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 Open CourseWare 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 --> 00:00:18,550 at ocw.mit.edu. 8 00:00:25,810 --> 00:00:28,690 ELIZABETH NOLAN: Where we left off yesterday 9 00:00:28,690 --> 00:00:32,170 was beginning to discuss methods for unnatural amino acid 10 00:00:32,170 --> 00:00:35,860 incorporation into proteins using the ribosome. 11 00:00:35,860 --> 00:00:39,400 And the methodology that was introduced 12 00:00:39,400 --> 00:00:40,990 and where we need to continue today 13 00:00:40,990 --> 00:00:46,390 is the Schultz method of using the native ribosome 14 00:00:46,390 --> 00:00:49,330 to play some tricks and get unnatural amino acids 15 00:00:49,330 --> 00:00:51,470 into proteins. 16 00:00:51,470 --> 00:00:55,150 So we'll work through this further to show 17 00:00:55,150 --> 00:00:57,760 how the rest of the machinery was generated 18 00:00:57,760 --> 00:01:00,180 and then we'll consider some of the limitations 19 00:01:00,180 --> 00:01:02,890 and some of that came up in questions last time. 20 00:01:02,890 --> 00:01:04,480 And then we'll close with a discussion 21 00:01:04,480 --> 00:01:09,430 of one strategy that's a different strategy that 22 00:01:09,430 --> 00:01:12,250 uses actually an orthogonal ribosome, which 23 00:01:12,250 --> 00:01:14,830 is really, really neat here. 24 00:01:14,830 --> 00:01:20,140 So where we left off last time in terms of the Schultz Method 25 00:01:20,140 --> 00:01:28,710 was that we needed a unique codon 26 00:01:28,710 --> 00:01:31,020 for the unnatural amino acid, right? 27 00:01:31,020 --> 00:01:33,420 And a stop codon was reassigned. 28 00:01:40,820 --> 00:01:45,830 So TAG or Amber stop. 29 00:01:45,830 --> 00:01:51,560 And the other thing that we need that we'll discuss now 30 00:01:51,560 --> 00:01:55,940 involves the requirement of an orthogonal tRNA 31 00:01:55,940 --> 00:01:59,600 and aminoacyl-tRNA synthetase pair that 32 00:01:59,600 --> 00:02:02,090 can be used in this method. 33 00:02:15,460 --> 00:02:19,700 So the question is, where does this come from? 34 00:02:19,700 --> 00:02:23,020 So where do we get a tRNA and an aaRS 35 00:02:23,020 --> 00:02:25,960 that can be used for this unnatural amino acid 36 00:02:25,960 --> 00:02:27,910 of interest. 37 00:02:27,910 --> 00:02:32,170 And one way to think about this in terms of a search 38 00:02:32,170 --> 00:02:35,500 is to think about different tRNAs and aaRS 39 00:02:35,500 --> 00:02:37,360 from different organisms. 40 00:02:37,360 --> 00:02:40,930 And so what's found if tRNAs are compared 41 00:02:40,930 --> 00:02:45,050 between bacteria, eukaryotes, [INAUDIBLE],, 42 00:02:45,050 --> 00:02:47,990 there's evolutionary divergence. 43 00:02:47,990 --> 00:02:50,080 And so can that evolutionary divergence 44 00:02:50,080 --> 00:02:51,940 be taken advantage of? 45 00:02:51,940 --> 00:02:55,030 And effectively, is it possible to find 46 00:02:55,030 --> 00:02:59,320 some tRNA and its aminoacyl-tRNA synthetase 47 00:02:59,320 --> 00:03:03,520 from one organism that's orthogonal to the corresponding 48 00:03:03,520 --> 00:03:07,630 tRNA and aminoacyl-tRNA synthetase 49 00:03:07,630 --> 00:03:09,460 in the organism of interest. 50 00:03:09,460 --> 00:03:12,320 So effectively, if we want to use E. coli, 51 00:03:12,320 --> 00:03:16,570 we want to find a pair from another organism that's 52 00:03:16,570 --> 00:03:21,200 completely independent of the endogenous E. coli machinery. 53 00:03:21,200 --> 00:03:23,530 So what does this mean? 54 00:03:23,530 --> 00:03:25,690 A lot of trial and error was done 55 00:03:25,690 --> 00:03:30,730 to identify a pair from another organism. 56 00:03:30,730 --> 00:03:39,890 And where they ended up finding one is from a methanogen. 57 00:03:39,890 --> 00:03:42,750 So methanococcus jannaschii here. 58 00:03:48,910 --> 00:03:53,140 OK, and this initial pair was for tyrosine. 59 00:04:09,740 --> 00:04:13,820 And so, there's some features of this pair that 60 00:04:13,820 --> 00:04:15,440 are noteworthy to bring up. 61 00:04:15,440 --> 00:04:24,890 So first if we think about the aminoacyl-tRNA synthetase here. 62 00:04:24,890 --> 00:04:26,570 This one has an unusual feature. 63 00:04:26,570 --> 00:04:28,880 So when we discuss these aaRS, remember 64 00:04:28,880 --> 00:04:30,800 we discussed the mechanism and we also 65 00:04:30,800 --> 00:04:34,640 discussed what happens if a wrong amino acid is selected. 66 00:04:34,640 --> 00:04:37,520 And we learned that they have editing function 67 00:04:37,520 --> 00:04:39,860 and that there's editing domains. 68 00:04:39,860 --> 00:04:41,450 What also came up in those discussions 69 00:04:41,450 --> 00:04:43,580 is that we need to take every one of these enzymes 70 00:04:43,580 --> 00:04:45,320 as a case-by-case basis. 71 00:04:45,320 --> 00:04:48,650 And as it turns out, this particular enzyme 72 00:04:48,650 --> 00:04:51,290 does not have an editing domain. 73 00:04:51,290 --> 00:04:53,390 So then the thing to think about is, 74 00:04:53,390 --> 00:04:57,440 why would that be useful from the standpoint of incorporating 75 00:04:57,440 --> 00:05:00,590 an unnatural amino acid? 76 00:05:00,590 --> 00:05:02,040 So what's the benefit there? 77 00:05:15,770 --> 00:05:17,490 So what does this editing domain do? 78 00:05:26,068 --> 00:05:27,610 AUDIENCE: It's one less thing to have 79 00:05:27,610 --> 00:05:29,950 to fix if you're assuming that the editing domain would 80 00:05:29,950 --> 00:05:32,550 recognize and hydrolyze an unnatural amino acid 81 00:05:32,550 --> 00:05:35,320 that you put in even if you got the binding site to recognize 82 00:05:35,320 --> 00:05:37,988 it, or the first binding site to recognize it. 83 00:05:37,988 --> 00:05:39,280 ELIZABETH NOLAN: Exactly right. 84 00:05:39,280 --> 00:05:42,430 There's no deacylation happening, so no hydrolosis. 85 00:05:42,430 --> 00:05:45,280 So it's just more likely this unnatural amino acid can 86 00:05:45,280 --> 00:05:47,080 be a successful substrate and there's 87 00:05:47,080 --> 00:05:49,180 less engineering that has to be done in terms 88 00:05:49,180 --> 00:05:53,470 of modifying the enzyme here. 89 00:05:53,470 --> 00:05:55,510 So, another point in terms of-- 90 00:05:58,030 --> 00:06:05,400 they found that it does not acylate E. coli tRNA. 91 00:06:05,400 --> 00:06:09,340 OK, and that's important for trying to use this in E. coli 92 00:06:09,340 --> 00:06:11,357 here. 93 00:06:11,357 --> 00:06:12,565 What's the potential problem? 94 00:06:17,240 --> 00:06:20,750 So is this going to be specific for the unnatural amino acid 95 00:06:20,750 --> 00:06:23,010 of interest? 96 00:06:23,010 --> 00:06:24,810 No way, right? 97 00:06:24,810 --> 00:06:27,570 Unlikely at least, and depending on what type 98 00:06:27,570 --> 00:06:30,300 of a natural amino acid you're thinking about, 99 00:06:30,300 --> 00:06:32,770 it may definitely be a no way. 100 00:06:32,770 --> 00:06:35,820 So there's some experimental work 101 00:06:35,820 --> 00:06:39,450 to do to make this specific for the unnatural amino acid 102 00:06:39,450 --> 00:06:42,000 of interest, which means there has to be some mutagenesis 103 00:06:42,000 --> 00:06:43,800 and selection, which we're not going 104 00:06:43,800 --> 00:06:47,100 to talk about in detail here. 105 00:06:47,100 --> 00:06:48,690 So what about the tRNA? 106 00:06:56,850 --> 00:07:00,040 So we need to think about the tRNA structure right 107 00:07:00,040 --> 00:07:04,050 and how tRNAs interact with aaRS, right? 