1 00:00:00,500 --> 00:00:02,810 The following content is provided under a Creative 2 00:00:02,810 --> 00:00:04,380 Commons license. 3 00:00:04,380 --> 00:00:06,670 Your support will help MIT OpenCourseWare 4 00:00:06,670 --> 00:00:11,010 continue to offer high quality educational resources for free. 5 00:00:11,010 --> 00:00:13,670 To make a donation or view additional materials 6 00:00:13,670 --> 00:00:17,119 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,119 --> 00:00:18,110 at ocw.mit.edu. 8 00:00:25,100 --> 00:00:28,870 ELIZABETH NOLAN: Today we should be completing the translation 9 00:00:28,870 --> 00:00:30,230 cycle. 10 00:00:30,230 --> 00:00:32,140 And the next topic that will come up 11 00:00:32,140 --> 00:00:36,250 is use of antibiotics as tools to study the ribosome 12 00:00:36,250 --> 00:00:38,020 and translation. 13 00:00:38,020 --> 00:00:42,220 So just a recap from last time, we went over the delivery 14 00:00:42,220 --> 00:00:47,830 of aminoacyl--tRNAs by EF-TU and looked at this model 15 00:00:47,830 --> 00:00:50,950 for understanding how that happens, OK? 16 00:00:50,950 --> 00:00:55,090 So recall, we discussed the initial binding 17 00:00:55,090 --> 00:01:00,610 of the ternary complex of EF-TU GTP and the aminoacyl-tRNA. 18 00:01:00,610 --> 00:01:02,740 And that's codon independent. 19 00:01:02,740 --> 00:01:09,010 When a codon/anticodon match occurs, 20 00:01:09,010 --> 00:01:11,850 we have a push in the forward direction. 21 00:01:11,850 --> 00:01:13,870 EF-TU's a GTPase. 22 00:01:13,870 --> 00:01:16,000 There's activation of the GTP center. 23 00:01:16,000 --> 00:01:20,020 So conformational changes, GTP hydrolysis. 24 00:01:20,020 --> 00:01:22,970 So we have EF-TU and the GTP bound form. 25 00:01:22,970 --> 00:01:25,270 There's conformational change, and ultimately 26 00:01:25,270 --> 00:01:29,220 accommodation of this tRNA in the A site. 27 00:01:29,220 --> 00:01:32,320 And that allows for peptide bond formation. 28 00:01:32,320 --> 00:01:35,890 So where we left off last time was 29 00:01:35,890 --> 00:01:37,960 discussing the conformational changes 30 00:01:37,960 --> 00:01:40,420 that occur in the decoding center 31 00:01:40,420 --> 00:01:44,490 and also in the GTP center of EF-TU. 32 00:01:44,490 --> 00:01:46,810 And just to highlight, I mentioned 33 00:01:46,810 --> 00:01:51,430 that there are conformational changes within the 16S rRNA, 34 00:01:51,430 --> 00:01:54,070 in particular three nucleotides that 35 00:01:54,070 --> 00:01:58,390 occur when it's a cognate codon/anticodon interaction. 36 00:01:58,390 --> 00:02:00,280 And these are just shown here. 37 00:02:00,280 --> 00:02:04,390 And effectively what we're looking at in these three 38 00:02:04,390 --> 00:02:10,509 panels are the 16S rRNA in the absence of the tRNA, 39 00:02:10,509 --> 00:02:12,910 in the presence of the tRNA, but the tRNA 40 00:02:12,910 --> 00:02:16,480 is removed from this image for simplicity, 41 00:02:16,480 --> 00:02:18,940 and then with the tRNA bound. 42 00:02:18,940 --> 00:02:22,360 So some of the easiest changes to see here 43 00:02:22,360 --> 00:02:26,300 are with A1492 and A1493. 44 00:02:26,300 --> 00:02:28,210 So if we look in the absence of tRNA, 45 00:02:28,210 --> 00:02:30,650 they're pointing down the bases. 46 00:02:30,650 --> 00:02:35,560 And here as a result of tRNA binding in the A site, 47 00:02:35,560 --> 00:02:41,450 we see that A1492 and 1493 are flipped, flipped up. 48 00:02:41,450 --> 00:02:41,950 OK. 49 00:02:41,950 --> 00:02:49,180 And if we look here, you can see how these are interacting 50 00:02:49,180 --> 00:02:51,020 with the bound tRNA. 51 00:02:51,020 --> 00:02:51,520 OK. 52 00:02:51,520 --> 00:02:53,530 So this conformational change helps 53 00:02:53,530 --> 00:02:56,020 to accelerate the forward steps. 54 00:02:56,020 --> 00:02:57,640 So that's in the decoding center. 55 00:02:57,640 --> 00:03:00,910 And then also just remember 70 angstroms away 56 00:03:00,910 --> 00:03:03,580 in the GTP center of EF-TU, there's 57 00:03:03,580 --> 00:03:06,580 conformational change of these hydrophobic residues that 58 00:03:06,580 --> 00:03:09,790 are thought to be a hydrophobic gate that allows histamine 59 00:03:09,790 --> 00:03:16,660 84 to activate a water molecule for attack in GTP hydrolysis. 60 00:03:16,660 --> 00:03:20,050 So at this stage we're finally ready to have a peptide bond 61 00:03:20,050 --> 00:03:21,940 formed by the ribosome. 62 00:03:25,920 --> 00:03:34,420 And so we need to think about that mechanism and then 63 00:03:34,420 --> 00:03:36,440 what happens after. 64 00:03:36,440 --> 00:03:42,130 I'll say, so effectively what we have in the P site 65 00:03:42,130 --> 00:03:51,055 is the tRNA with some growing peptide chain. 66 00:03:51,055 --> 00:03:54,236 [WRITING ON BOARD] 67 00:04:04,930 --> 00:04:10,160 And then we have the aminoacyl-tRNA in the A site. 68 00:04:10,160 --> 00:04:13,597 [WRITING ON BOARD] 69 00:04:28,435 --> 00:04:38,230 And so what happens effectively, we have attack from here, 70 00:04:38,230 --> 00:04:53,490 release such that we end up with a P site 71 00:04:53,490 --> 00:04:54,810 with a deacylated tRNA. 72 00:05:02,500 --> 00:05:11,120 And in the A site we now have the peptidyl-tRNA that 73 00:05:11,120 --> 00:05:14,355 has grown by one amino acid monomer. 74 00:05:14,355 --> 00:05:17,590 [WRITING ON BOARD] 75 00:05:27,190 --> 00:05:27,830 Here, OK? 76 00:05:34,790 --> 00:05:35,290 OK. 77 00:05:35,290 --> 00:05:37,960 And so this is the N- terminal end 78 00:05:37,960 --> 00:05:40,210 of the protein or the polypeptide, 79 00:05:40,210 --> 00:05:44,710 and here's the C terminal end here. 80 00:05:44,710 --> 00:05:51,620 So thinking about this mechanism and having nucleophilic attack 81 00:05:51,620 --> 00:05:56,620 from this alpha amino group of the aminoacyl-tRNA in the A 82 00:05:56,620 --> 00:06:01,140 site, what do we need to think about? 83 00:06:01,140 --> 00:06:02,940 Is there anything surprising or unusual? 84 00:06:18,884 --> 00:06:20,850 AUDIENCE: Think about protonation state. 85 00:06:20,850 --> 00:06:21,650 ELIZABETH NOLAN: Yeah, right. 86 00:06:21,650 --> 00:06:22,200 Exactly. 87 00:06:22,200 --> 00:06:24,540 We need to think about the pKa. 88 00:06:24,540 --> 00:06:27,840 So typically do we think about an alpha amino group being 89 00:06:27,840 --> 00:06:32,400 protonated or deprotonated, that physiological pH. 90 00:06:32,400 --> 00:06:32,900 Yeah. 91 00:06:32,900 --> 00:06:38,170 Protonated we typically think about an H3+, not an H2 here. 92 00:06:38,170 --> 00:06:41,070 So what does that tell us? 93 00:06:41,070 --> 00:06:44,160 There has to be a general base somewhere 94 00:06:44,160 --> 00:06:47,520 that deprotonates this alpha amino group, 95 00:06:47,520 --> 00:06:51,960 such that we have this species that can attack, 96 00:06:51,960 --> 00:06:54,270 and then can imagine just formation and collapse 97 00:06:54,270 --> 00:06:57,640 of a tetrahedral intermediate here. 98 00:06:57,640 --> 00:07:02,310 So what is the mechanism of catalysis? 99 00:07:05,840 --> 00:07:06,340 OK. 100 00:07:06,340 --> 00:07:07,420 Our room's possessed. 101 00:07:07,420 --> 00:07:12,050 So what is the mechanism of catalysis here? 102 00:07:12,050 --> 00:07:13,720 What do we know? 103 00:07:13,720 --> 00:07:15,760 So we know from looking at the structure 104 00:07:15,760 --> 00:07:19,920 that the ribosome is a ribozyme. 105 00:07:19,920 --> 00:07:25,185 So no proteins in the catalytic center. 