein at a specific 404 00:23:06,610 --> 00:23:09,340 location-- so exactly where we want it-- 405 00:23:09,340 --> 00:23:14,140 that opens up many possibilities for experiments. 406 00:23:14,140 --> 00:23:16,090 And we can think about those experiments 407 00:23:16,090 --> 00:23:20,440 both happening within a cell or outside of a cell. 408 00:23:20,440 --> 00:23:22,750 And these are experiments that just wouldn't be 409 00:23:22,750 --> 00:23:25,150 so easy or feasible otherwise. 410 00:23:25,150 --> 00:23:29,500 So maybe we'd like to study protein structure. 411 00:23:29,500 --> 00:23:31,150 What could we do? 412 00:23:31,150 --> 00:23:34,990 So fluorine is used in NMR quite a bit. 413 00:23:34,990 --> 00:23:36,730 Imagine if you could site-specifically 414 00:23:36,730 --> 00:23:39,340 label an unnatural amino acid that 415 00:23:39,340 --> 00:23:42,130 has a CF3 group, for example, and use 416 00:23:42,130 --> 00:23:44,040 that in your NMR studies. 417 00:23:44,040 --> 00:23:46,540 So that's something you'll get to think about in the context 418 00:23:46,540 --> 00:23:49,140 of problem set two. 419 00:23:49,140 --> 00:23:53,810 Ways to study protein function, protein localization. 420 00:23:53,810 --> 00:23:56,260 So for instance, instead of attaching 421 00:23:56,260 --> 00:24:00,490 GFP, which is big, to a protein of interest, 422 00:24:00,490 --> 00:24:02,260 maybe it's possible to incorporate 423 00:24:02,260 --> 00:24:04,330 a fluorescent amino acid that lets you 424 00:24:04,330 --> 00:24:07,000 see that protein in the cell. 425 00:24:07,000 --> 00:24:09,987 Protein-protein interactions. 426 00:24:09,987 --> 00:24:12,070 And maybe we'd like to make a new protein that has 427 00:24:12,070 --> 00:24:14,450 some desired characteristic. 428 00:24:14,450 --> 00:24:16,510 So there's a lot of possibilities 429 00:24:16,510 --> 00:24:20,140 to such technology. 430 00:24:20,140 --> 00:24:24,190 Just to keep in mind, what do many of us do? 431 00:24:24,190 --> 00:24:28,240 Many of us are familiar with site-directed mutagenesis, 432 00:24:28,240 --> 00:24:33,270 where we can change an amino acid in a protein. 433 00:24:33,270 --> 00:24:36,020 And we learn many, many things from this, 434 00:24:36,020 --> 00:24:39,350 but it is limited to naturally occurring amino acids. 435 00:24:39,350 --> 00:24:39,850 Right? 436 00:24:39,850 --> 00:24:44,920 So we'd like something more versatile. 437 00:24:44,920 --> 00:24:48,070 If we think about strategies also just a little 438 00:24:48,070 --> 00:24:58,500 bit, backing up here. 439 00:24:58,500 --> 00:25:01,070 OK, the first thing I'll just point out 440 00:25:01,070 --> 00:25:12,900 is that how I'm going to divide this, 441 00:25:12,900 --> 00:25:15,900 just in case this wasn't clear, is considering 442 00:25:15,900 --> 00:25:22,650 the native ribosome and then considering 443 00:25:22,650 --> 00:25:23,940 engineered ribosomes. 444 00:25:29,475 --> 00:25:31,350 And this is where we're going to focus today. 445 00:25:34,350 --> 00:25:44,880 And if we consider strategies, other strategies 446 00:25:44,880 --> 00:25:53,610 to incorporate unnatural amino acids, 447 00:25:53,610 --> 00:26:00,860 and I guess I'll call these standard, 448 00:26:00,860 --> 00:26:14,690 we can imagine chemical and biosynthetic. 449 00:26:14,690 --> 00:26:18,710 And I'm not going to go over a plethora of examples 450 00:26:18,710 --> 00:26:19,580 for either route. 451 00:26:19,580 --> 00:26:22,430 There'll be some slides included in the posted 452 00:26:22,430 --> 00:26:26,210 lecture notes that gives examples and pros and cons. 453 00:26:26,210 --> 00:26:28,460 But one example I will give here is just 454 00:26:28,460 --> 00:26:32,420 thinking from the standpoint of a chemical modification, what's 455 00:26:32,420 --> 00:26:38,790 an example and why we might want to do better. 