108 00:07:04,050 --> 00:07:06,780 And recall, we had in an earlier lecture one 109 00:07:06,780 --> 00:07:09,420 example of a crystal structure of this complex. 110 00:07:09,420 --> 00:07:13,530 And we saw there's many positions where they interact. 111 00:07:13,530 --> 00:07:22,000 What was known in this system here is that they figured-- 112 00:07:22,000 --> 00:07:24,310 OK, and also keep in mind, just backing up a minute, 113 00:07:24,310 --> 00:07:27,670 this tRNA, as we know, based on this nomenclature 114 00:07:27,670 --> 00:07:30,940 has an anticodon for tyrosine. 115 00:07:30,940 --> 00:07:32,650 So that's going to have to be mutated 116 00:07:32,650 --> 00:07:35,140 to be the anticodon on the Amber stop 117 00:07:35,140 --> 00:07:37,010 in order to use in this method. 118 00:07:37,010 --> 00:07:37,510 Right? 119 00:07:37,510 --> 00:07:47,840 So this is going to have to be mutated 120 00:07:47,840 --> 00:07:54,080 to give us tRNA(CUA) where this is indicating 121 00:07:54,080 --> 00:08:01,720 the anticodon here. 122 00:08:01,720 --> 00:08:04,780 So that mutation, we don't want that mutation 123 00:08:04,780 --> 00:08:07,240 to disrupt the interaction between the tRNA 124 00:08:07,240 --> 00:08:10,120 and aminoacyl-tRNA synthetase, right? 125 00:08:10,120 --> 00:08:12,580 And it turns out there were minimal interactions 126 00:08:12,580 --> 00:08:14,950 in that area for the native system. 127 00:08:14,950 --> 00:08:17,650 So the thinking was that these mutations 128 00:08:17,650 --> 00:08:19,120 could be tolerated here. 129 00:08:22,960 --> 00:08:27,180 So with that said, what is the potential problem 130 00:08:27,180 --> 00:08:27,940 with this tRNA? 131 00:08:36,799 --> 00:08:40,700 So if we want to put this tRNA into E. coli, 132 00:08:40,700 --> 00:08:44,660 it can't be recognized by any of the E. coli aaRS. 133 00:08:44,660 --> 00:08:49,440 So all of these recognition issues come up. 134 00:08:49,440 --> 00:08:54,050 So, here again with another example, 135 00:08:54,050 --> 00:08:58,130 where there needed to be some mutagenesis and selection 136 00:08:58,130 --> 00:09:01,580 to prevent interactions between this tRNA 137 00:09:01,580 --> 00:09:05,690 from the methanogen and the aaRS of E. coli. 138 00:09:05,690 --> 00:09:15,920 And effectively what they did is to pick 11 positions 139 00:09:15,920 --> 00:09:18,150 on the tRNA, which I'll just chart out. 140 00:09:23,290 --> 00:09:25,730 OK, so here's our tRNA. 141 00:09:25,730 --> 00:09:28,580 Here's our CUA anticodon here. 142 00:09:28,580 --> 00:09:33,410 And effectively these ends are positions where they randomized 143 00:09:33,410 --> 00:09:38,300 and did mutagenesis here. 144 00:09:48,070 --> 00:09:55,720 So they identified these 11 positions. 145 00:10:00,780 --> 00:10:01,760 OK? 146 00:10:01,760 --> 00:10:06,260 And these 11 positions do not interact 147 00:10:06,260 --> 00:10:09,770 with the aaRS here of the pair. 148 00:10:09,770 --> 00:10:13,040 So the idea is to maintain this interaction, 149 00:10:13,040 --> 00:10:16,040 but prevent any interaction of this tRNA with E. coli 150 00:10:16,040 --> 00:10:18,390 machinery here. 151 00:10:18,390 --> 00:10:20,030 So effectively, they used a method 152 00:10:20,030 --> 00:10:26,240 called directed evolution to do selection. 153 00:10:26,240 --> 00:10:28,400 And what might happen out of that, 154 00:10:28,400 --> 00:10:31,970 imagine you have some large pool of mutant tRNA, 155 00:10:31,970 --> 00:10:33,710 what might happen? 156 00:10:33,710 --> 00:10:58,660 So here OK, so the end result is that the tRNA 157 00:10:58,660 --> 00:11:04,210 might be non-functional. 158 00:11:07,310 --> 00:11:07,810 Right? 159 00:11:07,810 --> 00:11:11,030 So the mutation was not helpful. 160 00:11:11,030 --> 00:11:11,530 OK? 161 00:11:11,530 --> 00:11:18,690 It might be non-orthogonal, meaning 162 00:11:18,690 --> 00:11:23,460 that it's recognized by the endogenous E. coli machinery 163 00:11:23,460 --> 00:11:29,940 or it may be orthogonal here. 164 00:11:29,940 --> 00:11:48,710 OK, so recognized only OK? 165 00:11:48,710 --> 00:11:53,690 And so this is what needs to be selected for here. 166 00:11:58,340 --> 00:12:02,640 And so assays need to be done that allows these 167 00:12:02,640 --> 00:12:04,170 to be differentiated here. 168 00:12:07,280 --> 00:12:10,820 So the end result is an orthogonal pair. 169 00:12:10,820 --> 00:12:12,800 But the point is, you can't just take this pair 170 00:12:12,800 --> 00:12:14,360 from the other organism. 171 00:12:14,360 --> 00:12:16,910 It needs to be further modified. 172 00:12:16,910 --> 00:12:18,740 So where does that put us in terms 173 00:12:18,740 --> 00:12:23,120 of the cartoon we saw yesterday without some of these details? 174 00:12:23,120 --> 00:12:28,220 So here we have the tRNA that has this amber anticodon. 175 00:12:28,220 --> 00:12:30,100 So that's our orthogonal tRNA. 176 00:12:30,100 --> 00:12:33,110 We have an unnatural amino acid that's 177 00:12:33,110 --> 00:12:36,680 able to get into the organism of interest. 178 00:12:36,680 --> 00:12:40,280 And we have the orthogonal tRNA synthetase. 179 00:12:40,280 --> 00:12:44,480 So these give us this aminoacyl-tRNA 180 00:12:44,480 --> 00:12:46,740 with the unnatural amino acid. 181 00:12:46,740 --> 00:12:50,060 And then that can be incorporated into the A 182 00:12:50,060 --> 00:12:51,590 site of the ribosome. 183 00:12:51,590 --> 00:12:54,350 Right so this is a case where we have a plasma DNA. 184 00:12:54,350 --> 00:12:57,350 Here's the gene of interest in red. 185 00:12:57,350 --> 00:13:00,650 And somewhere in that gene, a stop codon 186 00:13:00,650 --> 00:13:04,010 has been placed to allow for incorporation 187 00:13:04,010 --> 00:13:06,620 of this unnatural amino acid somewhere 188 00:13:06,620 --> 00:13:08,750 within the polypeptide chain as shown here. 189 00:13:12,160 --> 00:13:17,310 So if we think about the scope of this methodology, 190 00:13:17,310 --> 00:13:19,380 where does this take us? 191 00:13:19,380 --> 00:13:22,110 So, it's quite broad. 192 00:13:22,110 --> 00:13:26,250 This type of work has been applied beyond E. coli, 193 00:13:26,250 --> 00:13:29,040 so in yeast and mammalian cells. 194 00:13:29,040 --> 00:13:32,730 At present, there is many, many different unnatural amino acids 195 00:13:32,730 --> 00:13:36,390 that can be incorporated and it's used by many labs. 196 00:13:36,390 --> 00:13:37,890 So that's something to keep in mind. 197 00:13:37,890 --> 00:13:39,750 If you're developing a new method, 198 00:13:39,750 --> 00:13:41,940 you'd really like other folks in other labs 199 00:13:41,940 --> 00:13:44,310 to be able to use your method. 200 00:13:44,310 --> 00:13:47,100 There's a lot of troubleshooting to do experimentally 201 00:13:47,100 --> 00:13:48,510 to get it up and running. 202 00:13:48,510 --> 00:13:51,180 And Joanne's a wonderful person to talk about that 203 00:13:51,180 --> 00:13:54,540 if you're curious for details. 