1 00:00:00,500 --> 00:00:02,830 The following content is provided under a Creative 2 00:00:02,830 --> 00:00:04,370 Commons license. 3 00:00:04,370 --> 00:00:06,670 Your support will help MIT Open Courseware 4 00:00:06,670 --> 00:00:11,030 continue to offer high quality educational resources for free. 5 00:00:11,030 --> 00:00:13,660 To make a donation or view additional materials 6 00:00:13,660 --> 00:00:17,610 from hundreds of MIT courses, visit MIT Open Courseware 7 00:00:17,610 --> 00:00:18,520 at ocw.mit.edu. 8 00:00:25,510 --> 00:00:27,010 ELIZABETH NOLAN: What we'll do today 9 00:00:27,010 --> 00:00:30,730 is have an overview looking at the ribosome structure, 10 00:00:30,730 --> 00:00:33,190 and also an overview of translation 11 00:00:33,190 --> 00:00:36,670 to get everyone on the same page for the discussions 12 00:00:36,670 --> 00:00:39,100 we'll start next week on the elongation 13 00:00:39,100 --> 00:00:41,290 cycle of translation. 14 00:00:41,290 --> 00:00:44,380 So I'll post, within lecture notes, 15 00:00:44,380 --> 00:00:47,110 reading as it applies to a given module 16 00:00:47,110 --> 00:00:49,610 and information about the problem sets, so you have that 17 00:00:49,610 --> 00:00:50,110 here. 18 00:00:53,510 --> 00:00:57,050 So before we get into some more molecular level 19 00:00:57,050 --> 00:00:59,510 details about ribosome structure, 20 00:00:59,510 --> 00:01:02,690 it's important to appreciate how we've 21 00:01:02,690 --> 00:01:06,290 gotten to where we are now in terms of our understanding, 22 00:01:06,290 --> 00:01:10,130 and so where we'll start is back in some early studies 23 00:01:10,130 --> 00:01:12,360 of electron microscopy. 24 00:01:12,360 --> 00:01:17,330 And this is in the '50s, and this researcher, Palade, 25 00:01:17,330 --> 00:01:20,130 obtained images, that looked like this, 26 00:01:20,130 --> 00:01:22,460 of rat pancreas tissue. 27 00:01:22,460 --> 00:01:24,140 And what was seen in these images 28 00:01:24,140 --> 00:01:26,750 were a lot of dark spheres. 29 00:01:26,750 --> 00:01:29,480 You can see them throughout, and they were 30 00:01:29,480 --> 00:01:32,630 called the particles of Palade. 31 00:01:32,630 --> 00:01:34,580 And one thing the scientists questioned 32 00:01:34,580 --> 00:01:37,490 is whether these black spheres or dots 33 00:01:37,490 --> 00:01:41,960 were something real or an artifact from his methods, 34 00:01:41,960 --> 00:01:43,790 so the perennial and arduous question 35 00:01:43,790 --> 00:01:46,100 of artifact versus reality. 36 00:01:46,100 --> 00:01:48,890 So this is something that we all question everyday when 37 00:01:48,890 --> 00:01:51,720 doing our experiments as well. 38 00:01:51,720 --> 00:01:54,770 And so he was quite a thorough scientist and experimentalist, 39 00:01:54,770 --> 00:01:56,510 and he repeated these experiments 40 00:01:56,510 --> 00:01:59,480 using different procedures to fix the tissues. 41 00:01:59,480 --> 00:02:02,690 And he observed these types of features 42 00:02:02,690 --> 00:02:05,090 in many different types of samples, 43 00:02:05,090 --> 00:02:08,389 and what was determined later on is that these black spheres are 44 00:02:08,389 --> 00:02:10,430 actually ribosomes. 