456 00:26:38,790 --> 00:26:40,550 OK, so this is independent of something 457 00:26:40,550 --> 00:26:42,410 like site-directed mutagenesis, where you're 458 00:26:42,410 --> 00:26:45,950 having an organism do the work. 459 00:26:45,950 --> 00:26:48,080 So if we just consider an example of a chemical 460 00:26:48,080 --> 00:26:57,710 modification, there's certain amino acid side chains that 461 00:26:57,710 --> 00:26:59,960 are amenable to modification. 462 00:26:59,960 --> 00:27:03,320 So imagine you purify a protein, and you 463 00:27:03,320 --> 00:27:07,670 want to somehow tag that or label it, right? 464 00:27:07,670 --> 00:27:10,355 One option is to modify cysteine residues. 465 00:27:17,070 --> 00:27:21,840 And so iodoacetamide and related reagents are commonly employed, 466 00:27:21,840 --> 00:27:31,035 so imagine that you have some cysteine. 467 00:27:33,630 --> 00:27:39,670 You can react this with iodoacetamide 468 00:27:39,670 --> 00:27:42,670 that has some R group, right? 469 00:27:42,670 --> 00:27:43,270 What happens? 470 00:28:11,440 --> 00:28:12,470 Here, OK. 471 00:28:12,470 --> 00:28:15,260 You can get a covalent modification, 472 00:28:15,260 --> 00:28:26,400 and maybe this is a fluorophore or something else, right? 473 00:28:26,400 --> 00:28:31,474 So this is terrific, but what are some potential problems? 474 00:28:31,474 --> 00:28:34,460 AUDIENCE: Sorry, would this be a way 475 00:28:34,460 --> 00:28:36,380 to modify the amino acid before it's 476 00:28:36,380 --> 00:28:37,630 incorporated into the protein? 477 00:28:37,630 --> 00:28:39,130 Or would this be something you would 478 00:28:39,130 --> 00:28:41,400 do to modify the cysteine in an assembled protein? 479 00:28:41,400 --> 00:28:43,567 ELIZABETH NOLAN: Yeah, this would be after the fact. 480 00:28:43,567 --> 00:28:46,320 So imagine you have some protein. 481 00:28:46,320 --> 00:28:51,300 You've isolated your protein, and you have some cysteine. 482 00:28:51,300 --> 00:28:52,530 Right, and you'd like-- 483 00:28:52,530 --> 00:28:54,900 for some reason, you'd like to modify this protein. 484 00:28:54,900 --> 00:28:58,170 So maybe a fluorophore to see it. 485 00:28:58,170 --> 00:29:09,420 Maybe you know, a CF3 group for NMR here, 486 00:29:09,420 --> 00:29:11,310 which then gets to the point, what are 487 00:29:11,310 --> 00:29:12,725 possible problems with this? 488 00:29:12,725 --> 00:29:15,225 AUDIENCE: Do you have to use a mild base to be deprotonated, 489 00:29:15,225 --> 00:29:16,730 or is it maybe deprotonated based 490 00:29:16,730 --> 00:29:17,980 on where it is in the protein? 491 00:29:17,980 --> 00:29:21,130 ELIZABETH NOLAN: Yeah, so that gets to an initial issue, which 492 00:29:21,130 --> 00:29:24,010 is what's required to have this chemistry to happen? 493 00:29:24,010 --> 00:29:24,510 Right? 494 00:29:24,510 --> 00:29:27,210 The cysteine needs to be deprotonated. 495 00:29:27,210 --> 00:29:29,070 So probably the pH of your buffer 496 00:29:29,070 --> 00:29:31,230 is going to be elevated some. 497 00:29:31,230 --> 00:29:33,450 Does your protein or enzyme like that or not? 498 00:29:33,450 --> 00:29:34,750 Maybe, maybe not. 499 00:29:34,750 --> 00:29:35,380 Yeah? 500 00:29:35,380 --> 00:29:37,580 AUDIENCE: You can also run into selectivity issues-- 501 00:29:37,580 --> 00:29:39,640 I mean, having free cysteine residues isn't common, 502 00:29:39,640 --> 00:29:41,140 but it could be a potential problem. 503 00:29:41,140 --> 00:29:43,200 ELIZABETH NOLAN: Yeah, so you need-- 504 00:29:43,200 --> 00:29:45,180 well, it will depend on the protein, right? 505 00:29:45,180 --> 00:29:47,910 Is the cysteine free or a disulfide? 506 00:29:47,910 --> 00:29:50,130 Is it a native cysteine, or have you 507 00:29:50,130 --> 00:29:53,460 done site-directed mutagenesis first to put this cysteine 508 00:29:53,460 --> 00:29:55,330 in the position you want? 509 00:29:55,330 --> 00:29:56,490 Right? 510 00:29:56,490 --> 00:30:00,150 And then what happens if your protein has multiple cysteines 511 00:30:00,150 --> 00:30:02,160 building on what Rebecca said, and you 512 00:30:02,160 --> 00:30:07,050 want to have this label at a site-specific location? 