204 00:13:54,540 --> 00:13:57,810 Just some amino acid scope, and you 205 00:13:57,810 --> 00:13:59,610 know what maybe we could do. 206 00:13:59,610 --> 00:14:02,520 So these are some earlier examples 207 00:14:02,520 --> 00:14:06,880 of unnatural amino acids that can be incorporated. 208 00:14:06,880 --> 00:14:08,460 And what are some of the neat things? 209 00:14:08,460 --> 00:14:11,880 If we look just here for example, there's an azide. 210 00:14:11,880 --> 00:14:14,431 Why might we want an azide? 211 00:14:14,431 --> 00:14:15,514 AUDIENCE: Click chemistry. 212 00:14:15,514 --> 00:14:17,431 ELIZABETH NOLAN: Yeah, click chemistry, right. 213 00:14:17,431 --> 00:14:20,740 Some chemistry that could be done after protein expression 214 00:14:20,740 --> 00:14:22,210 or maybe in a cell. 215 00:14:22,210 --> 00:14:24,070 Here we have a benzophenone. 216 00:14:24,070 --> 00:14:26,380 So they're useful for cross linking experiments 217 00:14:26,380 --> 00:14:29,200 and we'll likely talk about benzophenone cross linking 218 00:14:29,200 --> 00:14:32,620 in recitation five in detail. 219 00:14:32,620 --> 00:14:35,470 We see some sugars here. 220 00:14:35,470 --> 00:14:36,700 This is the damsel group. 221 00:14:36,700 --> 00:14:39,100 That's a fluorophore. 222 00:14:39,100 --> 00:14:42,280 So there's many possibilities here. 223 00:14:42,280 --> 00:14:45,160 Just looking at these molecules, what's something 224 00:14:45,160 --> 00:14:48,130 similar about all of them? 225 00:14:54,100 --> 00:14:57,505 We think about them compared to a native amino acid. 226 00:14:57,505 --> 00:14:59,630 AUDIENCE: I was just that they're small. 227 00:14:59,630 --> 00:15:01,782 ELIZABETH NOLAN: OK, they're quite small. 228 00:15:01,782 --> 00:15:04,190 AUDIENCE: It's a kind of modified tyrosine. 229 00:15:04,190 --> 00:15:06,782 It will have some sort of benzo group that's modified. 230 00:15:06,782 --> 00:15:08,240 ELIZABETH NOLAN: So they're sort of 231 00:15:08,240 --> 00:15:11,170 phenylalinine or tyrosine like, right? 232 00:15:11,170 --> 00:15:15,220 And does that make sense from the standpoint 233 00:15:15,220 --> 00:15:17,010 of using this machinery initially? 234 00:15:17,010 --> 00:15:19,690 Yes, and you can imagine looking for other pairs 235 00:15:19,690 --> 00:15:23,260 to put in other types of unnatural amino acids. 236 00:15:23,260 --> 00:15:25,240 So that's reflective there. 237 00:15:25,240 --> 00:15:28,930 Just as some further examples, So this 238 00:15:28,930 --> 00:15:33,940 is another example of using an unnatural amino acid that can 239 00:15:33,940 --> 00:15:37,450 be useful for click chemistry. 240 00:15:37,450 --> 00:15:41,500 And I picked this in part, for one, this unnatural amino acid 241 00:15:41,500 --> 00:15:45,370 looks very different than the ones we saw on the prior slide. 242 00:15:45,370 --> 00:15:47,770 But there's aminoacyl-tRNA synthetase 243 00:15:47,770 --> 00:15:50,890 and the tRNA for this alkyne. 244 00:15:50,890 --> 00:15:54,400 And so you can imagine expressing a protein with this 245 00:15:54,400 --> 00:15:56,320 at a specific location. 246 00:15:56,320 --> 00:15:59,050 And then after the fact, clicking on a molecule 247 00:15:59,050 --> 00:16:01,390 like this fluorophore here. 248 00:16:01,390 --> 00:16:04,840 So just thinking about this process, 249 00:16:04,840 --> 00:16:10,000 why maybe was this put on later rather than in the cell? 250 00:16:21,550 --> 00:16:23,800 AUDIENCE: Do you mean clicking it on or synthesizing-- 251 00:16:23,800 --> 00:16:25,440 or putting that whole thing on? 252 00:16:25,440 --> 00:16:26,950 ELIZABETH NOLAN: Yeah, as you can imagine someone 253 00:16:26,950 --> 00:16:28,825 could have thought, rather than clicking this 254 00:16:28,825 --> 00:16:34,120 on after the fact, why not just use this whole moiety here 255 00:16:34,120 --> 00:16:36,970 as the unnatural amino acid? 256 00:16:36,970 --> 00:16:39,082 So this fluorophore. 257 00:16:39,082 --> 00:16:41,040 AUDIENCE: It would be hard to find a synthetase 258 00:16:41,040 --> 00:16:42,550 to accommodate that fluorophore. 259 00:16:42,550 --> 00:16:45,143 ELIZABETH NOLAN: It might be hard to find a synthetase. 260 00:16:45,143 --> 00:16:47,060 AUDIENCE: Might just be too much [INAUDIBLE].. 261 00:16:47,060 --> 00:16:49,998 It might not fit physically within the ribosome machinery. 262 00:16:49,998 --> 00:16:51,290 ELIZABETH NOLAN: That could be. 263 00:16:51,290 --> 00:16:55,107 AUDIENCE: Are you asking why we would not put it in? 264 00:16:55,107 --> 00:16:56,690 ELIZABETH NOLAN: Yeah, I'm just asking 265 00:16:56,690 --> 00:16:58,010 you to think about this, right? 266 00:16:58,010 --> 00:17:01,190 So you know, what needs to be thought about, right? 267 00:17:01,190 --> 00:17:03,320 So here, there's still a chemical step 268 00:17:03,320 --> 00:17:07,040 after this unnatural amino acid was put in. 269 00:17:07,040 --> 00:17:09,990 And in this case, why might that be? 270 00:17:09,990 --> 00:17:12,530 Maybe it's a permeability issue. 271 00:17:12,530 --> 00:17:16,010 We don't know if that molecule readily taken up 272 00:17:16,010 --> 00:17:17,510 by the organism. 273 00:17:17,510 --> 00:17:20,180 Is it a size issue, that it's hard to get machinery 274 00:17:20,180 --> 00:17:25,829 to accommodate this type of molecule here. 275 00:17:25,829 --> 00:17:27,740 AUDIENCE: Is it folding? 276 00:17:27,740 --> 00:17:28,948 ELIZABETH NOLAN: Folding of-- 277 00:17:28,948 --> 00:17:30,865 AUDIENCE: If you had it, is the question like, 278 00:17:30,865 --> 00:17:32,560 you put it on the floor, which is after 279 00:17:32,560 --> 00:17:34,080 like it's been processed-- 280 00:17:34,080 --> 00:17:36,455 ELIZABETH NOLAN: Yeah, maybe it messed up. 281 00:17:36,455 --> 00:17:37,830 AUDIENCE: If it's a floppy thing, 282 00:17:37,830 --> 00:17:41,110 it might interfere with folding, or folding might 283 00:17:41,110 --> 00:17:42,697 interfere with its, like-- 284 00:17:42,697 --> 00:17:44,780 ELIZABETH NOLAN: Right, so can the the polypeptide 285 00:17:44,780 --> 00:17:47,780 breach its native confirmation with this perturbation. 286 00:17:47,780 --> 00:17:49,280 Just to think about. 287 00:17:49,280 --> 00:17:52,760 And here are just some examples of unnatural amino acids 288 00:17:52,760 --> 00:17:58,180 that can be used for fluorine NMR as was mentioned last time. 289 00:17:58,180 --> 00:17:59,060 OK. 290 00:17:59,060 --> 00:18:04,430 So this is all really exciting, but what is the limitation? 291 00:18:04,430 --> 00:18:08,270 And there is a major limitation of this methodology 292 00:18:08,270 --> 00:18:11,430 as it was first described. 293 00:18:11,430 --> 00:18:21,770 So the major limitation is that the efficiency is low. 294 00:18:26,800 --> 00:18:27,400 OK? 295 00:18:27,400 --> 00:18:30,400 And if we consider wanting to incorporate 296 00:18:30,400 --> 00:18:33,310 one unnatural amino acid into a polypeptide, 297 00:18:33,310 --> 00:18:37,780 so there is one amber stop codon put in, what was found 298 00:18:37,780 --> 00:18:48,010 is that about 20% to 30% efficiency for incorporation 299 00:18:48,010 --> 00:18:51,420 of one unnatural amino acid. 300 00:18:51,420 --> 00:18:51,970 OK? 301 00:18:51,970 --> 00:18:56,740 And then this value plummeted to less than 1% 302 00:18:56,740 --> 00:19:03,560 for incorporation of two unnatural amino acids. 