45 00:02:10,430 --> 00:02:12,500 So one of the things we're going to look at today 46 00:02:12,500 --> 00:02:14,570 is how did we get from an image like this, 47 00:02:14,570 --> 00:02:17,900 just seeing some black dots, to the crystal 48 00:02:17,900 --> 00:02:20,720 structures we have today and the atomic resolution 49 00:02:20,720 --> 00:02:22,910 and understanding. 50 00:02:22,910 --> 00:02:28,390 And so he received the Nobel Prize back in 1974 51 00:02:28,390 --> 00:02:30,680 for this contribution. 52 00:02:30,680 --> 00:02:32,690 So just to keep in mind the hypothesis 53 00:02:32,690 --> 00:02:34,910 of translation, which is easy for us 54 00:02:34,910 --> 00:02:38,270 to take for granted these days. 55 00:02:38,270 --> 00:02:41,570 Goes back into the '60s, so there were studies 56 00:02:41,570 --> 00:02:45,170 during the '60s that resulted in the discovery 57 00:02:45,170 --> 00:02:48,260 that the 50S subunit of E. coli ribosomes 58 00:02:48,260 --> 00:02:51,220 catalyzes peptide bond formation. 59 00:02:51,220 --> 00:02:54,860 And it was discovered that the anticodon of the tRNA 60 00:02:54,860 --> 00:02:57,290 interacts with the 30S subunit, and that 61 00:02:57,290 --> 00:03:00,330 was important for translation. 62 00:03:00,330 --> 00:03:02,180 So this decoding problem-- 63 00:03:02,180 --> 00:03:05,570 effectively, how do we get from mRNA to protein-- 64 00:03:05,570 --> 00:03:08,660 was also articulated in the early '60s, 65 00:03:08,660 --> 00:03:13,160 and this was a puzzle for basically four decades. 66 00:03:13,160 --> 00:03:15,830 If we think about this from the standpoint of structure 67 00:03:15,830 --> 00:03:18,950 analysis and crystal structure-- so we'll 68 00:03:18,950 --> 00:03:22,250 look at images and data from crystal structures 69 00:03:22,250 --> 00:03:24,560 of the ribosome subunits today. 70 00:03:24,560 --> 00:03:28,130 If you take a look, what's important to appreciate here 71 00:03:28,130 --> 00:03:31,580 is that there was huge amounts of effort over many, many years 72 00:03:31,580 --> 00:03:33,720 to get where we are now. 73 00:03:33,720 --> 00:03:39,290 So in 1980, first crystals of the ribosome were obtained, 74 00:03:39,290 --> 00:03:43,670 but these crystals weren't of suitable quality for analysis. 75 00:03:43,670 --> 00:03:48,290 If we look at 20 years later, in 2000, the first crystal 76 00:03:48,290 --> 00:03:51,890 structure of the 50S subunit was reported, 77 00:03:51,890 --> 00:03:54,690 and since then, there's been a flurry of activity. 78 00:03:54,690 --> 00:03:57,990 So in 2001, first crystal structure of the 30 subunit-- 79 00:03:57,990 --> 00:04:02,690 30S subunit with mRNA bound, and in this time, 80 00:04:02,690 --> 00:04:06,290 too, single molecule spectroscopy 81 00:04:06,290 --> 00:04:08,960 was well on its way, and so there were studies 82 00:04:08,960 --> 00:04:11,960 beginning of ribosome dynamics. 83 00:04:11,960 --> 00:04:15,050 And later, 2011, we have a crystal structure 84 00:04:15,050 --> 00:04:18,950 of a eukaryotic 60S subunit here. 85 00:04:18,950 --> 00:04:21,170 And so we're going to focus our discussions 86 00:04:21,170 --> 00:04:24,110 on the prokaryotic ribosome, their similarities 87 00:04:24,110 --> 00:04:26,570 and differences between prokaryotic ribosomes 88 00:04:26,570 --> 00:04:28,910 and eukaryotic ribosomes, just to keep 89 00:04:28,910 --> 00:04:31,430 in mind if you've heard about eukaryotic ribosomes 90 00:04:31,430 --> 00:04:32,180 in other classes. 