106 00:07:25,185 --> 00:07:26,060 What else do we know? 107 00:07:26,060 --> 00:07:33,670 There's no metal ions there and there's no covalent catalysis. 108 00:07:33,670 --> 00:07:35,740 So really what is a paradigm here? 109 00:07:35,740 --> 00:07:39,640 We have a paradigm of conformational change 110 00:07:39,640 --> 00:07:43,480 and effectively we have substrate positioning. 111 00:07:43,480 --> 00:07:45,520 You can imagine there's some protons shuttling 112 00:07:45,520 --> 00:07:49,510 in an electrostatic network that allows this to happen. 113 00:07:49,510 --> 00:07:52,870 And so as soon as this aminoacyl-tRNA 114 00:07:52,870 --> 00:07:55,750 enters the A site, we have formulation 115 00:07:55,750 --> 00:07:58,400 of this peptide bond. 116 00:07:58,400 --> 00:08:03,190 So what needs to happen next-- and once the screen gets fixed, 117 00:08:03,190 --> 00:08:05,320 we'll look at an actual depiction 118 00:08:05,320 --> 00:08:09,520 of these players in the PTC. 119 00:08:09,520 --> 00:08:12,250 What needs to happen after the peptide bond forms-- 120 00:08:12,250 --> 00:08:17,480 and we have now this peptidyl tRNA in the A site 121 00:08:17,480 --> 00:08:20,320 is that before the next round of elongation effectively 122 00:08:20,320 --> 00:08:24,850 we need to reset, and the mRNA and the tRNAs 123 00:08:24,850 --> 00:08:26,930 need to move relative to the ribosome. 124 00:08:26,930 --> 00:08:27,430 OK. 125 00:08:27,430 --> 00:08:34,030 So effectively we need to get this deacylated tRNA to the E 126 00:08:34,030 --> 00:08:40,630 site, and we need to get this peptidyl tRNA to the P site 127 00:08:40,630 --> 00:08:43,480 such that the A site is empty. 128 00:08:43,480 --> 00:08:45,280 Is it not going down? 129 00:08:45,280 --> 00:08:46,920 Pardon? 130 00:08:46,920 --> 00:08:48,400 OK, that's fine. 131 00:08:48,400 --> 00:08:59,390 So this process is called translocation here, 132 00:08:59,390 --> 00:09:02,360 and effectively we can just consider the three sites. 133 00:09:02,360 --> 00:09:08,495 We have the E site, the P site, and the A site. 134 00:09:08,495 --> 00:09:11,780 [WRITING ON BOARD] 135 00:09:24,395 --> 00:09:25,480 OK. 136 00:09:25,480 --> 00:09:29,280 And in this process another elongation factor, 137 00:09:29,280 --> 00:09:33,240 this time elongation factor G is involved. 138 00:09:33,240 --> 00:09:41,160 And the outcome is that we end up 139 00:09:41,160 --> 00:09:50,270 with the deacylated tRNA in the E site, 140 00:09:50,270 --> 00:09:57,270 the peptidyl tRNA in the P site. 141 00:09:57,270 --> 00:09:57,950 OK. 142 00:09:57,950 --> 00:10:00,300 And then the A site is empty such 143 00:10:00,300 --> 00:10:03,710 that the next aminoacyl-tRNA can come in. 144 00:10:03,710 --> 00:10:04,210 OK. 145 00:10:04,210 --> 00:10:08,130 So immediately after peptide bond formation, and this 146 00:10:08,130 --> 00:10:12,810 is the state after the process called translocation. 147 00:10:12,810 --> 00:10:25,100 So EFG is also a GTPase here. 148 00:10:25,100 --> 00:10:30,050 And effectively what happens is that EFG 149 00:10:30,050 --> 00:10:45,380 bound to GTP binds near the A site and GTP hydrolysis occurs. 150 00:10:45,380 --> 00:10:45,890 Bless you. 151 00:10:48,930 --> 00:10:49,750 OK. 152 00:10:49,750 --> 00:10:52,450 And as a result of GTP hydrolysis, 153 00:10:52,450 --> 00:10:53,815 there's conformational change. 154 00:10:59,200 --> 00:11:00,000 OK. 155 00:11:00,000 --> 00:11:08,630 And this results in translocation and then EFG 156 00:11:08,630 --> 00:11:09,130 is released. 157 00:11:14,820 --> 00:11:18,120 And in thinking about translocation 158 00:11:18,120 --> 00:11:19,260 we think about two steps. 159 00:11:30,930 --> 00:11:31,760 OK. 160 00:11:31,760 --> 00:11:35,390 And so the first step is something called formation 161 00:11:35,390 --> 00:11:47,130 of hybrid states, and then the second step 162 00:11:47,130 --> 00:11:58,370 is the actual movement, the mRNA and tRNA 163 00:11:58,370 --> 00:12:06,090 relative to the ribosome here. 164 00:12:11,480 --> 00:12:15,420 So we'll take a look at this. 165 00:12:15,420 --> 00:12:18,770 So here's just an overview of peptide bond formation 166 00:12:18,770 --> 00:12:23,510 backtracking a little bit, and then back to here. 167 00:12:23,510 --> 00:12:27,110 Thinking about confirmations and what 168 00:12:27,110 --> 00:12:32,390 this looks like, what we're seeing here in this depiction 169 00:12:32,390 --> 00:12:38,500 is the P site tRNA in green, we have the A site tRNA 170 00:12:38,500 --> 00:12:43,250 in this red color, and then we see the 23S rRNA shaded 171 00:12:43,250 --> 00:12:45,050 in light blue in the back. 172 00:12:45,050 --> 00:12:45,710 OK. 173 00:12:45,710 --> 00:12:51,530 So here's A76, here is an attached amino acid, 174 00:12:51,530 --> 00:12:54,870 and we see the nucleophile here, and attack there. 175 00:12:54,870 --> 00:12:55,370 OK. 176 00:12:55,370 --> 00:12:57,780 So substrate positioning within this active site. 177 00:13:01,510 --> 00:13:05,890 Here, as I talked about this translocation process, 178 00:13:05,890 --> 00:13:10,120 we're now at this stage as shown with a depiction 179 00:13:10,120 --> 00:13:11,320 of the ribosome. 180 00:13:11,320 --> 00:13:16,840 So this is where we're going using EFG in complex with GTP 181 00:13:16,840 --> 00:13:21,360 to allow the translocation process to occur. 182 00:13:21,360 --> 00:13:25,810 Here is another cartoon depiction. 183 00:13:25,810 --> 00:13:31,620 And so if we begin after peptide bond formation, 184 00:13:31,620 --> 00:13:36,250 we see the incoming EFG in complex with GTP. 185 00:13:36,250 --> 00:13:38,160 And if we look in this cartoon, it 186 00:13:38,160 --> 00:13:42,150 shows EFG binding near the A site. 187 00:13:42,150 --> 00:13:46,500 So it's not binding exactly in the A site, but nearby. 188 00:13:46,500 --> 00:13:47,160 OK. 189 00:13:47,160 --> 00:13:49,020 There's GTP hydrolysis. 190 00:13:49,020 --> 00:13:52,880 We now have EFG in the GTP bound form. 191 00:13:52,880 --> 00:13:53,380 OK. 192 00:13:53,380 --> 00:13:57,300 And now we see translocation, and that these tRNAs 193 00:13:57,300 --> 00:13:58,560 have moved. 194 00:13:58,560 --> 00:14:02,430 And after this step, EFG is released. 195 00:14:02,430 --> 00:14:04,200 And at this stage the ribosome is 196 00:14:04,200 --> 00:14:11,610 ready for its next round of the translation cycle. 197 00:14:11,610 --> 00:14:14,420 So let's take a look at what we know 198 00:14:14,420 --> 00:14:17,030 about the structure of EFG. 199 00:14:17,030 --> 00:14:22,160 So this is a really beautiful example of molecular mimicry, 200 00:14:22,160 --> 00:14:26,150 and EFG is shown here. 201 00:14:26,150 --> 00:14:34,040 And if we compare EFG to the complex of EF-TU 202 00:14:34,040 --> 00:14:38,870 with tRNA like the ternary complex shown here, what we see 203 00:14:38,870 --> 00:14:44,090 is they look very similar, and this domain IV of EFG 204 00:14:44,090 --> 00:14:46,730 resembles the tRNA quite well. 205 00:14:46,730 --> 00:14:48,350 So just looking at that we can begin 206 00:14:48,350 --> 00:14:50,240 to think maybe its domain IV that's 207 00:14:50,240 --> 00:14:54,140 coming in near that A site to have some interactions 208 00:14:54,140 --> 00:14:56,340 and cause this translocation event. 209 00:14:56,340 --> 00:15:02,390 Here is just a comparison of the ribosomes with either EF-TU 210 00:15:02,390 --> 00:15:04,850 and tRNA bound, which we've seen before, 211 00:15:04,850 --> 00:15:09,440 and here we see EFG in this red color. 