303 00:19:03,560 --> 00:19:05,680 So imagine there's two amber stop 304 00:19:05,680 --> 00:19:09,760 codons put within the gene. 305 00:19:09,760 --> 00:19:11,851 So why is this? 306 00:19:11,851 --> 00:19:14,530 This is because what's observed is 307 00:19:14,530 --> 00:19:27,870 that only a small amount of the protein or polypeptide 308 00:19:27,870 --> 00:19:33,210 synthesized reaches completion. 309 00:19:39,090 --> 00:19:40,890 And so, how can we think about this? 310 00:19:40,890 --> 00:19:45,630 Imagine here, I'm just going to draw some polypeptide chain 311 00:19:45,630 --> 00:19:48,150 going from end to C terminus. 312 00:19:48,150 --> 00:19:52,950 Let's imagine this is 20 kilodaltons in size. 313 00:19:52,950 --> 00:19:57,480 And maybe this unnatural amino acid 314 00:19:57,480 --> 00:20:00,950 is being placed right in the middle. 315 00:20:00,950 --> 00:20:01,740 OK? 316 00:20:01,740 --> 00:20:08,010 So we want to put an unnatural amino acid here. 317 00:20:08,010 --> 00:20:08,890 OK? 318 00:20:08,890 --> 00:20:12,000 So, imagine you make your plasma DNA to do this. 319 00:20:12,000 --> 00:20:16,410 You have the tRNA and aaRS and the unnatural amino acid, 320 00:20:16,410 --> 00:20:18,420 and you do your expression, and then 321 00:20:18,420 --> 00:20:21,840 you take a look by SDS page, so gel electrophoresis, 322 00:20:21,840 --> 00:20:22,615 what you see? 323 00:20:27,740 --> 00:20:39,990 So imagine here we have 20, 10, five, so kilodaltons here. 324 00:20:39,990 --> 00:20:40,490 Right? 325 00:20:40,490 --> 00:20:43,280 So we have some molecular weight markers let's just say here. 326 00:20:47,600 --> 00:20:51,720 If you do this, say for the native sequence. 327 00:20:51,720 --> 00:20:53,360 So you haven't put in the stop codon. 328 00:20:56,680 --> 00:20:59,330 Imagine there's your protein. 329 00:20:59,330 --> 00:21:09,320 If we have the unnatural amino acid, what do we see? 330 00:21:16,350 --> 00:21:18,280 Something like this. 331 00:21:18,280 --> 00:21:19,660 So what does this tell you? 332 00:21:27,540 --> 00:21:29,700 First of all, why do you look at the native one? 333 00:21:40,880 --> 00:21:43,430 Effectively, you want some positive control 334 00:21:43,430 --> 00:21:46,040 because if you can't express your polypeptide 335 00:21:46,040 --> 00:21:47,870 with the native sequence, you're not 336 00:21:47,870 --> 00:21:50,430 going to want to go try to stick in an unnatural amino acid, 337 00:21:50,430 --> 00:21:50,930 right? 338 00:21:50,930 --> 00:21:52,020 There's a problem. 339 00:21:52,020 --> 00:21:53,960 So that's your positive control. 340 00:21:53,960 --> 00:21:57,560 So we see in this make believe gel, 341 00:21:57,560 --> 00:22:00,650 there's one band at 20 kilodaltons, which 342 00:22:00,650 --> 00:22:02,642 is the size of that. 343 00:22:02,642 --> 00:22:04,100 If that ever happens to you, you've 344 00:22:04,100 --> 00:22:06,770 had an instant gratification protein trap. 345 00:22:06,770 --> 00:22:11,360 So, what about this lane with the unnatural amino acid? 346 00:22:11,360 --> 00:22:14,130 What do we see and what does this data tell us? 347 00:22:14,130 --> 00:22:14,630 Lindsey. 348 00:22:14,630 --> 00:22:16,350 AUDIENCE: It's like early truncation. 349 00:22:16,350 --> 00:22:18,100 ELIZABETH NOLAN: Yeah, something happened. 350 00:22:18,100 --> 00:22:20,930 So early truncation, and why are you saying that? 351 00:22:20,930 --> 00:22:22,580 We see two bands. 352 00:22:22,580 --> 00:22:26,420 There's one band with the expected migration 353 00:22:26,420 --> 00:22:28,400 to about 20 kilodaltons. 354 00:22:28,400 --> 00:22:31,430 And then there's the second band that's coming up 355 00:22:31,430 --> 00:22:33,560 around 10 kilodaltons. 356 00:22:33,560 --> 00:22:35,810 And based on what I sketched out here, 357 00:22:35,810 --> 00:22:37,790 that unnatural amino acid is roughly 358 00:22:37,790 --> 00:22:40,780 around the 10 kilodalton mark. 359 00:22:40,780 --> 00:22:41,540 OK? 360 00:22:41,540 --> 00:22:43,700 What about the relative intensity of these bands? 361 00:22:47,030 --> 00:22:49,004 What do we see more of? 362 00:22:49,004 --> 00:22:50,510 AUDIENCE: The truncated one. 363 00:22:50,510 --> 00:22:53,030 ELIZABETH NOLAN: We see more of the truncated form. 364 00:22:53,030 --> 00:22:54,710 So what's going on? 365 00:22:58,110 --> 00:22:58,610 we? 366 00:22:58,610 --> 00:23:04,870 Need to think about our ribosome. 367 00:23:04,870 --> 00:23:08,050 And there's some polypeptide being made. 368 00:23:08,050 --> 00:23:11,470 And then what's coming here? 369 00:23:11,470 --> 00:23:20,350 We either have our tRNA with the unnatural amino acid 370 00:23:20,350 --> 00:23:22,450 or the release factor, right? 371 00:23:22,450 --> 00:23:24,220 So there's going to be competition 372 00:23:24,220 --> 00:23:27,250 for binding in the A site between the tRNA 373 00:23:27,250 --> 00:23:28,335 and the release factor. 374 00:23:28,335 --> 00:23:29,710 And so this is getting back to, I 375 00:23:29,710 --> 00:23:33,190 believe, Max's question from last time about using the stop 376 00:23:33,190 --> 00:23:34,400 codon, right? 377 00:23:34,400 --> 00:23:38,200 There's fundamentally a problem here. 378 00:23:38,200 --> 00:23:39,317 So, yeah. 379 00:23:39,317 --> 00:23:41,400 AUDIENCE: How does the release time test different 380 00:23:41,400 --> 00:23:44,668 for different stop codons? 381 00:23:44,668 --> 00:23:46,210 ELIZABETH NOLAN: Yes, so we discussed 382 00:23:46,210 --> 00:23:47,950 that I think in lecture four. 383 00:23:47,950 --> 00:23:50,783 So there's a release factor one and release factor two, 384 00:23:50,783 --> 00:23:52,450 and there's three different stop codons. 385 00:23:52,450 --> 00:23:56,200 So they both recognize one of the same and two different. 386 00:23:56,200 --> 00:24:00,100 And in this case release factor one recognizes the amber stop 387 00:24:00,100 --> 00:24:02,230 codon here. 388 00:24:02,230 --> 00:24:05,320 So we're not worrying about release factor two 389 00:24:05,320 --> 00:24:07,930 competing with this stop codon because it doesn't 390 00:24:07,930 --> 00:24:11,490 recognize this stop codon here. 391 00:24:11,490 --> 00:24:12,340 Right? 392 00:24:12,340 --> 00:24:15,970 So if release factor one goes in, 393 00:24:15,970 --> 00:24:17,740 we get premature termination. 394 00:24:26,330 --> 00:24:28,335 And that results in truncated protein. 395 00:24:36,760 --> 00:24:37,690 So is this a problem? 396 00:24:42,150 --> 00:24:45,004 And how much of a problem is it? 397 00:24:45,004 --> 00:24:51,820 AUDIENCE: So you're saying that the release factors comes in 398 00:24:51,820 --> 00:24:54,800 because it's recognizing the codon that's trying to-- 399 00:24:54,800 --> 00:24:56,328 or that originally was a stop codon? 400 00:24:56,328 --> 00:24:58,370 ELIZABETH NOLAN: Yeah, because the codon is still 401 00:24:58,370 --> 00:25:00,070 a stop codon. 402 00:25:00,070 --> 00:25:01,445 AUDIENCE: So in the wild type, it 403 00:25:01,445 --> 00:25:03,790 wasn't that we replaced-- sorry. 404 00:25:03,790 --> 00:25:05,718 So we replaced it with a stop. 405 00:25:05,718 --> 00:25:07,260 But the stop wasn't there originally. 406 00:25:07,260 --> 00:25:10,195 And so that's why you get the full 20 length, right? 407 00:25:10,195 --> 00:25:11,070 ELIZABETH NOLAN: Yes. 408 00:25:11,070 --> 00:25:11,775 So you have-- 409 00:25:11,775 --> 00:25:12,400 AUDIENCE: Yeah, 410 00:25:12,400 --> 00:25:13,070 ELIZABETH NOLAN: OK, continue. 