513 00:30:07,050 --> 00:30:07,860 Right? 514 00:30:07,860 --> 00:30:10,380 What are you going to do about that? 515 00:30:10,380 --> 00:30:12,550 Are you going to have non-specific labeling? 516 00:30:12,550 --> 00:30:15,510 Are you going to mutate out the other cysteines? 517 00:30:15,510 --> 00:30:18,480 If you do that, what could that mean for your protein 518 00:30:18,480 --> 00:30:19,950 fold or function? 519 00:30:19,950 --> 00:30:24,360 There's a number of caveats that need to be considered. 520 00:30:24,360 --> 00:30:28,680 Nonetheless, it's a possibility to do. 521 00:30:28,680 --> 00:30:31,410 In terms of time, this is a pretty extreme example, 522 00:30:31,410 --> 00:30:33,390 but I'll just show one example here 523 00:30:33,390 --> 00:30:36,330 in thinking about this whole process and what you do, 524 00:30:36,330 --> 00:30:38,970 which also builds upon Rebecca's question. 525 00:30:38,970 --> 00:30:42,750 So imagine a protein with two subunits. 526 00:30:42,750 --> 00:30:47,010 And subunit 1 has a cysteine, and subunit 2 doesn't. 527 00:30:47,010 --> 00:30:49,530 So for some reason, you want to do this labeling. 528 00:30:49,530 --> 00:30:52,448 This is actually a protein from my group. 529 00:30:52,448 --> 00:30:54,240 And we wanted to stick a fluorophore on it. 530 00:30:54,240 --> 00:30:57,360 So we have a cysteine on one of the two subunits. 531 00:30:57,360 --> 00:31:00,540 You can run this reaction and get this fluorophore modified 532 00:31:00,540 --> 00:31:02,980 form here. 533 00:31:02,980 --> 00:31:07,720 And then you can see that's the case, looking at SDS-PAGE. 534 00:31:07,720 --> 00:31:09,900 So here we're looking at Coomassie stain that 535 00:31:09,900 --> 00:31:12,510 shows us total protein, and we see 536 00:31:12,510 --> 00:31:14,670 there's two subunits, 1 and 2. 537 00:31:14,670 --> 00:31:16,800 So the molecular weights are a little different, 538 00:31:16,800 --> 00:31:18,940 and we can separate them on this gel. 539 00:31:18,940 --> 00:31:20,940 And then if we look in the fluorescence channel, 540 00:31:20,940 --> 00:31:22,470 what do we see? 541 00:31:22,470 --> 00:31:25,920 We only see fluorescence associated with subunit 1 542 00:31:25,920 --> 00:31:29,490 and not subunit 2, which tells us our labeling strategy 543 00:31:29,490 --> 00:31:31,500 has worked well. 544 00:31:31,500 --> 00:31:35,550 Like, what we're showing in this equation. 545 00:31:35,550 --> 00:31:38,010 But what's everything that needs to be done? 546 00:31:38,010 --> 00:31:41,670 Well, we need to overexpress the protein in some organism. 547 00:31:41,670 --> 00:31:43,500 In this case, E. coli. 548 00:31:43,500 --> 00:31:45,900 We need to purify the protein. 549 00:31:45,900 --> 00:31:48,270 And once we have this purified protein in hand, 550 00:31:48,270 --> 00:31:51,810 we need to do the chemical reaction for the labeling. 551 00:31:51,810 --> 00:31:56,430 And then we need to purify that product somehow, 552 00:31:56,430 --> 00:31:59,190 and that's going to depend on the system you're working at. 553 00:31:59,190 --> 00:32:01,070 And then it needs to be analyzed, right? 554 00:32:01,070 --> 00:32:03,510 You always want to know what you're working with, right? 555 00:32:03,510 --> 00:32:06,690 So was this reaction to 100%? 556 00:32:06,690 --> 00:32:08,650 Did we end up with a mixture? 557 00:32:08,650 --> 00:32:11,220 If it's a mixture, what to do about that? 558 00:32:11,220 --> 00:32:13,500 So what does this mean in terms of time? 559 00:32:13,500 --> 00:32:15,690 And this is not for all cases, OK? 560 00:32:15,690 --> 00:32:19,440 This is for this exact case involving this protein shown 561 00:32:19,440 --> 00:32:21,390 as a cartoon here. 562 00:32:21,390 --> 00:32:24,690 So it takes about six days from start to finish to overexpress 563 00:32:24,690 --> 00:32:26,760 and purify it. 564 00:32:26,760 --> 00:32:29,700 Steps 2 to 4, based on the purification, 565 00:32:29,700 --> 00:32:32,190 we do another four days, right? 