212 00:15:09,440 --> 00:15:13,280 So quite similar, but are they the same? 213 00:15:13,280 --> 00:15:15,260 And the answer is no. 214 00:15:15,260 --> 00:15:18,140 So quite recently a crystal structure 215 00:15:18,140 --> 00:15:23,060 was obtained with EFG bound to the ribosome, 216 00:15:23,060 --> 00:15:25,910 and it was determined that in this structure 217 00:15:25,910 --> 00:15:30,660 EFG is bound in the post translational state. 218 00:15:30,660 --> 00:15:32,300 So what do we see? 219 00:15:32,300 --> 00:15:34,700 If we take a look here, we have a tRNA 220 00:15:34,700 --> 00:15:38,440 in the E site, a tRNA in the P site, 221 00:15:38,440 --> 00:15:42,320 and here in red we see EFG. 222 00:15:42,320 --> 00:15:45,470 And if you take a look, recall that ribosomal protein 223 00:15:45,470 --> 00:15:48,800 L12 that was involved in recruiting the ternary complex, 224 00:15:48,800 --> 00:15:51,650 we're seeing that there's some interaction here with EFG 225 00:15:51,650 --> 00:15:53,760 in that protein as well. 226 00:15:53,760 --> 00:15:55,550 If we look in this panel B-- 227 00:15:55,550 --> 00:15:59,390 so we look at a close up view near the A site, 228 00:15:59,390 --> 00:16:01,300 what's happening? 229 00:16:01,300 --> 00:16:08,600 Here's a P site tRNA, here is the mRNA, 230 00:16:08,600 --> 00:16:11,270 and here is EFG bound, and it's domain IV 231 00:16:11,270 --> 00:16:13,180 that's sticking its way in. 232 00:16:13,180 --> 00:16:15,010 OK. 233 00:16:15,010 --> 00:16:19,380 And so what are we seeing here? 234 00:16:19,380 --> 00:16:22,990 Basically these tRNAs have moved at this stage 235 00:16:22,990 --> 00:16:29,310 and we still have EFG bound here. 236 00:16:29,310 --> 00:16:40,770 So is EFG interacting with the A site codon of the mRNA 237 00:16:40,770 --> 00:16:42,684 based on this view? 238 00:16:42,684 --> 00:16:43,764 AUDIENCE: Not really. 239 00:16:43,764 --> 00:16:44,681 ELIZABETH NOLAN: Yeah. 240 00:16:44,681 --> 00:16:45,190 No. 241 00:16:45,190 --> 00:16:46,420 So not really. 242 00:16:46,420 --> 00:16:48,675 So it's not interacting in the same way as the tRNA. 243 00:16:48,675 --> 00:16:50,290 That's the take home here. 244 00:16:50,290 --> 00:16:53,880 They look the same, but the details are different here. 245 00:16:53,880 --> 00:16:54,380 OK. 246 00:16:57,260 --> 00:17:01,240 So what about these hybrid states? 247 00:17:01,240 --> 00:17:04,119 They have two different views, and the slides 248 00:17:04,119 --> 00:17:06,220 for looking at this-- 249 00:17:06,220 --> 00:17:09,040 effectively what the hybrid states are-- 250 00:17:22,589 --> 00:17:23,089 OK. 251 00:17:23,089 --> 00:17:27,349 These describe basically the orientation of the tRNAs. 252 00:17:27,349 --> 00:17:35,360 So effectively they can be P/E or A/P here. 253 00:17:35,360 --> 00:17:47,810 And the P/E state talks about having the anticodon end in P 254 00:17:47,810 --> 00:17:56,210 and the deacylated three prime end in E. OK. 255 00:17:56,210 --> 00:17:59,900 So here we're referring to this tRNA 256 00:17:59,900 --> 00:18:03,770 that ultimately needs to get ejected from the ribosome. 257 00:18:03,770 --> 00:18:14,385 And A/P we have the anticodon end in A 258 00:18:14,385 --> 00:18:21,840 and the peptidyl three prime end in P 259 00:18:21,840 --> 00:18:28,100 for the tRNA with the growing peptide chain. 260 00:18:30,800 --> 00:18:34,520 So effectively these hybrid states 261 00:18:34,520 --> 00:18:36,440 are describing the movement of the three 262 00:18:36,440 --> 00:18:41,540 prime ends of the tRNAs with respect to the 50S subunit. 263 00:18:48,590 --> 00:19:06,070 And that's shown in cartoon form on the slide here. 264 00:19:06,070 --> 00:19:06,570 OK. 265 00:19:06,570 --> 00:19:15,060 So effectively if we take a look, we have accommodation, 266 00:19:15,060 --> 00:19:18,120 so the aminoacyl-tRNA we see there's 267 00:19:18,120 --> 00:19:20,020 formation of a peptide bond. 268 00:19:20,020 --> 00:19:22,830 So there's some color coding here, and then look, 269 00:19:22,830 --> 00:19:26,320 rather than having these tRNAs straight up and down, 270 00:19:26,320 --> 00:19:28,890 we see that the three prime ends have shifted. 271 00:19:28,890 --> 00:19:33,960 So here we have the P anticodon end in P, three prime end in E, 272 00:19:33,960 --> 00:19:38,640 here AP with anticodon end in A, and peptidyl three prime end 273 00:19:38,640 --> 00:19:39,550 in P. 274 00:19:39,550 --> 00:19:40,050 OK. 275 00:19:40,050 --> 00:19:43,020 So first the three prime ends move, and then 276 00:19:43,020 --> 00:19:46,410 what do we see after the help of EFG? 277 00:19:46,410 --> 00:19:48,870 The five prime ends move and the A site 278 00:19:48,870 --> 00:19:52,980 is empty and able to take the next aminoacyl-tRNA. 279 00:19:55,510 --> 00:19:59,230 Here's just another view of the process 280 00:19:59,230 --> 00:20:02,470 for you to look at here. 281 00:20:02,470 --> 00:20:03,570 Yes? 282 00:20:03,570 --> 00:20:07,215 AUDIENCE: It's kind of from a few slides back, if that's OK. 283 00:20:07,215 --> 00:20:08,340 ELIZABETH NOLAN: That's OK. 284 00:20:08,340 --> 00:20:12,520 AUDIENCE: So on EFG where you have that anti-codon looking 285 00:20:12,520 --> 00:20:14,800 blue, there's no hydrogen bond interacting 286 00:20:14,800 --> 00:20:17,140 with the transcript. 287 00:20:17,140 --> 00:20:18,223 Is that the take away? 288 00:20:18,223 --> 00:20:20,140 ELIZABETH NOLAN: Can we say that based on what 289 00:20:20,140 --> 00:20:22,482 I'm showing you on this slide? 290 00:20:22,482 --> 00:20:23,440 AUDIENCE: I don't know. 291 00:20:23,440 --> 00:20:25,000 It just looks like it's around there, 292 00:20:25,000 --> 00:20:29,955 but I can't tell what the actual hydrogen bond interaction part. 293 00:20:29,955 --> 00:20:31,330 ELIZABETH NOLAN: So those details 294 00:20:31,330 --> 00:20:33,460 are outside of the scope. 295 00:20:33,460 --> 00:20:39,190 EFG will interact with that mRNA, the peptidyl tRNA, 296 00:20:39,190 --> 00:20:43,345 but it's interacting differently than a standard aminoacyl-tRNA. 297 00:20:43,345 --> 00:20:45,220 So it's not really interacting with the codon 298 00:20:45,220 --> 00:20:47,320 at this level of depiction. 299 00:20:47,320 --> 00:20:51,080 We're not seeing individual bonds or hydrogen bonds. 300 00:20:51,080 --> 00:20:53,530 So we can't make a conclusion about that 301 00:20:53,530 --> 00:20:55,670 based on this depiction here. 302 00:20:59,860 --> 00:21:02,220 AUDIENCE: What's the resolution of this structure? 303 00:21:02,220 --> 00:21:03,803 Is it high resolution or is it-- 304 00:21:03,803 --> 00:21:04,720 ELIZABETH NOLAN: Yeah. 305 00:21:04,720 --> 00:21:06,640 That's another issue here. 306 00:21:06,640 --> 00:21:09,670 I don't recall the resolution, but they're not great. 307 00:21:09,670 --> 00:21:12,340 So if you have a four angstrom resolution structure, 308 00:21:12,340 --> 00:21:17,140 for instance, is that type of information even available 309 00:21:17,140 --> 00:21:20,720 versus the resolution of maybe 1.5 or 1? 310 00:21:20,720 --> 00:21:21,220 Yeah. 311 00:21:21,220 --> 00:21:24,050 This resolution I don't recall, but that's a very good point 312 00:21:24,050 --> 00:21:24,550 to bring up. 313 00:21:24,550 --> 00:21:26,380 I don't think it's high enough to know 314 00:21:26,380 --> 00:21:30,430 that would be my guess here. 315 00:21:30,430 --> 00:21:32,920 Pardon? 