411 00:25:13,070 --> 00:25:14,528 AUDIENCE: There was no stop before. 412 00:25:14,528 --> 00:25:17,450 Now there's a stop, but it's not supposed to act like a stop, 413 00:25:17,450 --> 00:25:18,442 right? 414 00:25:18,442 --> 00:25:19,400 ELIZABETH NOLAN: Right. 415 00:25:19,400 --> 00:25:22,285 AUDIENCE: So here it is acting like a stop kind of? 416 00:25:22,285 --> 00:25:24,410 ELIZABETH NOLAN: It depends what enters the A-site. 417 00:25:24,410 --> 00:25:27,050 So a stop codon is a stop codon. 418 00:25:27,050 --> 00:25:30,710 But the idea is that this tRNA has 419 00:25:30,710 --> 00:25:36,740 been tweaked to allow a tRNA to recognize the stop. 420 00:25:36,740 --> 00:25:38,840 But there's going to be competition 421 00:25:38,840 --> 00:25:40,910 because you have the tRNA that's going to deliver 422 00:25:40,910 --> 00:25:42,560 the unnatural amino acid. 423 00:25:42,560 --> 00:25:46,300 But you also have release factor around. 424 00:25:46,300 --> 00:25:49,730 So this release factor one is in the endogenous pool. 425 00:25:49,730 --> 00:25:54,330 So the question is, which one gets there and does the job? 426 00:25:54,330 --> 00:25:54,830 Right? 427 00:25:54,830 --> 00:25:57,690 And so what that gel is telling you is that there's a mixture. 428 00:25:57,690 --> 00:25:58,190 Right? 429 00:25:58,190 --> 00:26:00,470 Sometimes the tRNA will get there 430 00:26:00,470 --> 00:26:04,730 and translation continues until you get to the desired stop 431 00:26:04,730 --> 00:26:07,940 where you want translation to stop, in terms of stopping. 432 00:26:07,940 --> 00:26:11,450 Or if the release factor gets there, you get termination. 433 00:26:11,450 --> 00:26:13,550 So you get some truncated protein. 434 00:26:13,550 --> 00:26:14,967 AUDIENCE: How do you know, though, 435 00:26:14,967 --> 00:26:16,884 that you've got in the end-- that you actually 436 00:26:16,884 --> 00:26:20,610 got the unnatural amino acid in the 20 [INAUDIBLE] 437 00:26:20,610 --> 00:26:22,394 and not just the original? 438 00:26:22,394 --> 00:26:24,140 Is that fluorescing? 439 00:26:24,140 --> 00:26:25,190 ELIZABETH NOLAN: No. 440 00:26:25,190 --> 00:26:27,950 I mean, just imagine we're just looking at protein here-- 441 00:26:27,950 --> 00:26:29,450 I mean, where this came from. 442 00:26:29,450 --> 00:26:30,950 AUDIENCE: So it would look the same? 443 00:26:30,950 --> 00:26:32,630 ELIZABETH NOLAN: If you had a fluorescent amino acid, 444 00:26:32,630 --> 00:26:33,672 you'd see something-- no. 445 00:26:33,672 --> 00:26:37,400 Because if you didn't have the unnatural amino acid there, 446 00:26:37,400 --> 00:26:39,440 what else could be there? 447 00:26:39,440 --> 00:26:42,500 AUDIENCE: Just like the native. 448 00:26:42,500 --> 00:26:46,100 ELIZABETH NOLAN: But what native amino acid can be incorporated 449 00:26:46,100 --> 00:26:47,460 if there is a stop? 450 00:26:47,460 --> 00:26:49,613 AUDIENCE: Oh, because you also put in the mRNA. 451 00:26:49,613 --> 00:26:50,530 ELIZABETH NOLAN: Yeah. 452 00:26:50,530 --> 00:26:51,030 Right. 453 00:26:51,030 --> 00:26:54,110 So there has to be a stop. 454 00:26:54,110 --> 00:26:56,063 Now, that's also backtracking why 455 00:26:56,063 --> 00:26:57,980 you need to make sure everything's orthogonal. 456 00:26:57,980 --> 00:27:00,920 Because you don't want one of the endogenous amino 457 00:27:00,920 --> 00:27:04,790 aminoacyl-tRNA synthetases to put some endogenous amino acid 458 00:27:04,790 --> 00:27:07,350 on this tRNA. 459 00:27:07,350 --> 00:27:08,810 OK? 460 00:27:08,810 --> 00:27:12,200 So either full length with the unnatural amino acid 461 00:27:12,200 --> 00:27:17,670 or truncated because RF1 came along here. 462 00:27:17,670 --> 00:27:18,170 Right? 463 00:27:18,170 --> 00:27:21,695 So in terms of how much of a problem this is, 464 00:27:21,695 --> 00:27:23,570 in some respects, it depends on what you need 465 00:27:23,570 --> 00:27:25,910 and what you want to do. 466 00:27:25,910 --> 00:27:28,200 If you're over expressing protein 467 00:27:28,200 --> 00:27:29,750 and you can deal with this mixture 468 00:27:29,750 --> 00:27:33,860 and get enough full length, maybe that's OK. 469 00:27:33,860 --> 00:27:37,010 If you're doing an experiment in cells, 470 00:27:37,010 --> 00:27:39,860 you have to ask, what is the consequence of also having 471 00:27:39,860 --> 00:27:42,200 some truncated protein around? 472 00:27:42,200 --> 00:27:43,860 What does that mean for the cell? 473 00:27:43,860 --> 00:27:48,870 What does that mean for your measurement there for that? 474 00:27:48,870 --> 00:27:53,600 So how can we get around this problem of RF1? 475 00:27:53,600 --> 00:27:58,130 So effectively, we want to diminish RF1 mediated chain 476 00:27:58,130 --> 00:27:59,720 termination. 477 00:27:59,720 --> 00:28:01,160 What are some possibilities? 478 00:28:03,870 --> 00:28:06,463 Is that feasible? 479 00:28:06,463 --> 00:28:08,630 So we could do that and we could get a better yield. 480 00:28:08,630 --> 00:28:11,000 That would be great for protein overexpression. 481 00:28:11,000 --> 00:28:13,250 If we could minimize truncated phenotypes, 482 00:28:13,250 --> 00:28:15,790 that would be great for an experiment in cells. 483 00:28:15,790 --> 00:28:19,730 You don't need to worry about what this truncated protein 484 00:28:19,730 --> 00:28:20,270 might do. 485 00:28:22,950 --> 00:28:24,290 So what are possibilities? 486 00:28:27,060 --> 00:28:29,750 So can we knock down or knock out our RF1? 487 00:28:32,620 --> 00:28:38,602 AUDIENCE: [INAUDIBLE] 488 00:28:38,602 --> 00:28:40,810 ELIZABETH NOLAN: So this is a wonderful little story. 489 00:28:40,810 --> 00:28:42,245 I'll just tell a little bit about, 490 00:28:42,245 --> 00:28:43,870 we're not going to go into huge detail. 491 00:28:43,870 --> 00:28:46,960 But for quite some time, it was thought that RF1 492 00:28:46,960 --> 00:28:49,000 was essential in E. coli. 493 00:28:49,000 --> 00:28:51,910 So a lot of experiments were done with E. coli K12 494 00:28:51,910 --> 00:28:53,980 and even if you go look on a website about all 495 00:28:53,980 --> 00:28:58,570 the genes in E. coli K12, it will tell you RF1 is essential. 496 00:28:58,570 --> 00:29:01,000 But then in 2012, a paper came out 497 00:29:01,000 --> 00:29:02,950 in ACS Chemical Biology, where they 498 00:29:02,950 --> 00:29:05,500 were doing some work in a different strain of E. coli. 499 00:29:05,500 --> 00:29:07,390 So there's many different E. coli's. 500 00:29:07,390 --> 00:29:10,210 And K12 is a laboratory workhorse. 501 00:29:10,210 --> 00:29:12,550 And there's also strains, E. coli B. 502 00:29:12,550 --> 00:29:14,920 And they're also laboratory workhorses. 503 00:29:14,920 --> 00:29:18,430 So maybe many of you have used BL 21DE3 cells 504 00:29:18,430 --> 00:29:19,900 for protein expression. 505 00:29:19,900 --> 00:29:23,410 So this lab was working in E. coli B strain, 506 00:29:23,410 --> 00:29:26,510 and found that RF1 could be knocked out; 507 00:29:26,510 --> 00:29:28,360 that it's not essential. 508 00:29:28,360 --> 00:29:32,090 So then the question is, what's going on? 509 00:29:32,090 --> 00:29:37,630 And as it turns out, the essentiality of RF1 in E. coli 510 00:29:37,630 --> 00:29:40,510 turned out to be due to an issue with RF2. 