566 00:32:32,190 --> 00:32:34,680 So that's 10 days from start to finish, just 567 00:32:34,680 --> 00:32:38,140 to get this protein you'd like to use in your experiment. 568 00:32:38,140 --> 00:32:38,640 Right? 569 00:32:38,640 --> 00:32:40,800 And you can imagine if somehow a label 570 00:32:40,800 --> 00:32:44,130 could be put on in vivo, during this initial step 571 00:32:44,130 --> 00:32:47,670 here, that that would save some time at the end of the day. 572 00:32:50,290 --> 00:32:55,740 So before moving on to what's done 573 00:32:55,740 --> 00:32:57,990 for unnatural amino acid incorporation 574 00:32:57,990 --> 00:33:00,540 by what we'll call the Schultz method out of Professor Peter 575 00:33:00,540 --> 00:33:04,980 Schultz's group, just to think about biosynthetic methods 576 00:33:04,980 --> 00:33:06,300 for a minute. 577 00:33:06,300 --> 00:33:10,350 So some common ones are done for structural studies. 578 00:33:10,350 --> 00:33:14,250 So for instance, you can imagine feeding an organism something 579 00:33:14,250 --> 00:33:18,570 like selenocysteine or selenomethionine. 580 00:33:18,570 --> 00:33:22,370 Another example is labeling nitrogens or carbons 581 00:33:22,370 --> 00:33:25,250 for NMR, where the organism is fed, 582 00:33:25,250 --> 00:33:31,220 say, a labeled amino acid, maybe with N15 or C13 there, right? 583 00:33:31,220 --> 00:33:33,470 So that's just a biosynthetic method, 584 00:33:33,470 --> 00:33:36,800 where you're changing the growth conditions, rather 585 00:33:36,800 --> 00:33:41,870 than doing something to manipulate the genetic code 586 00:33:41,870 --> 00:33:44,510 or the ribosome. 587 00:33:44,510 --> 00:33:46,800 So what's the conclusion here? 588 00:33:53,340 --> 00:34:18,719 What we want is we want a method of site-specific incorporation 589 00:34:18,719 --> 00:34:22,185 of unnatural amino acids in vivo. 590 00:34:25,170 --> 00:34:37,409 So in a cell and in a desired organism, depending on what 591 00:34:37,409 --> 00:34:49,420 you want to do with high efficiency and also fidelity, 592 00:34:49,420 --> 00:34:54,040 so getting back to that idea and before. 593 00:34:54,040 --> 00:34:57,170 OK, so why do we want to do this in vivo? 594 00:35:00,410 --> 00:35:03,230 It allows for studies within cells, 595 00:35:03,230 --> 00:35:06,290 and you also can purify protein from cells, 596 00:35:06,290 --> 00:35:09,518 so you can do in vitro experiments as well. 597 00:35:09,518 --> 00:35:11,060 OK, and you can imagine, if you could 598 00:35:11,060 --> 00:35:14,540 have all of the pieces of this machinery in a cell, 599 00:35:14,540 --> 00:35:19,310 maybe there's some technical advantage to that. 600 00:35:19,310 --> 00:35:23,930 So this is what we're going to consider here. 601 00:35:23,930 --> 00:35:26,120 So to this question, can the ribosome 602 00:35:26,120 --> 00:35:29,600 incorporate unnatural amino acids into proteins? 603 00:35:29,600 --> 00:35:33,500 Effectively, what do we need to think about? 604 00:35:33,500 --> 00:35:37,130 One, we need to think about relaxing 605 00:35:37,130 --> 00:35:39,890 the substrate specificity of the aminoacyl tRNA 606 00:35:39,890 --> 00:35:44,150 synthetase to accommodate some unnatural amino acid, right? 607 00:35:44,150 --> 00:35:46,040 Somehow that unnatural amino acid 608 00:35:46,040 --> 00:35:48,300 needs to get to the ribosome. 609 00:35:48,300 --> 00:35:50,990 So if this can be done, and we can 610 00:35:50,990 --> 00:35:55,070 make a tRNA that has an unnatural amino acid attached 611 00:35:55,070 --> 00:36:03,710 to it, can this aminoacyl tRNA get to the A-site 612 00:36:03,710 --> 00:36:05,340 and do the work? 613 00:36:05,340 --> 00:36:08,390 So this is the method we're going to talk about 614 00:36:08,390 --> 00:36:11,510 in some detail for the rest of today 615 00:36:11,510 --> 00:36:14,540 and into Wednesday, this Schultz method. 