316 00:21:32,920 --> 00:21:34,390 Oh, the resolution? 317 00:21:34,390 --> 00:21:40,810 So rewinding back to recitation last week. 318 00:21:40,810 --> 00:21:43,690 So crystal structures have a resolution. 319 00:21:43,690 --> 00:21:49,030 And so what does one angstrom resolution versus two 320 00:21:49,030 --> 00:21:50,575 versus four allow us to see? 321 00:21:53,577 --> 00:21:54,077 Oh. 322 00:21:54,077 --> 00:21:54,577 Oh. 323 00:21:54,577 --> 00:21:58,360 The question is, are there hydrogen bonding interactions 324 00:21:58,360 --> 00:22:00,540 between EFG, and say, the mRNA? 325 00:22:11,160 --> 00:22:14,040 Well, there's definitely going to be hydrogens 326 00:22:14,040 --> 00:22:16,260 because you're going to have C-H bonds or C-N bonds. 327 00:22:16,260 --> 00:22:16,530 But-- 328 00:22:16,530 --> 00:22:17,654 AUDIENCE: I can't see any of them, at least 329 00:22:17,654 --> 00:22:18,086 from this picture. 330 00:22:18,086 --> 00:22:20,294 And also, is this picture actually a picture or is it 331 00:22:20,294 --> 00:22:23,253 just like a [INAUDIBLE]? 332 00:22:23,253 --> 00:22:25,420 ELIZABETH NOLAN: This is from the crystal structure. 333 00:22:25,420 --> 00:22:29,690 AUDIENCE: But it's not itself a crystal structure. 334 00:22:29,690 --> 00:22:31,709 They take out all the hydrogens, wouldn't you, 335 00:22:31,709 --> 00:22:35,607 and you wouldn't see anything, right? 336 00:22:35,607 --> 00:22:36,440 ELIZABETH NOLAN: OK. 337 00:22:36,440 --> 00:22:38,210 So this is from the crystal structure 338 00:22:38,210 --> 00:22:40,700 and you can make choices as to what information you 339 00:22:40,700 --> 00:22:43,070 put in your depiction, whether or not 340 00:22:43,070 --> 00:22:46,610 you're going to show certain residues say, 341 00:22:46,610 --> 00:22:50,095 or just the backbone there. 342 00:22:50,095 --> 00:22:54,350 AUDIENCE: I'm not sure what we are expecting to see, 343 00:22:54,350 --> 00:22:55,610 but don't see. 344 00:22:55,610 --> 00:22:57,660 We will get the heteroatom distances. 345 00:22:57,660 --> 00:22:58,530 Yeah. 346 00:22:58,530 --> 00:23:00,730 And so the question is, how much error is there, 347 00:23:00,730 --> 00:23:02,150 and if there's one error, you can't tell 348 00:23:02,150 --> 00:23:03,400 where the analide regions are. 349 00:23:03,400 --> 00:23:05,170 AUDIENCE: So we're looking at distances 350 00:23:05,170 --> 00:23:10,520 like between potential hydrogen bonding sites as our measure-- 351 00:23:10,520 --> 00:23:13,040 ELIZABETH NOLAN: Or heteroatoms because you may not 352 00:23:13,040 --> 00:23:16,460 be able to see that hydrogen. But you can know something 353 00:23:16,460 --> 00:23:19,220 like, oh, if this heteroatom and that heteroatom 354 00:23:19,220 --> 00:23:22,610 are so many angstroms apart, is it likely that there's 355 00:23:22,610 --> 00:23:26,210 a hydrogen bonding interaction or not based on knowledge 356 00:23:26,210 --> 00:23:28,280 of bond distances here? 357 00:23:28,280 --> 00:23:31,900 So one thing I'll note and will come up 358 00:23:31,900 --> 00:23:33,800 as we're discussing antibiotics, I 359 00:23:33,800 --> 00:23:37,070 said nothing about how they've obtained the structure. 360 00:23:37,070 --> 00:23:39,290 And that's just something to keep in mind. 361 00:23:39,290 --> 00:23:41,690 And this also gets to the question of resolution, 362 00:23:41,690 --> 00:23:42,950 and what can you see? 363 00:23:42,950 --> 00:23:45,110 But they had tremendous difficulties 364 00:23:45,110 --> 00:23:49,400 getting this structure, and they had to use a mutant ribosome, 365 00:23:49,400 --> 00:23:51,680 and they strategically used an antibiotic 366 00:23:51,680 --> 00:23:55,520 to stall the ribosome here. 367 00:23:55,520 --> 00:23:57,920 So many, many attempts to get crystals 368 00:23:57,920 --> 00:24:02,170 that are even good enough to get some information here. 369 00:24:06,240 --> 00:24:07,800 OK. 370 00:24:07,800 --> 00:24:12,030 So back to these hybrid states and the formation 371 00:24:12,030 --> 00:24:14,250 of the hybrid states. 372 00:24:14,250 --> 00:24:16,470 Something important to know about-- 373 00:24:16,470 --> 00:24:19,360 and this is something I have a lot of time seeing 374 00:24:19,360 --> 00:24:21,780 in any cartoon that's presented is 375 00:24:21,780 --> 00:24:26,850 that the 30S subunit undergoes some conformational change 376 00:24:26,850 --> 00:24:28,440 called ratcheting. 377 00:24:28,440 --> 00:24:30,870 And effectively the ribosome can exist 378 00:24:30,870 --> 00:24:37,140 at this stage in either an unratcheted or ratcheted state 379 00:24:37,140 --> 00:24:39,960 and EFG selects from one over the other. 380 00:24:39,960 --> 00:24:43,980 So EFG will bind this ratcheted ribosome. 381 00:24:43,980 --> 00:24:46,800 And effectively what that terminology 382 00:24:46,800 --> 00:24:48,720 is describing is a small rotation 383 00:24:48,720 --> 00:24:54,780 of about 6 degrees of the 30S relative to the 50S 384 00:24:54,780 --> 00:24:56,160 in one direction. 385 00:24:56,160 --> 00:24:58,200 So the ribosome will be going between 386 00:24:58,200 --> 00:24:59,790 unratcheted and ratcheted. 387 00:24:59,790 --> 00:25:02,410 EFG can bind the ratcheted form. 388 00:25:02,410 --> 00:25:02,910 OK. 389 00:25:02,910 --> 00:25:04,600 And after that occurs, they'll be 390 00:25:04,600 --> 00:25:10,250 GTP hydrolysis on these translocation events here. 391 00:25:10,250 --> 00:25:12,950 So an awful lot is going on to get 392 00:25:12,950 --> 00:25:15,380 that one peptide bond formed and the ribosome 393 00:25:15,380 --> 00:25:18,380 ready to do it again. 394 00:25:18,380 --> 00:25:20,630 Where we're going to go at this stage 395 00:25:20,630 --> 00:25:23,240 is a brief discussion of the termination 396 00:25:23,240 --> 00:25:28,670 process in translation and the players that come up there. 397 00:25:28,670 --> 00:25:31,100 So effectively the elongation cycle 398 00:25:31,100 --> 00:25:37,370 is going to continue until a stop codon enters the A site. 399 00:25:37,370 --> 00:25:41,360 That's making the assumption some unforeseen circumstance 400 00:25:41,360 --> 00:25:42,980 hasn't happened to this ribosome. 401 00:25:42,980 --> 00:25:47,000 It hasn't stalled or prematurely stopped translation. 402 00:25:47,000 --> 00:25:52,230 So what happens when a stop codon enters the A site? 403 00:25:52,230 --> 00:25:52,730 OK. 404 00:25:52,730 --> 00:25:55,280 Again, we have translation factors. 405 00:25:55,280 --> 00:25:58,790 These translation factors are release factors 406 00:25:58,790 --> 00:26:01,850 that recognize the stop codon, and they 407 00:26:01,850 --> 00:26:05,990 have the responsibility of cleaving the polypeptide chain 408 00:26:05,990 --> 00:26:09,050 from the P site tRNA. 409 00:26:09,050 --> 00:26:13,280 And so there are two different classes of release factors. 410 00:26:13,280 --> 00:26:18,230 We have class 1, which are release factors 1 and 2. 411 00:26:18,230 --> 00:26:20,600 Release factor 1 and release factor 2 412 00:26:20,600 --> 00:26:23,810 each recognize they're in stop codons. 413 00:26:23,810 --> 00:26:28,640 So, for instance, RF1 recognizes UAA and UAG. 414 00:26:28,640 --> 00:26:34,790 Whereas RF2 recognizes UAA and UGA here. 415 00:26:34,790 --> 00:26:37,850 There's a class 3 release factor RF3. 416 00:26:37,850 --> 00:26:41,480 This one is a GTPase and it has the job 417 00:26:41,480 --> 00:26:44,930 of accelerating dissociation of RF1 or RF2 418 00:26:44,930 --> 00:26:46,610 after peptide release. 