511 00:29:40,510 --> 00:29:45,340 And in the K12 release factor 2 has a single point mutation 512 00:29:45,340 --> 00:29:52,670 that makes it less able to stop at certain stop codons. 513 00:29:52,670 --> 00:29:56,290 So when you had both of those together, it was deleterious. 514 00:29:56,290 --> 00:29:58,570 So RF1 can be knocked out. 515 00:29:58,570 --> 00:30:00,116 Would you want to do that? 516 00:30:00,116 --> 00:30:04,300 AUDIENCE: So, RF1 can be knocked out without RF2 or RF3, 517 00:30:04,300 --> 00:30:06,908 I don't remember. 518 00:30:06,908 --> 00:30:09,200 ELIZABETH NOLAN: Yeah, there are three release factors. 519 00:30:09,200 --> 00:30:10,780 RF3 is a GTPase. 520 00:30:10,780 --> 00:30:13,520 It's a little different. 521 00:30:13,520 --> 00:30:17,650 AUDIENCE: There's redundant kind of behavior. 522 00:30:17,650 --> 00:30:20,210 ELIZABETH NOLAN: There's some redundancy. 523 00:30:20,210 --> 00:30:22,090 And I mean, something too just to ask 524 00:30:22,090 --> 00:30:26,710 is, if you can knock it out and the cell is viable, 525 00:30:26,710 --> 00:30:31,180 viability is different than normal healthy cell. 526 00:30:31,180 --> 00:30:34,960 So those E. coli B, without RF1 will grow, 527 00:30:34,960 --> 00:30:36,580 but are they growing and replicating 528 00:30:36,580 --> 00:30:41,380 as well as the wild type? 529 00:30:41,380 --> 00:30:42,030 No. 530 00:30:42,030 --> 00:30:42,530 No. 531 00:30:42,530 --> 00:30:43,640 But is it good enough? 532 00:30:43,640 --> 00:30:45,880 And I think again, it comes down to asking what is it 533 00:30:45,880 --> 00:30:47,350 that you want to do? 534 00:30:47,350 --> 00:30:49,390 So maybe if you're over expressing protein 535 00:30:49,390 --> 00:30:52,190 and you're going to purify that, it's not such a big deal. 536 00:30:52,190 --> 00:30:55,020 But again, if you're looking at some cellular process, 537 00:30:55,020 --> 00:30:56,770 you're going to need to think about what's 538 00:30:56,770 --> 00:31:01,750 happening if RF1 can't terminate translation for, you know, 539 00:31:01,750 --> 00:31:05,110 its repertoire of proteins and genes there. 540 00:31:05,110 --> 00:31:08,470 There will be some consequence of that perturbation 541 00:31:08,470 --> 00:31:11,450 just to keep in mind. 542 00:31:11,450 --> 00:31:14,170 But there's certainly work going on with that now that it 543 00:31:14,170 --> 00:31:17,410 was found not to be essential. 544 00:31:17,410 --> 00:31:21,640 So in vitro translation, just something to think about. 545 00:31:21,640 --> 00:31:23,950 If you're going to work in a test tube, 546 00:31:23,950 --> 00:31:26,300 could you just do this outside of the cell? 547 00:31:26,300 --> 00:31:30,220 And then, the possibility we're going to discuss in closing 548 00:31:30,220 --> 00:31:35,360 is this one of a new ribosome, which I think is pretty cool. 549 00:31:35,360 --> 00:31:41,470 So, is it possible to have an orthogonal ribosome here 550 00:31:41,470 --> 00:31:44,110 to get around this problem? 551 00:31:44,110 --> 00:31:48,700 So effectively, can we make a new ribosome 552 00:31:48,700 --> 00:31:54,250 that only translates the message encoded in a plasmid that 553 00:31:54,250 --> 00:31:56,050 has the gene of interest where you want 554 00:31:56,050 --> 00:31:59,260 the unnatural amino acid to go? 555 00:31:59,260 --> 00:32:02,120 And so thinking about this in cartoon form, 556 00:32:02,120 --> 00:32:05,320 imagine we have E. Coli or some organism, 557 00:32:05,320 --> 00:32:09,520 and there's the native ribosome, and this native ribosome 558 00:32:09,520 --> 00:32:11,860 translates all of the native wild type 559 00:32:11,860 --> 00:32:15,790 mRNAs and gives synthesis of the proteome. 560 00:32:15,790 --> 00:32:18,670 But then imagine we can put in an orthogonal ribosome 561 00:32:18,670 --> 00:32:20,440 into this organism. 562 00:32:20,440 --> 00:32:23,620 And this orthogonal ribosome only 563 00:32:23,620 --> 00:32:27,310 recognizes an orthogonal mRNA, which 564 00:32:27,310 --> 00:32:33,340 means it only translates off of this orthogonal mRNA 565 00:32:33,340 --> 00:32:37,180 and only gives you synthesis of the protein 566 00:32:37,180 --> 00:32:40,960 you want with the unnatural amino acid. 567 00:32:40,960 --> 00:32:44,290 So how to think about doing this? 568 00:32:44,290 --> 00:32:48,160 Need to think back about the initiation process, 569 00:32:48,160 --> 00:32:52,540 and that mRNAs have a ribosome binding site. 570 00:32:52,540 --> 00:32:59,200 So effectively, it's necessary to engineer an mRNA that 571 00:32:59,200 --> 00:33:03,190 contains a ribosome binding site that will not 572 00:33:03,190 --> 00:33:07,510 direct translation by the endogenous ribosome, 573 00:33:07,510 --> 00:33:10,310 so some new ribosome binding site. 574 00:33:10,310 --> 00:33:10,810 OK? 575 00:33:10,810 --> 00:33:12,760 And then this orthogonal ribosome 576 00:33:12,760 --> 00:33:16,090 needs to be engineered such that it's specifically 577 00:33:16,090 --> 00:33:18,820 binding to the orthogonal mRNA. 578 00:33:18,820 --> 00:33:24,880 And it doesn't bind to the wild type mRNAs there. 579 00:33:24,880 --> 00:33:26,920 So no translation of the cellular message 580 00:33:26,920 --> 00:33:30,250 because this ribosome binding site and orthogonal ribosome 581 00:33:30,250 --> 00:33:31,280 are a match. 582 00:33:31,280 --> 00:33:32,020 OK? 583 00:33:32,020 --> 00:33:35,290 So a unique binding site. 584 00:33:35,290 --> 00:33:38,810 So in thinking about how to do this, 585 00:33:38,810 --> 00:33:41,660 you want to think about the ribosome structure. 586 00:33:41,660 --> 00:33:45,440 And we know that the 16S rRNA is involved 587 00:33:45,440 --> 00:33:51,040 in binding to the mRNA at the beginning of the initiation 588 00:33:51,040 --> 00:33:51,620 step. 589 00:33:51,620 --> 00:33:56,810 So what was done was to mutate the 16S 590 00:33:56,810 --> 00:34:00,950 and come up with an orthogonal ribosome. 591 00:34:00,950 --> 00:34:03,980 So this has been done. 592 00:34:03,980 --> 00:34:06,680 That's not a solution to the problem of RF1 593 00:34:06,680 --> 00:34:10,620 terminating translation on its own. 594 00:34:10,620 --> 00:34:12,860 So then the next question is, if we 595 00:34:12,860 --> 00:34:16,790 can have just this orthogonal ribosome and orthogonal mRNA, 596 00:34:16,790 --> 00:34:22,280 can we improve that system to minimize RF1 mediated chain 597 00:34:22,280 --> 00:34:23,750 termination? 598 00:34:23,750 --> 00:34:26,750 So effectively what we want to do 599 00:34:26,750 --> 00:34:29,750 is prevent RF1 from binding to the A-site 600 00:34:29,750 --> 00:34:31,580 of the orthogonal ribosome. 601 00:34:31,580 --> 00:34:35,610 But it's still going to do its job for the endogenous ribosome 602 00:34:35,610 --> 00:34:36,110 here. 603 00:34:38,870 --> 00:34:41,628 So what needs to happen? 604 00:34:41,628 --> 00:34:42,920 And we'll go through the steps. 605 00:34:42,920 --> 00:34:48,370 This is just the schematic in cartoon form. 606 00:34:48,370 --> 00:34:51,080 So imagine we're starting with native ribosomes 607 00:34:51,080 --> 00:34:53,989 and orthogonal ribosomes. 608 00:34:53,989 --> 00:34:57,890 And we have tRNAs and RF1. 