91 00:04:35,100 --> 00:04:39,740 So also to note, in 2009, the Nobel Prize in chemistry 92 00:04:39,740 --> 00:04:41,930 was awarded for structural studies 93 00:04:41,930 --> 00:04:46,580 of the ribosome to these three researchers here. 94 00:04:46,580 --> 00:04:48,560 And their contributions are shown 95 00:04:48,560 --> 00:04:52,820 ranging from basically the first low quality crystals of the 50S 96 00:04:52,820 --> 00:04:56,990 ribosomal subunit to understand how important that was, 97 00:04:56,990 --> 00:04:59,000 to the first crystal structures. 98 00:04:59,000 --> 00:05:01,320 And something Professor Stubbe and I 99 00:05:01,320 --> 00:05:04,100 like to remind everyone and keep in mind is, 100 00:05:04,100 --> 00:05:07,130 with these types of problems and areas, 101 00:05:07,130 --> 00:05:09,980 there's often many contributors, and they can't all 102 00:05:09,980 --> 00:05:12,710 be recognized by this prize because it's 103 00:05:12,710 --> 00:05:15,470 limited to three individuals at maximum. 104 00:05:15,470 --> 00:05:18,620 And so other folks like Harry Noller, Peter Moore, 105 00:05:18,620 --> 00:05:21,320 and Joaquim Frank made really seminal contributions 106 00:05:21,320 --> 00:05:26,810 to our understanding of this macromolecular machine. 107 00:05:26,810 --> 00:05:28,700 So what are the questions we're going 108 00:05:28,700 --> 00:05:31,462 to address in this module? 109 00:05:31,462 --> 00:05:33,170 And then we'll go over some of the basics 110 00:05:33,170 --> 00:05:35,370 in ribosome structure. 111 00:05:35,370 --> 00:05:38,390 So first, one is that we learn from structural studies 112 00:05:38,390 --> 00:05:40,880 of the ribosome, and really, what 113 00:05:40,880 --> 00:05:43,730 does ribosome structure at an atomic level 114 00:05:43,730 --> 00:05:46,160 tell us about its function? 115 00:05:46,160 --> 00:05:51,260 How does the ribosome recognize, bind, and decode mRNA? 116 00:05:51,260 --> 00:05:54,620 How are amino acids recognized and delivered, 117 00:05:54,620 --> 00:05:57,410 and how is the correct amino acid delivered? 118 00:05:57,410 --> 00:05:59,480 The genetic message needs to be read, 119 00:05:59,480 --> 00:06:01,910 and it needs to be read properly. 120 00:06:01,910 --> 00:06:05,330 And what happens if a wrong amino acid is delivered? 121 00:06:05,330 --> 00:06:06,620 So that's a possibility. 122 00:06:06,620 --> 00:06:08,660 How does the ribosome cope? 123 00:06:08,660 --> 00:06:11,270 So this brings up the notion of fidelity. 124 00:06:11,270 --> 00:06:14,000 How is fidelity of translation maintained? 125 00:06:14,000 --> 00:06:17,150 And we'll address that next week. 126 00:06:17,150 --> 00:06:20,180 How is translation initiated? 127 00:06:20,180 --> 00:06:23,420 How does the ribosome catalyze peptide bond formation? 128 00:06:23,420 --> 00:06:26,990 So we're interested in that mechanism within the context 129 00:06:26,990 --> 00:06:28,850 of this course. 130 00:06:28,850 --> 00:06:31,250 How does the polypeptide leave the ribosome, 131 00:06:31,250 --> 00:06:34,400 and what happens to that polypeptide after it exits? 132 00:06:34,400 --> 00:06:36,020 So that will be a transition for us 133 00:06:36,020 --> 00:06:39,470 into module 2 on protein folding. 134 00:06:39,470 --> 00:06:42,290 How is translation terminated, and what 135 00:06:42,290 --> 00:06:44,490 happens to the ribosome after? 