419 00:26:46,610 --> 00:26:49,400 So we'll look a little bit at structure and then one 420 00:26:49,400 --> 00:26:52,130 schematic for how this may all happen. 421 00:26:52,130 --> 00:26:55,700 So similar to EFG and the ternary complex 422 00:26:55,700 --> 00:27:00,080 of EF-TU GTP and the tRNA, we have another example 423 00:27:00,080 --> 00:27:04,190 of molecular mimicry with these release factors. 424 00:27:04,190 --> 00:27:09,290 And so initially when release factor 1 was crystallized, 425 00:27:09,290 --> 00:27:12,390 the structure shown here was obtained. 426 00:27:12,390 --> 00:27:14,390 So this was the protein crystallized 427 00:27:14,390 --> 00:27:20,060 in the absence of the ribosome, and it was a little difficult 428 00:27:20,060 --> 00:27:24,770 to reconcile this structure with function immediately. 429 00:27:24,770 --> 00:27:27,770 And then in later work RF1 was crystallized 430 00:27:27,770 --> 00:27:29,690 bound to the ribosome. 431 00:27:29,690 --> 00:27:32,910 And that structure is shown here. 432 00:27:32,910 --> 00:27:36,410 And so if we compare the left to the right 433 00:27:36,410 --> 00:27:39,320 or what's described as the closed to the open version 434 00:27:39,320 --> 00:27:41,750 of RF1, what we see is that there's 435 00:27:41,750 --> 00:27:45,740 a pretty substantial change in conformation 436 00:27:45,740 --> 00:27:49,040 when we're looking at RF1 on the ribosome. 437 00:27:49,040 --> 00:27:51,170 And if we use a little imagination 438 00:27:51,170 --> 00:27:55,340 we can think about RF1 resembling a tRNA. 439 00:27:55,340 --> 00:27:56,090 OK. 440 00:27:56,090 --> 00:27:59,230 We have this region here that's sticking out. 441 00:27:59,230 --> 00:28:02,570 And if we look at an overlay of RF1, 442 00:28:02,570 --> 00:28:04,040 so this structure of the ribosome 443 00:28:04,040 --> 00:28:08,420 bound structure in a tRNA, what do we see? 444 00:28:08,420 --> 00:28:14,300 So we have the tRNA, we have the anticodon end down here, 445 00:28:14,300 --> 00:28:18,710 we have the CCA end of the tRNA up here. 446 00:28:18,710 --> 00:28:20,180 And so what do we see? 447 00:28:20,180 --> 00:28:24,710 In terms of RF1, we have this PVT motif down here 448 00:28:24,710 --> 00:28:30,980 and we have this GGQ motif up here for that. 449 00:28:30,980 --> 00:28:34,880 And so this motif is important for hydrolysis 450 00:28:34,880 --> 00:28:37,850 of the peptidyl tRNA. 451 00:28:37,850 --> 00:28:39,560 And that's where it is. 452 00:28:42,340 --> 00:28:45,940 In terms of a schematic for termination 453 00:28:45,940 --> 00:28:48,040 as a way to thinking about this-- 454 00:28:48,040 --> 00:28:52,690 so here we have our ribosome that's then translating 455 00:28:52,690 --> 00:28:55,570 and now there's a stop codon in the A site. 456 00:28:55,570 --> 00:28:59,050 So here comes a release factor, either 1 or 2. 457 00:28:59,050 --> 00:29:02,890 It recognizes this stop and binds. 458 00:29:02,890 --> 00:29:05,590 So there's hydrolysis of this linkage-- 459 00:29:05,590 --> 00:29:08,890 and should think about that chemistry to what's happening. 460 00:29:08,890 --> 00:29:10,720 --peptide release. 461 00:29:10,720 --> 00:29:14,410 So what's shown in this depiction is that RF3 comes in 462 00:29:14,410 --> 00:29:16,780 and it was in GTP bound form. 463 00:29:16,780 --> 00:29:19,900 It binds in the region of the A site. 464 00:29:19,900 --> 00:29:22,510 There's some exchange. 465 00:29:22,510 --> 00:29:28,060 We have GTP coming in here and then some additional steps 466 00:29:28,060 --> 00:29:34,600 that involve GTP hydrolysis by RF3 involvement of the ribosome 467 00:29:34,600 --> 00:29:36,460 recycling factor. 468 00:29:36,460 --> 00:29:40,420 And we see that our friend EFG comes into play again here 469 00:29:40,420 --> 00:29:42,640 along with initiation factor 3. 470 00:29:42,640 --> 00:29:45,370 So some of these other translation factors 471 00:29:45,370 --> 00:29:49,760 seem to play a role in this termination cycle. 472 00:29:49,760 --> 00:29:51,400 And really, again, it's a question 473 00:29:51,400 --> 00:29:53,770 of looking at the data that's presented to you 474 00:29:53,770 --> 00:29:57,440 and interpreting that data and drawing some conclusions. 475 00:29:57,440 --> 00:29:59,950 So there's still a number of questions 476 00:29:59,950 --> 00:30:05,020 about this process and the ribosome recycling that remain. 477 00:30:05,020 --> 00:30:09,100 So if we look about this slide and where 478 00:30:09,100 --> 00:30:12,970 we've come in this discussion of translation effectively, 479 00:30:12,970 --> 00:30:16,510 all of the pieces are shown here for prokaryotes. 480 00:30:16,510 --> 00:30:19,540 So this is just a map to work your way through when 481 00:30:19,540 --> 00:30:21,310 studying the system. 482 00:30:21,310 --> 00:30:24,920 But we have initiation, we have elongation, 483 00:30:24,920 --> 00:30:29,380 and then this process of peptide release and ribosome recycling. 484 00:30:29,380 --> 00:30:29,920 OK. 485 00:30:29,920 --> 00:30:33,700 And so throughout this we're seeing the action of GTPases. 486 00:30:33,700 --> 00:30:37,450 So the power of GTP hydrolysis is needed. 487 00:30:37,450 --> 00:30:40,550 Conversion of chemical to mechanical energy. 488 00:30:40,550 --> 00:30:44,650 There's a lot of conformational change that's happening. 489 00:30:44,650 --> 00:30:49,450 The slides I've shown you don't do that justice but it's 490 00:30:49,450 --> 00:30:51,520 something to think about and keep in mind, 491 00:30:51,520 --> 00:30:55,390 and that this ribosome is amazingly dynamic. 492 00:30:55,390 --> 00:30:58,780 And so that is what's going to lead us 493 00:30:58,780 --> 00:31:03,160 into the next subtopic related to the ribosome, which 494 00:31:03,160 --> 00:31:06,790 is thinking about how have some of these observations been 495 00:31:06,790 --> 00:31:07,720 made? 496 00:31:07,720 --> 00:31:12,040 So how is it that we've obtained structural insights 497 00:31:12,040 --> 00:31:16,720 into the ribosome at different steps along this translation 498 00:31:16,720 --> 00:31:18,550 cycle? 499 00:31:18,550 --> 00:31:21,990 And just as for consideration, there's 500 00:31:21,990 --> 00:31:24,880 a little excerpt from a paper I like. 501 00:31:24,880 --> 00:31:26,890 So this was in 2010. 502 00:31:26,890 --> 00:31:30,700 So shortly after the Nobel Prize was awarded. 503 00:31:30,700 --> 00:31:33,940 And so there's a number of perspectives, 504 00:31:33,940 --> 00:31:36,490 retrospectives in the literature. 505 00:31:36,490 --> 00:31:39,590 And in this one called the Ribosome Comes Alive, 506 00:31:39,590 --> 00:31:42,940 Joachim Frank is talking about these pioneering work 507 00:31:42,940 --> 00:31:44,720 of the X-ray structure. 508 00:31:44,720 --> 00:31:49,600 And just in yellow here, he's stating, 509 00:31:49,600 --> 00:31:52,390 those who might have expected that the atomic resolution 510 00:31:52,390 --> 00:31:55,300 structure of this massive RNA protein complex 511 00:31:55,300 --> 00:31:57,250 would itself offer immediate insight 512 00:31:57,250 --> 00:31:58,930 into the mechanism of translation 513 00:31:58,930 --> 00:32:01,380 were thoroughly disappointed. 