609 00:34:57,890 --> 00:35:00,920 And nothing has been done to this orthogonal ribosome 610 00:35:00,920 --> 00:35:03,380 so RF1 can still bind there. 611 00:35:03,380 --> 00:35:07,280 And so we want to have some evolution. 612 00:35:07,280 --> 00:35:11,240 So mutagenesis and selection of the orthogonal ribosome 613 00:35:11,240 --> 00:35:15,140 such that only the tRNA goes to the A-site. 614 00:35:15,140 --> 00:35:17,990 And RF1 only goes to the wild type ribosome. 615 00:35:23,170 --> 00:35:24,860 So there's other possibilities. 616 00:35:24,860 --> 00:35:26,770 One possibility I'll just throw out there 617 00:35:26,770 --> 00:35:31,750 is using rather than a triplet, a quadruplet codon there, 618 00:35:31,750 --> 00:35:35,080 which we won't talk about. 619 00:35:35,080 --> 00:35:37,150 There's more than one solution to the problem. 620 00:35:37,150 --> 00:35:43,450 But where we're going to focus on is work done to minimize RF1 621 00:35:43,450 --> 00:35:45,112 and how to think about doing that 622 00:35:45,112 --> 00:35:46,570 from the standpoint of what we know 623 00:35:46,570 --> 00:35:50,060 of ribosome structure and the interactions. 624 00:35:50,060 --> 00:35:50,650 OK? 625 00:35:50,650 --> 00:35:54,790 So the name of this new O ribosome 626 00:35:54,790 --> 00:35:58,780 is Ribo-X and so what did they do? 627 00:35:58,780 --> 00:36:04,090 They started with the orthogonal ribosome. 628 00:36:07,170 --> 00:36:07,920 OK? 629 00:36:07,920 --> 00:36:10,140 And so, the first is that there needs 630 00:36:10,140 --> 00:36:15,780 to be some mutation to the ribosome, 631 00:36:15,780 --> 00:36:17,235 so libraries of mutants. 632 00:36:28,120 --> 00:36:30,205 There needs to be some selection process. 633 00:36:35,000 --> 00:36:45,090 So effectively, there is a requirement for of activity 634 00:36:45,090 --> 00:36:46,320 from the ribosome. 635 00:36:50,790 --> 00:36:51,290 and. 636 00:36:51,290 --> 00:36:56,030 When there's this, there needs to be some sequencing. 637 00:37:00,430 --> 00:37:03,100 Or identity determination, so where is the mutation? 638 00:37:09,800 --> 00:37:10,670 Here. 639 00:37:10,670 --> 00:37:14,630 And then with some mutant in hand, 640 00:37:14,630 --> 00:37:16,610 that looks like it's a good option, 641 00:37:16,610 --> 00:37:18,520 there needs to be assays to study it. 642 00:37:32,250 --> 00:37:36,360 And we're pretty much going to focus on step four. 643 00:37:36,360 --> 00:37:42,400 I'll briefly say something about steps one, two, and three here. 644 00:37:42,400 --> 00:37:49,110 So the first thing is, if we want to mutate this O ribosome, 645 00:37:49,110 --> 00:37:52,605 how do we think about designing a mutant library? 646 00:37:55,200 --> 00:37:58,800 And so, what we need to think about in this case 647 00:37:58,800 --> 00:38:05,790 because the goal is to minimize RF1 mediated chain termination 648 00:38:05,790 --> 00:38:10,050 and enhance tRNA getting into this A-site, 649 00:38:10,050 --> 00:38:14,550 we want to look at how the ribosome interacts 650 00:38:14,550 --> 00:38:19,530 with RF1, how it interacts with the tRNA, 651 00:38:19,530 --> 00:38:22,350 and also think about the mRNA there. 652 00:38:22,350 --> 00:38:25,440 And so, there's crystal structures available. 653 00:38:25,440 --> 00:38:28,860 There might be biochemical information available. 654 00:38:28,860 --> 00:38:31,200 But really to ask, where does that 655 00:38:31,200 --> 00:38:34,140 make sense to make mutations? 656 00:38:34,140 --> 00:38:37,740 And so if we think about the stop codon being recognized 657 00:38:37,740 --> 00:38:42,450 by the tRNA and RF1 in the A-site, 658 00:38:42,450 --> 00:38:48,270 somehow we want to mutate the ribosomal RNA in that region 659 00:38:48,270 --> 00:38:51,270 to give us the desired outcome. 660 00:38:51,270 --> 00:38:58,160 So what they did is mutate 16S rRNA 661 00:38:58,160 --> 00:39:02,160 to favor suppression of the amber stop codon by the tRNA. 662 00:39:02,160 --> 00:39:05,820 And crystal structures guided the library design. 663 00:39:05,820 --> 00:39:07,590 And so they looked at crystal structures 664 00:39:07,590 --> 00:39:10,380 where tRNAs are bound to the A-site 665 00:39:10,380 --> 00:39:14,190 or where RF1 is bound to the A-site. 666 00:39:14,190 --> 00:39:17,040 And from these, they selected seven different positions 667 00:39:17,040 --> 00:39:22,110 of the RNA and randomly mutated them. 668 00:39:22,110 --> 00:39:25,950 So that gives you some new mutants to study. 669 00:39:25,950 --> 00:39:28,620 Then there needs to be a selection process. 670 00:39:28,620 --> 00:39:32,220 So the mutant needs to be active. 671 00:39:32,220 --> 00:39:34,710 Some of these mutations might cause the ribosome 672 00:39:34,710 --> 00:39:38,560 to be inactive and that won't be very helpful. 673 00:39:38,560 --> 00:39:43,260 And so they developed an assay based on antibiotic resistance 674 00:39:43,260 --> 00:39:45,290 to select. 675 00:39:45,290 --> 00:39:50,550 And effectively, an enzyme that provides resistance 676 00:39:50,550 --> 00:39:53,850 to chloranfenicol, which is an antibiotic that 677 00:39:53,850 --> 00:39:56,940 blocks translation and was put under the control 678 00:39:56,940 --> 00:39:59,160 of the O ribosome. 679 00:39:59,160 --> 00:40:04,710 So you can imagine using antibiotic resistance 680 00:40:04,710 --> 00:40:07,290 as a selection there. 681 00:40:07,290 --> 00:40:09,720 And then the sequencing, once we've 682 00:40:09,720 --> 00:40:13,020 selected first some mutants, we have 683 00:40:13,020 --> 00:40:15,370 to ask where is the mutation? 684 00:40:15,370 --> 00:40:18,750 And so what they found after going through this work 685 00:40:18,750 --> 00:40:35,360 is that for Ribo-X it's only a double mutant in the 16 S rRNA. 686 00:40:35,360 --> 00:40:46,010 So two positions, U3531G and U534A. 687 00:40:46,010 --> 00:40:50,780 So these mutations in proved suppression of the amber stop 688 00:40:50,780 --> 00:40:57,370 codon, and I also point out these mutations 689 00:40:57,370 --> 00:40:58,130 are very unusual. 690 00:41:01,050 --> 00:41:03,470 So, at least at the time of this work, 691 00:41:03,470 --> 00:41:09,200 no sequenced natural ribosome had these two mutations here. 692 00:41:09,200 --> 00:41:15,060 And they're found in very few examples of sequenced RNase 693 00:41:15,060 --> 00:41:17,670 here. 694 00:41:17,670 --> 00:41:22,560 So, I mean, just to think about the ribosome's so huge 695 00:41:22,560 --> 00:41:25,950 and just two point mutations can make this change here. 696 00:41:25,950 --> 00:41:30,140 So what's seen in terms of some characterization. 697 00:41:36,180 --> 00:41:40,770 What do we need to ask in terms of characterization. 698 00:41:49,100 --> 00:41:49,640 Bless you. 699 00:41:54,740 --> 00:42:00,780 So something we want to ask about is fidelity here. 700 00:42:00,780 --> 00:42:09,920 So if we think about fidelity, one, we can ask, 701 00:42:09,920 --> 00:42:13,250 if we're using this to express some protein, 702 00:42:13,250 --> 00:42:16,370 what is the protein yield and how does that 703 00:42:16,370 --> 00:42:19,490 compare to the native ribosome? 704 00:42:19,490 --> 00:42:25,820 We want it to incorporate amino acids correctly 705 00:42:25,820 --> 00:42:27,380 with high fidelity and incorporation 706 00:42:27,380 --> 00:42:29,210 of the unnatural amino acid. 707 00:42:29,210 --> 00:42:31,400 So doesn't this incorporate amino acids? 