136 00:06:44,490 --> 00:06:47,090 So a given polypeptide chain is made. 137 00:06:47,090 --> 00:06:49,370 What happens after that? 138 00:06:49,370 --> 00:06:51,080 And where we'll close this module 139 00:06:51,080 --> 00:06:54,050 is thinking about how our understanding of the ribosome, 140 00:06:54,050 --> 00:06:56,810 from all of these basic and fundamental studies, 141 00:06:56,810 --> 00:06:59,700 allows for the development of new technologies. 142 00:06:59,700 --> 00:07:01,460 And we'll specifically think about how 143 00:07:01,460 --> 00:07:03,740 it's possible to use the ribosome 144 00:07:03,740 --> 00:07:08,170 to incorporate unnatural amino acids into proteins. 145 00:07:08,170 --> 00:07:09,920 So where we're going to move forward today 146 00:07:09,920 --> 00:07:14,150 is really structure-- focusing on ribosome structure 147 00:07:14,150 --> 00:07:16,700 and a general overview of translation, 148 00:07:16,700 --> 00:07:20,360 basically to have everyone here up to speed for the discussions 149 00:07:20,360 --> 00:07:22,190 to come next week. 150 00:07:22,190 --> 00:07:26,360 So first of all, we'll do an overview of key players 151 00:07:26,360 --> 00:07:29,302 in translation, a brief look at the cycle, 152 00:07:29,302 --> 00:07:31,010 and then we'll go into structural studies 153 00:07:31,010 --> 00:07:31,880 of the ribosome. 154 00:07:46,110 --> 00:07:48,360 And within this set of lecture notes 155 00:07:48,360 --> 00:07:50,580 are several tables that have lists 156 00:07:50,580 --> 00:07:53,700 of the players and detailed overall cycle 157 00:07:53,700 --> 00:07:55,530 that I encourage you to use, just as 158 00:07:55,530 --> 00:07:58,770 a reference throughout this module for keeping everything 159 00:07:58,770 --> 00:08:00,150 straight. 160 00:08:00,150 --> 00:08:03,300 So first, of course, we have the ribosome. 161 00:08:07,770 --> 00:08:16,785 So the ribosome, as we all know, reads the genetic code 162 00:08:16,785 --> 00:08:23,880 via the mRNA, and it catalyzes peptide bond formation. 163 00:08:39,950 --> 00:08:44,449 So in addition to the ribosome, we have the mRNA. 164 00:08:48,550 --> 00:09:08,070 So this mRNA delivers the genetic code to the ribosome, 165 00:09:08,070 --> 00:09:09,690 and it provides a template-- 166 00:09:16,382 --> 00:09:18,310 [AUDIENCE MEMBER SNEEZES] 167 00:09:18,310 --> 00:09:20,260 Bless you-- for protein synthesis. 168 00:09:27,380 --> 00:09:34,310 So effectively, we can think about this process 169 00:09:34,310 --> 00:09:37,280 as a template-driven polymerization. 170 00:09:44,010 --> 00:09:48,390 So somehow, the amino acids need to get to the ribosome, 171 00:09:48,390 --> 00:09:50,550 and so we need the help of the tRNAs. 172 00:09:57,920 --> 00:10:11,130 So these transfer RNAs deliver the amino acid monomers, 173 00:10:11,130 --> 00:10:25,660 to the ribosome, and they transfer the amino acids 174 00:10:25,660 --> 00:10:28,690 during synthesis of the polypeptide. 175 00:10:39,350 --> 00:10:43,730 So in addition to the ribosome, the mRNA, and the tRNAs, 176 00:10:43,730 --> 00:10:48,740 the ribosome needs some help, so we have translation factors. 177 00:10:48,740 --> 00:10:51,140 And there's translation factors that 178 00:10:51,140 --> 00:10:56,030 are involved in each step of the translation cycle. 179 00:10:56,030 --> 00:10:59,270 So these are proteins that are required at specific points 180 00:10:59,270 --> 00:11:00,790 during the translation process. 