514 00:32:01,380 --> 00:32:03,460 And in fact the mechanism proposed 515 00:32:03,460 --> 00:32:06,310 from some of this early study ended up not being 516 00:32:06,310 --> 00:32:08,090 the correct mechanism here. 517 00:32:08,090 --> 00:32:09,790 There's a note about that on an earlier 518 00:32:09,790 --> 00:32:13,630 slide where the peptide bond formation step is shown. 519 00:32:13,630 --> 00:32:15,070 So what does he say? 520 00:32:15,070 --> 00:32:18,160 "I'd like to compare this situation to a visit to Earth 521 00:32:18,160 --> 00:32:19,900 by a martian who wants to understand 522 00:32:19,900 --> 00:32:21,080 how an automobile works." 523 00:32:21,080 --> 00:32:21,580 OK. 524 00:32:21,580 --> 00:32:24,190 So we can all think about flipping up the hood of our car 525 00:32:24,190 --> 00:32:25,960 and what do we see? 526 00:32:25,960 --> 00:32:28,000 "She looks under the hood of a parked car, 527 00:32:28,000 --> 00:32:31,780 perhaps even takes the engine apart, but still has no clue. 528 00:32:31,780 --> 00:32:33,910 It's clear she'll have much better luck 529 00:32:33,910 --> 00:32:36,830 if she's able to see that engine in motion." 530 00:32:36,830 --> 00:32:40,120 And so that's been a major goal in terms 531 00:32:40,120 --> 00:32:41,860 of thinking about the ribosome as well as 532 00:32:41,860 --> 00:32:43,810 other micro-molecular machines. 533 00:32:43,810 --> 00:32:46,360 How can you actually see these in motion 534 00:32:46,360 --> 00:32:50,110 and see the dynamics and conformational changes here? 535 00:32:50,110 --> 00:32:51,190 Really critical. 536 00:32:51,190 --> 00:32:55,120 So the question I pose is, is it possible to see 537 00:32:55,120 --> 00:32:59,350 the ribosome stopped at various points in translation cycle? 538 00:32:59,350 --> 00:33:00,790 And if so, how? 539 00:33:00,790 --> 00:33:04,360 So maybe we can't see the dynamics continually, 540 00:33:04,360 --> 00:33:08,090 but can we sort of park it at different steps? 541 00:33:08,090 --> 00:33:10,660 And the answer to that is yes. 542 00:33:10,660 --> 00:33:16,690 And basically a huge part of our understanding of this 70S 543 00:33:16,690 --> 00:33:19,570 ribosome does come from crystal structures, 544 00:33:19,570 --> 00:33:22,660 and researchers have been able to trap 545 00:33:22,660 --> 00:33:26,830 the ribosome at various points in the translation cycle using 546 00:33:26,830 --> 00:33:28,570 small molecules. 547 00:33:28,570 --> 00:33:32,890 And these small molecules are antibiotics. 548 00:33:32,890 --> 00:33:35,950 So where we're going to focus on for the rest of today 549 00:33:35,950 --> 00:33:38,170 and probably the beginning of Monday 550 00:33:38,170 --> 00:33:41,710 is thinking about the use as antibiotics. 551 00:33:41,710 --> 00:33:46,180 So small molecules that inhibit bacterial growth as tools 552 00:33:46,180 --> 00:33:49,040 for studying ribosome function. 553 00:33:49,040 --> 00:33:52,450 So a few questions related to that. 554 00:33:52,450 --> 00:33:55,210 First of all, what types of antibiotics 555 00:33:55,210 --> 00:33:57,430 target the ribosome? 556 00:33:57,430 --> 00:34:00,280 Where do they bind to the ribosome, 557 00:34:00,280 --> 00:34:03,460 and how can we use them experimentally? 558 00:34:03,460 --> 00:34:05,240 And also something just to think about, 559 00:34:05,240 --> 00:34:07,840 we have a crisis in the clinic in terms 560 00:34:07,840 --> 00:34:12,350 of a lack of new antibiotics and emerging antibiotic resistance. 561 00:34:12,350 --> 00:34:15,550 So how can fundamental understanding of the ribosome 562 00:34:15,550 --> 00:34:18,070 help in terms of therapeutic development? 563 00:34:18,070 --> 00:34:22,600 And this came up in a bit of a different context in seminar 564 00:34:22,600 --> 00:34:25,540 on Monday for anyone that was at Biological Chemistry Seminar. 565 00:34:25,540 --> 00:34:29,020 So we had Professor Matt Disney with us who was looking 566 00:34:29,020 --> 00:34:31,659 at small molecules to target RNA's. 567 00:34:31,659 --> 00:34:34,570 And one question that can come from that is, are there 568 00:34:34,570 --> 00:34:36,610 unknown molecules out there that might target 569 00:34:36,610 --> 00:34:39,790 the ribosome in different ways from the examples 570 00:34:39,790 --> 00:34:40,585 we currently have? 571 00:34:43,239 --> 00:34:46,989 I was super excited this morning to learn about a new book. 572 00:34:46,989 --> 00:34:49,929 So if any of you are interested in antibiotics, 573 00:34:49,929 --> 00:34:53,730 Professor Chris Walsh and Professor Tim Wencewicz 574 00:34:53,730 --> 00:34:59,470 at St. Louis have written a new book looking at antibiotics 575 00:34:59,470 --> 00:35:02,460 from a very chemocentric perspective here, 576 00:35:02,460 --> 00:35:04,330 and our friends on the cover. 577 00:35:04,330 --> 00:35:10,240 So I suspect be a wonderful read if you're curious. 578 00:35:10,240 --> 00:35:15,310 So let's take a look as a segue into thinking about these 579 00:35:15,310 --> 00:35:17,550 at the structure I just showed you 580 00:35:17,550 --> 00:35:21,820 a VFG bound to the ribosome. 581 00:35:21,820 --> 00:35:26,950 So we talked about how EFG is helping in the translocation 582 00:35:26,950 --> 00:35:29,740 process, and we saw the structure, 583 00:35:29,740 --> 00:35:32,110 and I told you in passing that this structure was 584 00:35:32,110 --> 00:35:35,980 very difficult for the researchers to obtain. 585 00:35:35,980 --> 00:35:40,360 And at the end of the day, they needed to use a mutant ribosome 586 00:35:40,360 --> 00:35:42,280 for reasons I won't go into. 587 00:35:42,280 --> 00:35:44,500 It's not relevant for this discussion. 588 00:35:44,500 --> 00:35:48,790 And also, a natural product that has antibacterial activity 589 00:35:48,790 --> 00:35:51,050 shown here. 590 00:35:51,050 --> 00:35:57,040 And so this small molecule binds EFG 591 00:35:57,040 --> 00:36:02,860 and it binds to EFG when EFG is bound to the ribosome. 592 00:36:02,860 --> 00:36:08,360 And moreover, it binds to EFG after GTP hydrolysis occurs. 593 00:36:08,360 --> 00:36:08,860 OK. 594 00:36:08,860 --> 00:36:12,670 So the result is that this natural product 595 00:36:12,670 --> 00:36:18,000 can be used to trap the ribosome in this post translocational 596 00:36:18,000 --> 00:36:20,740 state where EFG is still bound. 597 00:36:20,740 --> 00:36:23,110 So it's hydrolyzed GTP. 598 00:36:23,110 --> 00:36:26,290 There's been movement of the mRNA and tRNAs, 599 00:36:26,290 --> 00:36:30,220 but EFG cannot dissociate as a result of use of this small 600 00:36:30,220 --> 00:36:31,970 molecule. 601 00:36:31,970 --> 00:36:34,360 So you can begin to imagine how including 602 00:36:34,360 --> 00:36:37,570 this molecule or maybe other antibiotics 603 00:36:37,570 --> 00:36:39,910 that stop the ribosome at different steps 604 00:36:39,910 --> 00:36:45,220 can be used to obtain crystals and crystal structures here. 605 00:36:45,220 --> 00:36:46,990 And furthermore, they can also be 606 00:36:46,990 --> 00:36:49,240 used in a number of biochemical studies-- 607 00:36:49,240 --> 00:36:51,370 and we'll look at an example of that 608 00:36:51,370 --> 00:36:53,620 in the context of this lecture. 609 00:36:53,620 --> 00:36:57,940 --and also in recitations and problem sets. 610 00:36:57,940 --> 00:37:09,000 So where do antibiotics bind to the ribosome, 611 00:37:09,000 --> 00:37:11,580 and how many of them are out there that 612 00:37:11,580 --> 00:37:14,940 can bind the ribosome here? 613 00:37:14,940 --> 00:37:16,290 There's many options. 614 00:37:16,290 --> 00:37:20,220 So many antibiotics target the ribosome. 