616 00:36:14,540 --> 00:36:19,220 So the idea is that there's a tRNA that's dedicated 617 00:36:19,220 --> 00:36:21,920 for this unnatural amino acid. 618 00:36:21,920 --> 00:36:25,160 We see this unnatural amino acid shown here, where 619 00:36:25,160 --> 00:36:26,915 the UAA is indicated by probe. 620 00:36:29,510 --> 00:36:32,540 We need an aminoacyl tRNA synthetase 621 00:36:32,540 --> 00:36:35,300 that will take this unnatural amino acid 622 00:36:35,300 --> 00:36:39,170 and attach it to the three-prime end of the tRNA to give us 623 00:36:39,170 --> 00:36:42,920 this aminoacylated tRNA. 624 00:36:42,920 --> 00:36:43,790 And then what? 625 00:36:43,790 --> 00:36:47,720 Imagine this tRNA can make its way to the ribosome. 626 00:36:47,720 --> 00:36:48,500 What happens? 627 00:36:48,500 --> 00:36:53,310 We need a codon for this aminoacyl tRNA. 628 00:36:53,310 --> 00:36:55,490 It needs to carry the anticodon, and we're 629 00:36:55,490 --> 00:36:58,190 going to talk about this in some more detail in a minute. 630 00:36:58,190 --> 00:37:02,030 So we can have a plasmid that has 631 00:37:02,030 --> 00:37:05,420 the DNA with the gene of interest in it, right? 632 00:37:05,420 --> 00:37:11,870 This plasmid DNA can be transformed into, say, E. coli 633 00:37:11,870 --> 00:37:15,050 that has this machinery here. 634 00:37:15,050 --> 00:37:19,820 We can have transcription to give the mRNA that 635 00:37:19,820 --> 00:37:24,230 is from this plasmid DNA. 636 00:37:24,230 --> 00:37:26,600 And then imagine translations such 637 00:37:26,600 --> 00:37:29,560 that this unnatural amino acid is incorporated. 638 00:37:35,680 --> 00:37:39,220 So effectively, where we're going 639 00:37:39,220 --> 00:37:43,270 is that we need a general method. 640 00:37:43,270 --> 00:37:47,560 We want this method to be broadly useful, 641 00:37:47,560 --> 00:37:54,010 where we can genetically encode this unnatural amino acid 642 00:37:54,010 --> 00:37:58,480 and have it incorporated in response to a unique triplet 643 00:37:58,480 --> 00:38:00,280 codon, here. 644 00:38:03,220 --> 00:38:09,610 So in thinking about that, what are the pieces that we need? 645 00:38:43,750 --> 00:38:47,560 And we'll think about E. coli for the moment, 646 00:38:47,560 --> 00:38:48,970 but this could be other. 647 00:38:48,970 --> 00:38:51,370 So yeast, mammalian cells, right? 648 00:38:51,370 --> 00:38:57,060 Let your imagination run wild with this here. 649 00:38:57,060 --> 00:39:11,750 When the incorporation of the UAA in response 650 00:39:11,750 --> 00:39:18,020 to a unique triplet codon. 651 00:39:22,080 --> 00:39:25,230 So if we're going to do this, what do we need? 652 00:39:32,060 --> 00:39:36,330 OK, effectively, we need some new components of the trans-- 653 00:39:36,330 --> 00:39:39,480 like protein biosynthetic translation machinery, right? 654 00:39:39,480 --> 00:39:42,725 So we need to rewind and think about the whole translation 655 00:39:42,725 --> 00:39:43,225 process. 656 00:40:01,380 --> 00:40:04,450 OK, so the first order of business 657 00:40:04,450 --> 00:40:06,790 is that we need a unique codon. 658 00:40:11,610 --> 00:40:12,110 Right? 659 00:40:12,110 --> 00:40:19,230 So this only designates or uniquely designates the UAA. 660 00:40:26,710 --> 00:40:29,930 And so we need to ask, where does this come from? 661 00:40:29,930 --> 00:40:31,520 Because we just went over the fact 662 00:40:31,520 --> 00:40:37,790 that the codons are used up for amino acid start and stop. 663 00:40:37,790 --> 00:40:38,900 We need a new tRNA. 664 00:40:43,620 --> 00:40:56,460 OK, so this tRNA needs to be specific for the unique codon. 665 00:41:02,810 --> 00:41:14,990 OK, and we need the corresponding aminoacyl tRNA 666 00:41:14,990 --> 00:41:18,480 synthetase, right? 667 00:41:18,480 --> 00:41:25,300 And we need this to load the unnatural amino acid 668 00:41:25,300 --> 00:41:35,370 onto the unique tRNA here. 669 00:41:35,370 --> 00:41:40,120 So what is a key feature of all of this? 670 00:41:40,120 --> 00:41:48,170 A key feature is that if we want to do this in some organism, 671 00:41:48,170 --> 00:41:51,410 we need this machinery to be orthogonal to the machinery 672 00:41:51,410 --> 00:41:52,145 in that organism. 