181 00:11:37,670 --> 00:11:41,150 And so in terms of translation factors, 182 00:11:41,150 --> 00:11:43,760 we can break the process of translation 183 00:11:43,760 --> 00:11:45,980 into three or four steps-- 184 00:11:45,980 --> 00:11:50,970 I prefer three-- which are initiation, elongation, 185 00:11:50,970 --> 00:11:53,580 and termination. 186 00:11:53,580 --> 00:11:56,450 Some review articles and papers will divide this 187 00:11:56,450 --> 00:11:59,390 into four steps, because termination, 188 00:11:59,390 --> 00:12:01,340 you can think about peptide release 189 00:12:01,340 --> 00:12:03,650 and then ribosome recycling. 190 00:12:03,650 --> 00:12:08,090 But regardless to that detail, at each of these stages, 191 00:12:08,090 --> 00:12:10,400 there are translation factors that help. 192 00:12:10,400 --> 00:12:18,980 So we have initiation factors that 193 00:12:18,980 --> 00:12:23,000 help with the process of initiation, and in prokaryotes, 194 00:12:23,000 --> 00:12:27,080 we have initiation factors 1, 2, and 3, 195 00:12:27,080 --> 00:12:31,530 so 3 translation factors that help during elongation. 196 00:12:31,530 --> 00:12:34,820 So the process of making the peptide bond-- 197 00:12:34,820 --> 00:12:38,820 there are elongation factors. 198 00:12:38,820 --> 00:12:41,580 EF for Elongation Factor. 199 00:12:41,580 --> 00:12:43,700 IF for Initiation Factor. 200 00:12:43,700 --> 00:12:52,310 We have EF-Tu, EF-G, and others, and we'll 201 00:12:52,310 --> 00:12:56,840 spend quite a bit of time thinking about EF-Tu and EF-G 202 00:12:56,840 --> 00:12:59,930 over the course of the next week and in recitation 203 00:12:59,930 --> 00:13:02,090 and in problem sets, thinking about how 204 00:13:02,090 --> 00:13:05,090 these factors are really facilitating the elongation 205 00:13:05,090 --> 00:13:07,420 process here. 206 00:13:07,420 --> 00:13:15,810 And during termination, there are release factors, 207 00:13:15,810 --> 00:13:21,410 so we have release factors 1, 2, and 3. 208 00:13:21,410 --> 00:13:22,370 And we can also-- 209 00:13:22,370 --> 00:13:25,250 these are involved in release of the polypeptide that's 210 00:13:25,250 --> 00:13:27,800 been synthesized from the ribosome, 211 00:13:27,800 --> 00:13:29,390 and there's other players as well 212 00:13:29,390 --> 00:13:33,560 that I'll list here, including ribosome recycling 213 00:13:33,560 --> 00:13:40,070 factor, so the subunits get recycled, as we'll see. 214 00:13:40,070 --> 00:13:43,730 And we can also include a protein called trigger factor 215 00:13:43,730 --> 00:13:45,140 here. 216 00:13:45,140 --> 00:13:48,140 That is involved in folding of nascent polypeptide 217 00:13:48,140 --> 00:13:51,770 chains or the polypeptide chain as it's 218 00:13:51,770 --> 00:13:53,120 coming off the ribosome. 219 00:13:58,220 --> 00:14:02,270 And then just to summarize in terms 220 00:14:02,270 --> 00:14:10,180 of three stages of translation as I'll present them 221 00:14:10,180 --> 00:14:18,430 to you within this course, we have the initiation process; 222 00:14:18,430 --> 00:14:27,430 two, elongation; and three, termination. 223 00:14:30,800 --> 00:14:34,580 And where we'll be focusing the lectures next week, and really, 224 00:14:34,580 --> 00:14:41,340 this whole module, is here on elongation. 225 00:14:41,340 --> 00:14:43,730 And I'll just note that the elongation 226 00:14:43,730 --> 00:14:47,030 cycle is highly conserved. 