708 00:42:35,850 --> 00:42:39,250 So that's the question we need to ask. 709 00:42:39,250 --> 00:42:39,750 OK? 710 00:42:39,750 --> 00:42:48,090 And then of course, we need to ask about amber stop codon 711 00:42:48,090 --> 00:42:49,440 suppression efficiency. 712 00:42:58,100 --> 00:43:00,350 And so, in thinking about this what 713 00:43:00,350 --> 00:43:01,850 is the point of comparison? 714 00:43:05,300 --> 00:43:08,360 So we can imagine in all of these comparing 715 00:43:08,360 --> 00:43:11,270 this new orthogonal ribosome Ribo-X 716 00:43:11,270 --> 00:43:13,840 to the starting orthogonal ribosome. 717 00:43:13,840 --> 00:43:15,750 Right? 718 00:43:15,750 --> 00:43:18,300 Here. 719 00:43:18,300 --> 00:43:20,190 So what are the experiments? 720 00:43:24,160 --> 00:43:26,550 So first let's think about protein yield. 721 00:43:32,780 --> 00:43:34,640 And I'll just say, I have a pet peeve 722 00:43:34,640 --> 00:43:37,880 when people don't report their protein yields in experimental. 723 00:43:37,880 --> 00:43:41,240 So if you're doing biochemistry, always 724 00:43:41,240 --> 00:43:43,880 think about doing that there. 725 00:43:43,880 --> 00:43:47,030 So what they did is an experiment 726 00:43:47,030 --> 00:43:48,410 where they made a plasmid. 727 00:43:48,410 --> 00:43:52,540 So we have an orthogonal DNA that will give orthogonal mRNA. 728 00:44:07,240 --> 00:44:08,610 So this gets transcribed... 729 00:44:13,940 --> 00:44:25,850 to give the orthogonal mRNA and then it gets translated 730 00:44:25,850 --> 00:44:27,590 by either the O ribosome... 731 00:44:33,060 --> 00:44:38,550 or Ribo-X. And the result of this 732 00:44:38,550 --> 00:44:42,120 is a fusion protein where we have a protein called 733 00:44:42,120 --> 00:44:47,610 GST, glutathione S-transferase, and then MBP, which 734 00:44:47,610 --> 00:44:48,915 is maltose-binding protein. 735 00:44:53,133 --> 00:44:55,050 And as we move forward, it will become clearer 736 00:44:55,050 --> 00:44:57,800 why they use this fusion. 737 00:44:57,800 --> 00:45:00,330 OK so just the first question is, how does 738 00:45:00,330 --> 00:45:03,660 the yield of protein compare? 739 00:45:03,660 --> 00:45:07,470 Are they doing a similar job or were these two mutations 740 00:45:07,470 --> 00:45:10,350 detrimental? 741 00:45:10,350 --> 00:45:13,680 So here's the result from this experiment 742 00:45:13,680 --> 00:45:16,110 one looking at protein yield. 743 00:45:16,110 --> 00:45:20,220 OK, so again we're looking at an SDS page gel that's 744 00:45:20,220 --> 00:45:23,340 being stained for the protein. 745 00:45:23,340 --> 00:45:28,350 And we see that this GST and BP fusion has a molecular weight 746 00:45:28,350 --> 00:45:31,170 of 71 kilodaltons, right? 747 00:45:31,170 --> 00:45:33,930 And what we see up here are the components 748 00:45:33,930 --> 00:45:36,720 that were in each of the experiments for each 749 00:45:36,720 --> 00:45:38,340 of the lanes. 750 00:45:38,340 --> 00:45:43,800 So here in this lane, we have no O ribosome, 751 00:45:43,800 --> 00:45:49,020 no Ribo-X but the plasmid was included. 752 00:45:49,020 --> 00:45:51,720 Here, we have the orthogonal ribosome in the plasmed, 753 00:45:51,720 --> 00:45:54,030 here Ribo-X in the plasmid. 754 00:45:54,030 --> 00:45:54,990 So what do we see? 755 00:46:02,426 --> 00:46:02,926 Pardon? 756 00:46:02,926 --> 00:46:04,635 AUDIENCE: Are they all the same yield? 757 00:46:04,635 --> 00:46:06,510 ELIZABETH NOLAN: Are they all the same yield? 758 00:46:06,510 --> 00:46:07,560 There's three lanes. 759 00:46:07,560 --> 00:46:12,260 AUDIENCE: [INAUDIBLE] 760 00:46:12,260 --> 00:46:14,880 ELIZABETH NOLAN: Yeah, so no orthogonal ribosome, 761 00:46:14,880 --> 00:46:16,470 no translation. 762 00:46:16,470 --> 00:46:18,240 And that's a good thing to see, right? 763 00:46:18,240 --> 00:46:21,720 That tells you that this orthogonal mRNA is not 764 00:46:21,720 --> 00:46:24,240 being translated by the endogenous ribosome. 765 00:46:24,240 --> 00:46:25,860 That's an important observation. 766 00:46:25,860 --> 00:46:27,900 And then I think what you meant to say, 767 00:46:27,900 --> 00:46:29,970 is that in these two lanes where we 768 00:46:29,970 --> 00:46:31,890 have either the starting orthogonal 769 00:46:31,890 --> 00:46:36,390 ribosome or Ribo-X, what we see is 770 00:46:36,390 --> 00:46:39,210 what appears to be a very similar amount of protein. 771 00:46:39,210 --> 00:46:45,180 So here, you know you assume and you look at the experimental, 772 00:46:45,180 --> 00:46:47,400 the same volumes were loaded, all of these things. 773 00:46:47,400 --> 00:46:49,800 We're getting the same amount of protein yield. 774 00:46:49,800 --> 00:46:53,550 So that's a great result here. 775 00:46:53,550 --> 00:46:55,450 So that's good news. 776 00:46:55,450 --> 00:46:59,290 What's the next experiment? 777 00:46:59,290 --> 00:47:01,690 And we'll close, I think on this experiment. 778 00:47:01,690 --> 00:47:07,300 So the next experiment is amino acid misincorporation. 779 00:47:14,910 --> 00:47:20,995 So again, what they did is they used this GST MBP fusion 780 00:47:20,995 --> 00:47:21,495 protein. 781 00:47:27,470 --> 00:47:31,070 And there's a linker region here. 782 00:47:31,070 --> 00:47:34,370 And in this linker region, they engineered a protease cleavage 783 00:47:34,370 --> 00:47:35,720 site here. 784 00:47:50,660 --> 00:47:54,000 So for thrombin here. 785 00:47:54,000 --> 00:47:58,950 And why did they do this to look at amino acid misincorporation, 786 00:47:58,950 --> 00:48:00,900 whether that's happening. 787 00:48:00,900 --> 00:48:04,710 Effectively, they took advantage of the fact 788 00:48:04,710 --> 00:48:15,320 that GST contains cystine, whereas maltose binding protein 789 00:48:15,320 --> 00:48:16,115 has no cystine. 790 00:48:21,470 --> 00:48:24,170 So their idea was let's use radio 791 00:48:24,170 --> 00:48:29,780 labeled cystine as a probe and monitor for radioactive cystine 792 00:48:29,780 --> 00:48:30,790 incorporation. 793 00:49:08,940 --> 00:49:11,760 So effectively, what can be done is 794 00:49:11,760 --> 00:49:15,120 that this can be expressed and purified 795 00:49:15,120 --> 00:49:18,380 in the presence of the radio labeled cystine. 796 00:49:18,380 --> 00:49:20,970 Thrombin can be used to cleave. 797 00:49:20,970 --> 00:49:23,730 And then you can look and ask is there radioactivity 798 00:49:23,730 --> 00:49:26,070 associated with GST? 799 00:49:26,070 --> 00:49:27,510 And we hope the answer is yes. 800 00:49:27,510 --> 00:49:29,100 And is there radioactive activity 801 00:49:29,100 --> 00:49:32,630 associated with maltose binding protein. 802 00:49:32,630 --> 00:49:36,120 And so where we'll begin on Friday is looking at the data 803 00:49:36,120 --> 00:49:36,990 from this assay. 804 00:49:36,990 --> 00:49:39,750 But until then, what I'd like you to think about 805 00:49:39,750 --> 00:49:43,830 is in terms of amino acid misincorporation, 806 00:49:43,830 --> 00:49:47,070 kind of strengths and limitations of this assay. 807 00:49:47,070 --> 00:49:49,380 Right so the choice of using one amino acid 808 00:49:49,380 --> 00:49:51,160 to take a look there. 809 00:49:51,160 --> 00:49:51,660 OK? 810 00:49:51,660 --> 00:49:53,720 So I'll see you Friday.