673 00:41:56,450 --> 00:42:00,080 We cannot have cross-reactivity, because then there's not going 674 00:42:00,080 --> 00:42:03,380 to be any selectivity of this incorporation. 675 00:42:38,580 --> 00:42:40,200 So no cross-reaction. 676 00:42:46,060 --> 00:42:51,420 So what do we need to consider, in terms of these? 677 00:42:51,420 --> 00:42:55,260 We need to think about all of the machinery, right? 678 00:42:55,260 --> 00:43:00,160 And I just list some considerations here. 679 00:43:00,160 --> 00:43:04,530 So this new tRNA can only allow for translation 680 00:43:04,530 --> 00:43:07,230 of the codon for the UAA. 681 00:43:07,230 --> 00:43:10,890 It can't be a substrate for any of the endogenous aaRS, 682 00:43:10,890 --> 00:43:13,230 because then it will become loaded, potentially, 683 00:43:13,230 --> 00:43:14,610 with the wrong amino acid. 684 00:43:14,610 --> 00:43:18,570 So think back to lectures 2 and 3. 685 00:43:18,570 --> 00:43:20,700 This new aminoacyl tRNA synthetase 686 00:43:20,700 --> 00:43:24,690 can only recognize the new tRNA and not endogenous tRNA. 687 00:43:24,690 --> 00:43:26,220 So cross-reactivity again. 688 00:43:28,740 --> 00:43:32,370 This unnatural amino acid also can't be a substrate 689 00:43:32,370 --> 00:43:35,130 for endogenous enzymes. 690 00:43:35,130 --> 00:43:36,600 And also keep in mind, there needs 691 00:43:36,600 --> 00:43:39,330 to be some way to get this unnatural amino acid 692 00:43:39,330 --> 00:43:43,050 into a cell if we want to do this in a cellular context. 693 00:43:43,050 --> 00:43:47,190 So there's just transport issue that needs to be kept in mind. 694 00:43:47,190 --> 00:43:51,570 Will this unnatural amino acid get into the cell? 695 00:43:51,570 --> 00:43:59,100 OK, so what we're going to do is consider these requirements 696 00:43:59,100 --> 00:44:02,070 and what was done to build up this methodology 697 00:44:02,070 --> 00:44:04,240 during initial work. 698 00:44:04,240 --> 00:44:07,980 So the first issue is this unique codon, 699 00:44:07,980 --> 00:44:09,960 and what is its identity here? 700 00:44:28,650 --> 00:44:33,860 And so if we consider the 64 codons, 701 00:44:33,860 --> 00:44:41,400 they're used up with the 20 common amino acids. 702 00:44:41,400 --> 00:44:47,220 We have the three stop codons and the one start codon. 703 00:44:50,850 --> 00:44:54,660 And so in thinking about this, can ask, 704 00:44:54,660 --> 00:44:57,690 do we really need three stop codons? 705 00:44:57,690 --> 00:45:00,030 We certainly need our start, and we need 706 00:45:00,030 --> 00:45:02,310 codons for the amino acids. 707 00:45:02,310 --> 00:45:05,490 But is there some wiggle room here? 708 00:45:05,490 --> 00:45:14,420 And so in terms of these stop codons, 709 00:45:14,420 --> 00:45:21,960 we have TAA, TAG, and TGA. 710 00:45:21,960 --> 00:45:23,165 And these all have names. 711 00:45:27,200 --> 00:45:31,710 Ochre, amber, and opal. 712 00:45:31,710 --> 00:45:37,220 OK, and so the idea we're going to see is just the question, 713 00:45:37,220 --> 00:45:39,860 can we reassign a stop codon? 714 00:45:39,860 --> 00:45:42,380 And can we reassign a stop codon such 715 00:45:42,380 --> 00:45:45,530 that it's the codon for the unnatural amino acid? 716 00:45:48,090 --> 00:45:52,340 And so basically, if we want to reassign a stop codon, 717 00:45:52,340 --> 00:45:54,440 how do we choose? 718 00:45:54,440 --> 00:45:55,910 Right? 719 00:45:55,910 --> 00:45:59,080 So two things to consider. 720 00:45:59,080 --> 00:46:04,160 One, how frequently is each stop codon used? 721 00:46:04,160 --> 00:46:06,920 So what do we know about that? 722 00:46:06,920 --> 00:46:13,340 And then does this stop codon terminate essential genes? 