615 00:37:20,220 --> 00:37:25,080 And if we just look at a 30S subunit and a 50S subunit 616 00:37:25,080 --> 00:37:27,360 and take a handful of antibiotics 617 00:37:27,360 --> 00:37:29,790 that target the ribosome and see what 618 00:37:29,790 --> 00:37:32,460 we know about where they bind, we 619 00:37:32,460 --> 00:37:38,950 can make maps like these ones here 620 00:37:38,950 --> 00:37:42,130 and we can consider larger lists. 621 00:37:42,130 --> 00:37:44,320 You're not responsible for these details at all. 622 00:37:44,320 --> 00:37:46,300 Just the take home message is that there's 623 00:37:46,300 --> 00:37:48,573 many options and an extensive toolkit. 624 00:37:48,573 --> 00:37:49,073 Yeah? 625 00:37:49,073 --> 00:37:51,898 AUDIENCE: Are the eukaryotic and prokaryotic ribosomes 626 00:37:51,898 --> 00:37:54,810 similar enough that most of these antibiotics 627 00:37:54,810 --> 00:37:56,633 also affect the eukaryotic ribosomes? 628 00:37:56,633 --> 00:37:57,550 ELIZABETH NOLAN: Yeah. 629 00:37:57,550 --> 00:38:01,540 So that's a great question and something to think about. 630 00:38:01,540 --> 00:38:05,200 So that will depend on the molecule. 631 00:38:05,200 --> 00:38:07,510 There are many differences between the prokaryotic and 632 00:38:07,510 --> 00:38:09,340 eukaryotic ribosomes. 633 00:38:09,340 --> 00:38:12,010 Some will bind both. 634 00:38:12,010 --> 00:38:13,600 There is an example thiostrepton I 635 00:38:13,600 --> 00:38:19,255 believe that's quite specific, not for eukaryotic ribosomes. 636 00:38:19,255 --> 00:38:21,130 I mean, that's something also to think about. 637 00:38:21,130 --> 00:38:24,430 If they interact, are they interacting in the same way? 638 00:38:24,430 --> 00:38:26,200 And if they do inhibit the ribosome, 639 00:38:26,200 --> 00:38:28,540 is it by the same mechanism? 640 00:38:28,540 --> 00:38:30,970 And you can imagine implications related 641 00:38:30,970 --> 00:38:34,916 to therapeutic development in terms of that exact issue. 642 00:38:34,916 --> 00:38:35,416 Yeah? 643 00:38:35,416 --> 00:38:37,333 AUDIENCE: Do we have like a lot more ribosomes 644 00:38:37,333 --> 00:38:38,310 than prokaryotes? 645 00:38:38,310 --> 00:38:41,510 Is that also [INAUDIBLE] to have a lot more [INAUDIBLE] 646 00:38:41,510 --> 00:38:50,450 a lot more antibiotic [INAUDIBLE] that for us too? 647 00:38:50,450 --> 00:38:51,450 Do you know what I mean? 648 00:38:51,450 --> 00:38:52,366 ELIZABETH NOLAN: Yeah. 649 00:38:52,366 --> 00:38:53,950 I mean I think that's a little outside 650 00:38:53,950 --> 00:38:56,500 of the scope of our discussion because how do you 651 00:38:56,500 --> 00:38:58,030 get to counting ribosomes? 652 00:38:58,030 --> 00:39:04,000 Is that per cell or organism by organism and microbiome 653 00:39:04,000 --> 00:39:04,645 versus person? 654 00:39:07,537 --> 00:39:08,620 Is there another question? 655 00:39:08,620 --> 00:39:12,160 AUDIENCE: The ratio of the number of ribosomes 656 00:39:12,160 --> 00:39:14,110 we have to the number of proteins 657 00:39:14,110 --> 00:39:16,990 that we need to be producing, eukaryotic cells 658 00:39:16,990 --> 00:39:18,110 are more complex-- 659 00:39:18,110 --> 00:39:20,110 ELIZABETH NOLAN: Eukaryotic cells are definitely 660 00:39:20,110 --> 00:39:24,170 more complex right there. 661 00:39:24,170 --> 00:39:34,290 So what I say is overall case by case basis. 662 00:39:34,290 --> 00:39:36,780 So what about structures? 663 00:39:39,600 --> 00:39:42,850 Here are just some examples. 664 00:39:42,850 --> 00:39:45,580 Structures are highly variable and the ways 665 00:39:45,580 --> 00:39:48,880 in which these molecules can inhibit translation 666 00:39:48,880 --> 00:39:50,470 are highly variable. 667 00:39:50,470 --> 00:39:52,270 These are some examples that you may 668 00:39:52,270 --> 00:39:55,150 have come up with some of these in terms of laboratory work 669 00:39:55,150 --> 00:39:57,340 or maybe even been prescribed. 670 00:39:57,340 --> 00:40:00,190 So, for instance, chloroamphenicol This molecule 671 00:40:00,190 --> 00:40:04,360 here binds the 30S, prevents peptidyl transfer. 672 00:40:04,360 --> 00:40:08,880 Tetracycline binds the 50s and blocks accommodation. 673 00:40:08,880 --> 00:40:14,980 Gentamicin binds the 30S, causes premature termination. 674 00:40:14,980 --> 00:40:17,710 Erythromycin is a macrolide that for a long time 675 00:40:17,710 --> 00:40:22,330 has been thought to block exit of the polypeptide 676 00:40:22,330 --> 00:40:24,340 because it binds in the exit tunnel. 677 00:40:24,340 --> 00:40:27,160 But there's some new recent work suggesting a revision 678 00:40:27,160 --> 00:40:31,340 to that mechanism here. 679 00:40:31,340 --> 00:40:33,860 What are some general observations we can make? 680 00:40:33,860 --> 00:40:38,420 And keep in mind, there's always exceptions to the rule. 681 00:40:38,420 --> 00:40:42,170 So most of the antibiotics targeting the ribosome 682 00:40:42,170 --> 00:40:46,620 that we know about interact with the RNA, 683 00:40:46,620 --> 00:40:49,200 but, of course, some can interact with proteins. 684 00:40:49,200 --> 00:40:53,520 And we just saw an example of that with EFG. 685 00:40:53,520 --> 00:40:58,500 These antibiotics primarily target the decoding center 686 00:40:58,500 --> 00:41:01,080 and peptidyl transfer A center. 687 00:41:01,080 --> 00:41:02,580 Which makes sense if you're thinking 688 00:41:02,580 --> 00:41:04,320 about inhibiting translation. 689 00:41:04,320 --> 00:41:05,940 But, again, there's some exceptions. 690 00:41:05,940 --> 00:41:09,450 So thiostrepton interacts with the ribosomal protein 691 00:41:09,450 --> 00:41:11,940 that's not in that region. 692 00:41:11,940 --> 00:41:15,520 Magnesium might be necessary for antibiotic binding. 693 00:41:15,520 --> 00:41:18,390 So this is something to think about if using antibiotics 694 00:41:18,390 --> 00:41:20,640 in experiments. 695 00:41:20,640 --> 00:41:24,450 And just related to the earlier question, 696 00:41:24,450 --> 00:41:28,770 a given antibiotic may bind ribosomes of different species 697 00:41:28,770 --> 00:41:30,390 differently there. 698 00:41:30,390 --> 00:41:33,450 And so what are the consequences of that 699 00:41:33,450 --> 00:41:35,350 is something to think about. 700 00:41:35,350 --> 00:41:40,170 So here we're just looking at an overview of various antibiotics 701 00:41:40,170 --> 00:41:41,370 bound to the 50S. 702 00:41:41,370 --> 00:41:44,370 So this is taken from multiple different structures 703 00:41:44,370 --> 00:41:48,210 and the ribosome itself has been removed. 704 00:41:48,210 --> 00:41:51,070 But imagine that the A site tRNA is around here, 705 00:41:51,070 --> 00:41:53,220 here P site tRNA. 706 00:41:53,220 --> 00:41:54,960 What do we see? 707 00:41:54,960 --> 00:41:57,690 We have antibiotics called puromycins 708 00:41:57,690 --> 00:41:59,700 that are down by the A site. 709 00:41:59,700 --> 00:42:02,160 Here we have chloroamphenicol bound. 710 00:42:02,160 --> 00:42:07,730 Here we have the macrolides here. 711 00:42:07,730 --> 00:42:12,090 And just as an example of an antibiotic binding to the exit 712 00:42:12,090 --> 00:42:13,890 tunnel-- bless you. 713 00:42:13,890 --> 00:42:16,300 --here we're looking at the 50S. 714 00:42:16,300 --> 00:42:18,960 We have a P site tRNA, and here we 715 00:42:18,960 --> 00:42:23,940 have a nascent polypeptide coming through the exit tunnel, 716 00:42:23,940 --> 00:42:27,960 and here we have some examples from structural information 717 00:42:27,960 --> 00:42:30,840 about erythromycin, chloroamphenicol 718 00:42:30,840 --> 00:42:32,370 bound in this region here. 719 00:42:35,360 --> 00:42:40,240 So we're going to look at a puromycin as a case study 720 00:42:40,240 --> 00:42:47,890 for using antibiotics as a tool in a biochemical experiment. 