227 00:14:47,030 --> 00:14:50,750 Termination and initiation vary quite a bit between prokaryotic 228 00:14:50,750 --> 00:14:54,830 and eukaryotes there in terms of the processes 229 00:14:54,830 --> 00:14:57,890 and involved players. 230 00:14:57,890 --> 00:15:01,070 So where are we going? 231 00:15:01,070 --> 00:15:03,710 Just as a brief overview of the cycle, 232 00:15:03,710 --> 00:15:07,020 and we'll come back to this later within today's lecture, 233 00:15:07,020 --> 00:15:08,750 or if not, on Monday. 234 00:15:08,750 --> 00:15:10,910 So we start with initiation, and we're 235 00:15:10,910 --> 00:15:13,310 going to have to ask ourselves, how 236 00:15:13,310 --> 00:15:18,450 is it that this 70S prokaryotic ribosome or initiation complex 237 00:15:18,450 --> 00:15:19,730 is assembled? 238 00:15:19,730 --> 00:15:23,510 And so there's a special tRNA involved, the initiator tRNA 239 00:15:23,510 --> 00:15:25,430 that we see binds here, and we'll 240 00:15:25,430 --> 00:15:29,150 talk more about these E, P, and A-sites in a moment. 241 00:15:29,150 --> 00:15:32,360 So we see the ribosome is assembled, the mRNA is bound, 242 00:15:32,360 --> 00:15:35,630 and there's an initiator tRNA bound. 243 00:15:35,630 --> 00:15:40,730 In order for the elongation cycle to be entered, 244 00:15:40,730 --> 00:15:43,170 an amino acid needs to be delivered, 245 00:15:43,170 --> 00:15:46,670 and that's delivered by an aminoacyl tRNA. 246 00:15:46,670 --> 00:15:50,730 That's in a ternary complex, so three components. 247 00:15:50,730 --> 00:15:56,270 We have the tRNA, the elongation factor Tu, and GTP. 248 00:15:56,270 --> 00:16:01,040 So this complex here somehow delivers an aminoacyl tRNA 249 00:16:01,040 --> 00:16:03,350 to the ribosome, and we're going to look 250 00:16:03,350 --> 00:16:05,800 at this process in detail next week. 251 00:16:05,800 --> 00:16:07,460 So this will be one of our case studies 252 00:16:07,460 --> 00:16:10,910 thinking about experiments and how experiments have supported 253 00:16:10,910 --> 00:16:14,090 a specific kinetic model here. 254 00:16:14,090 --> 00:16:19,400 So here, we have a complex where the tRNA 255 00:16:19,400 --> 00:16:21,680 is ready to occupy the A-site. 256 00:16:21,680 --> 00:16:25,310 What happens here-- we see that there's a GTP hydrolysis event. 257 00:16:25,310 --> 00:16:27,650 We'll talk about more as we go forward. 258 00:16:27,650 --> 00:16:30,320 Peptide transfer reaction-- so we 259 00:16:30,320 --> 00:16:32,600 have formation of a peptide bond, 260 00:16:32,600 --> 00:16:35,330 and then this elongation factor G 261 00:16:35,330 --> 00:16:38,930 comes in to facilitate the elongation cycle. 262 00:16:38,930 --> 00:16:42,950 And then this cycle will continue until some point that 263 00:16:42,950 --> 00:16:45,590 signals to stop synthesis, so a stop codon 264 00:16:45,590 --> 00:16:47,340 will enter the A-site. 265 00:16:47,340 --> 00:16:50,840 And there's a termination process, ribosome recycling, 266 00:16:50,840 --> 00:16:54,920 and you can imagine this whole cycle happening again. 267 00:16:54,920 --> 00:16:58,100 So how do we get to this cartoon to some more detailed 268 00:16:58,100 --> 00:16:59,060 understanding? 269 00:16:59,060 --> 00:17:00,390 That's where we're going. 270 00:17:00,390 --> 00:17:03,710 So come back to this cartoon at various stages 271 00:17:03,710 --> 00:17:05,940 throughout the course. 