723 00:46:13,340 --> 00:46:16,850 So we can imagine that if we were to reassign a stop 724 00:46:16,850 --> 00:46:21,860 codon that's used frequently by E. coli or another host, 725 00:46:21,860 --> 00:46:24,680 or if we were to reassign a stop codon that's 726 00:46:24,680 --> 00:46:28,640 important for terminating the synthesis of essential genes, 727 00:46:28,640 --> 00:46:32,480 in either case, the outcome could be pretty bad, right? 728 00:46:32,480 --> 00:46:36,710 So what was found in thinking about those issues is 729 00:46:36,710 --> 00:46:43,070 this amber stop codon, TAG, one, it's the least frequently used. 730 00:46:51,410 --> 00:47:01,460 And just for an example, about 9% in E. coli and about 23% 731 00:47:01,460 --> 00:47:05,000 in yeast for terminating genes. 732 00:47:05,000 --> 00:47:10,190 And additionally, it rarely terminates essential genes. 733 00:47:23,760 --> 00:47:29,310 OK, so based on this, it was decided 734 00:47:29,310 --> 00:47:47,220 to reassign TAG as the codon for the unnatural amino acid. 735 00:47:58,240 --> 00:48:03,270 OK, so we've gotten through to here. 736 00:48:03,270 --> 00:48:08,210 So the question is now, what about requirements 2 and 3? 737 00:48:08,210 --> 00:48:08,913 So yeah? 738 00:48:08,913 --> 00:48:10,080 AUDIENCE: I have a question. 739 00:48:10,080 --> 00:48:12,520 So it seems interesting to choose a stop codon 740 00:48:12,520 --> 00:48:14,580 to change because if the stop codon messes up, 741 00:48:14,580 --> 00:48:17,940 it seems more catastrophic to us all than one 742 00:48:17,940 --> 00:48:21,046 of the other redundant amino acids. 743 00:48:21,046 --> 00:48:22,760 Like, why use a stop codon? 744 00:48:22,760 --> 00:48:24,920 I think it's interesting. 745 00:48:24,920 --> 00:48:27,230 ELIZABETH NOLAN: Yeah, so there is a risk. 746 00:48:27,230 --> 00:48:29,400 There's certainly a risk, right? 747 00:48:29,400 --> 00:48:31,980 But these considerations were made 748 00:48:31,980 --> 00:48:35,280 to try to diminish that risk. 749 00:48:35,280 --> 00:48:35,970 Right? 750 00:48:35,970 --> 00:48:41,220 So you could make the argument that maybe all of these stop 751 00:48:41,220 --> 00:48:43,230 codons aren't essential, right? 752 00:48:43,230 --> 00:48:44,730 And what is more deleterious? 753 00:48:44,730 --> 00:48:49,590 Will it be to try to use a stop codon that's infrequently used, 754 00:48:49,590 --> 00:48:54,600 or to reassign a codon that's for an amino acid that comes up 755 00:48:54,600 --> 00:48:58,290 in many, many different proteins in the cellular pool? 756 00:48:58,290 --> 00:48:58,890 Right? 757 00:48:58,890 --> 00:49:03,490 So there's a judgment call there. 758 00:49:03,490 --> 00:49:07,980 But if we consider in E. coli, this TAG stop is for about 9% 759 00:49:07,980 --> 00:49:09,120 of proteins. 760 00:49:09,120 --> 00:49:13,620 How does that compare to, say, reassigning one of the codons 761 00:49:13,620 --> 00:49:16,650 to incorporate a lysine or a valine. 762 00:49:16,650 --> 00:49:18,690 I don't know, but that was just want 763 00:49:18,690 --> 00:49:21,480 to think about, how frequently is that codon used? 764 00:49:21,480 --> 00:49:23,490 Because certainly there's different codon usage 765 00:49:23,490 --> 00:49:24,750 in different organisms. 766 00:49:24,750 --> 00:49:25,958 Do you have something to say? 767 00:49:25,958 --> 00:49:27,458 JOANNE STUBBE: So it depends on what 768 00:49:27,458 --> 00:49:29,620 you want to put the unnatural amino acid in for. 769 00:49:29,620 --> 00:49:33,460 So if you want it in endogenous levels, it could be a problem. 770 00:49:33,460 --> 00:49:35,210 But if you're overproducing your protein-- 771 00:49:35,210 --> 00:49:36,895 ELIZABETH NOLAN: Yeah, it may not be a problem. 772 00:49:36,895 --> 00:49:39,353 JOANNE STUBBE: Then it's not a problem, because you induce, 773 00:49:39,353 --> 00:49:41,057 and then you flood it with that and you 774 00:49:41,057 --> 00:49:42,940 get high levels of cooperation. 775 00:49:42,940 --> 00:49:45,428 So it depends on what your purpose is. 776 00:49:45,428 --> 00:49:47,220 ELIZABETH NOLAN: Yeah, so is JoAnne's point 777 00:49:47,220 --> 00:49:49,290 clear to everyone? 