721 00:42:47,890 --> 00:42:50,530 So the first thing that we need to think about 722 00:42:50,530 --> 00:42:57,870 is the chemical structure of puromycin 723 00:42:57,870 --> 00:43:01,800 and how that structure relates to its ability 724 00:43:01,800 --> 00:43:03,450 to inhibit translation. 725 00:43:28,500 --> 00:43:31,590 So these puromycins are molecules 726 00:43:31,590 --> 00:43:33,360 that cause chain termination. 727 00:43:43,160 --> 00:43:45,135 And we'll look at an example of a structure. 728 00:43:45,135 --> 00:43:48,492 [WRITING ON BOARD] 729 00:44:30,452 --> 00:44:35,140 So basically just want to use a little imagination 730 00:44:35,140 --> 00:44:36,620 when looking at this structure. 731 00:44:40,750 --> 00:44:41,680 What do we see? 732 00:44:52,670 --> 00:44:56,460 So what is this small molecule mimicking? 733 00:44:56,460 --> 00:44:56,960 Yeah. 734 00:44:56,960 --> 00:45:00,950 So what do we have up here? 735 00:45:00,950 --> 00:45:03,530 We have something that's adenosine like. 736 00:45:03,530 --> 00:45:06,320 Not exactly the same structure. 737 00:45:06,320 --> 00:45:14,660 But this may be similar to A76 of the three prime end 738 00:45:14,660 --> 00:45:16,420 of the tRNA. 739 00:45:16,420 --> 00:45:22,110 We have these methyl groups rather than an H2, but similar. 740 00:45:22,110 --> 00:45:23,480 What's going on down here? 741 00:45:27,670 --> 00:45:30,250 Pardon? 742 00:45:30,250 --> 00:45:30,910 Yes. 743 00:45:30,910 --> 00:45:34,180 It's similar to one amino acid or a peptide. 744 00:45:34,180 --> 00:45:42,210 So we have something here that's amino acid like. 745 00:45:42,210 --> 00:45:44,670 So if we're thinking about this as a mimic of the three 746 00:45:44,670 --> 00:45:48,000 prime end of the tRNA with the amino acid bound, 747 00:45:48,000 --> 00:45:50,370 what's fundamentally different here that's 748 00:45:50,370 --> 00:45:53,115 going to result in different chemistry happening? 749 00:45:59,060 --> 00:46:03,320 So how are the amino acids attached to the three prime end 750 00:46:03,320 --> 00:46:04,250 of the tRNA? 751 00:46:04,250 --> 00:46:06,150 What kind of linkage? 752 00:46:06,150 --> 00:46:06,650 Yeah. 753 00:46:06,650 --> 00:46:09,050 We have an ester in the normal circumstance. 754 00:46:09,050 --> 00:46:11,840 And what do we have here? 755 00:46:11,840 --> 00:46:14,950 Here we have an amide. 756 00:46:14,950 --> 00:46:18,280 So this is non-hydrolyzable. 757 00:46:18,280 --> 00:46:21,490 And what else do we have? 758 00:46:21,490 --> 00:46:33,090 Right here we have a nucleophile for the P site ester. 759 00:46:40,060 --> 00:46:41,290 So what can happen? 760 00:46:41,290 --> 00:46:46,540 Imagine that puromycin somehow can enter the A site. 761 00:46:46,540 --> 00:46:51,930 There's a nucleophile that will allow for chain transfer, such 762 00:46:51,930 --> 00:46:56,310 that the peptide that's on the P site tRNA gets transferred. 763 00:46:56,310 --> 00:46:57,660 But then what? 764 00:46:57,660 --> 00:46:58,380 OK. 765 00:46:58,380 --> 00:47:02,020 We're stuck because of this MI bond here. 766 00:47:02,020 --> 00:47:06,610 So effectively chain termination. 767 00:47:06,610 --> 00:47:07,550 OK. 768 00:47:07,550 --> 00:47:11,960 And so what's known is this molecule 769 00:47:11,960 --> 00:47:15,710 and its analogs can bind to the 50s A site. 770 00:47:15,710 --> 00:47:18,770 And that's something kind of incredible to think about. 771 00:47:18,770 --> 00:47:21,350 We talked about this machinery EF-TU 772 00:47:21,350 --> 00:47:25,070 that's needed to deliver the aminoacyl-tRNA. 773 00:47:25,070 --> 00:47:28,220 Puromycin can get there on its own. 774 00:47:28,220 --> 00:47:31,100 Which means maybe for an experiment, 775 00:47:31,100 --> 00:47:33,590 that is easier to do if you're going 776 00:47:33,590 --> 00:47:37,010 to use this in your experiment. 777 00:47:37,010 --> 00:47:38,990 Moreover, people have synthesized 778 00:47:38,990 --> 00:47:40,820 more complex versions. 779 00:47:40,820 --> 00:47:43,400 Just an example is shown here. 780 00:47:43,400 --> 00:47:51,600 C-pmn where we also have C75 of the tRNA mimicked here. 781 00:47:51,600 --> 00:47:57,930 And so this is just an overview of elongation, 782 00:47:57,930 --> 00:48:00,800 and then effectively chain termination happening 783 00:48:00,800 --> 00:48:04,450 after thinking about having a puromycin in the A site, 784 00:48:04,450 --> 00:48:08,810 a peptidyl tRNA, or some other molecule in the P site 785 00:48:08,810 --> 00:48:10,900 and the chemistry that occurs here. 786 00:48:14,020 --> 00:48:20,170 So we'll think about and close with one experiment that's 787 00:48:20,170 --> 00:48:22,870 been done using puromycin. 788 00:48:22,870 --> 00:48:26,440 And we won't have time to go through all of it 789 00:48:26,440 --> 00:48:28,390 in the last few minutes of today, 790 00:48:28,390 --> 00:48:32,730 but I'll just introduce the problem 791 00:48:32,730 --> 00:48:34,940 and we'll continue with the experiment next time. 792 00:48:48,300 --> 00:48:48,800 OK. 793 00:48:48,800 --> 00:48:51,920 And so what we're going to think about 794 00:48:51,920 --> 00:48:56,440 is a translation factor that hasn't come up yet in class. 795 00:48:56,440 --> 00:49:00,650 And this is elongation factor P. OK. 796 00:49:00,650 --> 00:49:06,875 And for a long time its function was unclear. 797 00:49:06,875 --> 00:49:10,184 [WRITING ON BOARD] 798 00:49:27,030 --> 00:49:27,530 OK. 799 00:49:27,530 --> 00:49:31,430 And so over the years this translation factor 800 00:49:31,430 --> 00:49:34,370 was implicated in a variety of cellular processes, 801 00:49:34,370 --> 00:49:39,050 but there wasn't any clear answer in terms of really 802 00:49:39,050 --> 00:49:40,250 what is its role? 803 00:49:40,250 --> 00:49:43,790 And so about two years ago there were two back to back papers-- 804 00:49:43,790 --> 00:49:46,310 one of these papers by Rodina and co-workers. 805 00:49:46,310 --> 00:49:48,950 So they're the authors of the paper being studied 806 00:49:48,950 --> 00:49:51,980 in recitation this week. 807 00:49:51,980 --> 00:49:56,540 --published work reporting on why EFP 808 00:49:56,540 --> 00:49:59,250 is important for translation. 809 00:49:59,250 --> 00:50:03,050 So prior to their work there were some preliminary studies 810 00:50:03,050 --> 00:50:05,450 indicating that somehow this elongation 811 00:50:05,450 --> 00:50:09,740 factor helps to modulate and accelerate 812 00:50:09,740 --> 00:50:11,630 peptide bond formation. 813 00:50:11,630 --> 00:50:14,690 But the questions are, when? 814 00:50:14,690 --> 00:50:18,560 So under what circumstances does EFP accelerate peptide bond 815 00:50:18,560 --> 00:50:19,580 formation? 816 00:50:19,580 --> 00:50:22,340 And then you can think kind of a follow up of that, how? 817 00:50:22,340 --> 00:50:25,040 And so we'll look at some experiments that 818 00:50:25,040 --> 00:50:29,180 were designed and performed using puromycin as a tool 819 00:50:29,180 --> 00:50:30,440 to address this question. 820 00:50:30,440 --> 00:50:33,160 And that's where we'll start on Monday.