778 00:49:49,290 --> 00:49:55,260 So you could imagine expressing at an endogenous level, right? 779 00:49:55,260 --> 00:49:58,410 Or you could imagine causing the cell 780 00:49:58,410 --> 00:50:01,047 to overexpress the proteins, like off a plasmid, 781 00:50:01,047 --> 00:50:02,880 like what many of you have done in lab class 782 00:50:02,880 --> 00:50:05,310 or maybe in research there for that. 783 00:50:07,830 --> 00:50:10,340 Are there many examples of reassigning a different one? 784 00:50:10,340 --> 00:50:11,430 JOANNE STUBBE: I think it's really tough. 785 00:50:11,430 --> 00:50:12,888 I mean, inside the cell, you really 786 00:50:12,888 --> 00:50:15,840 do have problems if you don't re-engineer, 787 00:50:15,840 --> 00:50:17,820 because you get truncations also. 788 00:50:17,820 --> 00:50:20,070 ELIZABETH NOLAN: Yeah, we're going to talk about that. 789 00:50:20,070 --> 00:50:22,500 So there is a big problem about the stop 790 00:50:22,500 --> 00:50:24,210 that we're going to talk about, once we 791 00:50:24,210 --> 00:50:28,740 get to how we have this done, which is premature termination. 792 00:50:28,740 --> 00:50:32,435 AUDIENCE: I'm kind of confused more or less at that point, 793 00:50:32,435 --> 00:50:35,970 because stop codon, we're just using because it's not 794 00:50:35,970 --> 00:50:38,030 currently-- it doesn't go for anything, 795 00:50:38,030 --> 00:50:43,530 it just ends, like, endogenous sequences that 796 00:50:43,530 --> 00:50:46,350 are not the UAA so if we replace it, we might not get those. 797 00:50:46,350 --> 00:50:49,970 But we'll get the one that we're trying to synthesize. 798 00:50:49,970 --> 00:50:52,750 ELIZABETH NOLAN: Right, so we need a codon 799 00:50:52,750 --> 00:50:54,640 for the unnatural amino acid. 800 00:50:54,640 --> 00:50:56,320 And right now, we're limiting our space 801 00:50:56,320 --> 00:50:58,870 to triplet codons, which is what was initially 802 00:50:58,870 --> 00:51:02,020 done when this type of methodology was developed. 803 00:51:02,020 --> 00:51:08,398 So the question is, we have four options in terms of bases. 804 00:51:08,398 --> 00:51:09,940 AUDIENCE: That are not coding, right? 805 00:51:09,940 --> 00:51:12,232 ELIZABETH NOLAN: No, no, in terms of our codons, right? 806 00:51:12,232 --> 00:51:13,030 So three, right? 807 00:51:13,030 --> 00:51:15,010 Four, three, so there's 64 codons, 808 00:51:15,010 --> 00:51:16,395 and they're all used up. 809 00:51:16,395 --> 00:51:17,020 AUDIENCE: Yeah. 810 00:51:17,020 --> 00:51:18,890 ELIZABETH NOLAN: Right, so there's not some extra. 811 00:51:18,890 --> 00:51:19,515 AUDIENCE: Yeah. 812 00:51:19,515 --> 00:51:21,780 ELIZABETH NOLAN: So then what can be reassigned? 813 00:51:21,780 --> 00:51:23,560 AUDIENCE: These stop-- so the stop. 814 00:51:23,560 --> 00:51:24,310 ELIZABETH NOLAN: Well, yeah, well, we 815 00:51:24,310 --> 00:51:25,540 can't reassign the start. 816 00:51:25,540 --> 00:51:27,540 Then there's no proteins, right? 817 00:51:27,540 --> 00:51:29,500 There's only one start codon. 818 00:51:29,500 --> 00:51:34,370 So the thinking was, is a stop codon dispensable? 819 00:51:34,370 --> 00:51:34,870 Right? 820 00:51:34,870 --> 00:51:36,610 And then a decision was made, based 821 00:51:36,610 --> 00:51:40,750 on basically the frequency of use of the stop codon 822 00:51:40,750 --> 00:51:43,030 and whether or not the stop codon terminates 823 00:51:43,030 --> 00:51:44,470 essential genes. 824 00:51:44,470 --> 00:51:46,000 Is this something foolproof? 825 00:51:46,000 --> 00:51:48,130 No, there's major problems in terms 826 00:51:48,130 --> 00:51:49,990 of yields that come up as a result. 827 00:51:49,990 --> 00:51:52,090 And we'll see that on Wednesday, right? 828 00:51:52,090 --> 00:51:55,810 But you need a starting point to get a method underway. 829 00:51:55,810 --> 00:51:58,690 So where will we begin tomorrow is 830 00:51:58,690 --> 00:52:02,500 talking about where this tRNA and the aaRS 831 00:52:02,500 --> 00:52:04,980 come from to do this.