272 00:17:05,940 --> 00:17:14,420 So first, we'll do a cartoon overview of the prokaryotic 70S 273 00:17:14,420 --> 00:17:16,500 ribosome, and then we're going to look 274 00:17:16,500 --> 00:17:20,010 at some of the data from crystallography studies here. 275 00:17:34,660 --> 00:17:37,690 So as I think we all know the ribosome is comprised 276 00:17:37,690 --> 00:17:57,310 of RNA and proteins, and by mass, it's about 66% 277 00:17:57,310 --> 00:18:01,390 RNA and about 34% protein. 278 00:18:05,360 --> 00:18:09,310 And it's comprised of two subunits, 279 00:18:09,310 --> 00:18:12,655 and those are indicated in the cartoon by different colors. 280 00:18:23,150 --> 00:18:28,995 So in prokaryotes, we have the 50S, 281 00:18:28,995 --> 00:18:30,120 which is the large subunit. 282 00:18:33,710 --> 00:18:41,120 This is made up of 23S ribosomal RNA, a piece of 5S 283 00:18:41,120 --> 00:18:44,375 ribosomal RNA, and proteins. 284 00:18:48,200 --> 00:18:51,290 In terms of size this is huge, so it's 285 00:18:51,290 --> 00:18:53,580 approximately 1.5 megadaltons. 286 00:19:00,300 --> 00:19:02,820 And what we find within this subunit 287 00:19:02,820 --> 00:19:06,030 is the catalytic center, or peptidyl transferase center. 288 00:19:06,030 --> 00:19:08,085 This is sometimes abbreviated as PTC. 289 00:19:18,690 --> 00:19:22,380 And what we also find in the 50S subunit 290 00:19:22,380 --> 00:19:24,480 are three sites for tRNA binding. 291 00:19:55,500 --> 00:19:59,135 And so the other subunit in prokaryotes is the 30S. 292 00:20:04,620 --> 00:20:05,715 This is a small subunit. 293 00:20:11,590 --> 00:20:21,480 It's comprised of 16S rRNA and proteins, 294 00:20:21,480 --> 00:20:22,930 and it's also quite large. 295 00:20:22,930 --> 00:20:31,000 Just smaller than the 50S, so on the order of 0.8 megadaltons. 296 00:20:31,000 --> 00:20:34,750 And in terms of function, what we have in the 30S 297 00:20:34,750 --> 00:20:41,065 is the decoding center, so for decoding the mRNA, 298 00:20:41,065 --> 00:20:43,315 and the site of mRNA binding. 299 00:20:47,520 --> 00:20:51,590 So if we draw this in cartoon form-- 300 00:20:51,590 --> 00:20:55,220 and this is something I really encourage you all 301 00:20:55,220 --> 00:20:58,710 to do when thinking about the experiments and the problem 302 00:20:58,710 --> 00:21:00,710 sets because that's going to help you understand 303 00:21:00,710 --> 00:21:04,580 the experimental design and what actually happened. 304 00:21:04,580 --> 00:21:10,940 Here, what we have on top is the 50S subunit. 305 00:21:10,940 --> 00:21:14,090 On bottom, the 30S subunit. 306 00:21:14,090 --> 00:21:19,400 We have the mRNA, and note the directionality, so 5 prime end, 307 00:21:19,400 --> 00:21:22,820 5 prime end of the ribose, 3 prime end here. 308 00:21:22,820 --> 00:21:25,580 And then within this 50S, we can think 309 00:21:25,580 --> 00:21:31,640 about these sites for tRNA binding ordered as such, 310 00:21:31,640 --> 00:21:38,540 so E, P, and A here, so this is the catalytic center 311 00:21:38,540 --> 00:21:40,470 or peptidyl transferase center here. 312 00:21:43,250 --> 00:21:47,360 So overall, this assembled ribosome 313 00:21:47,360 --> 00:21:50,240 is on the order of 2.3 megadaltons 314 00:21:50,240 --> 00:21:53,780 and is about 200 Angstroms in diameter. 315 00:21:53,780 --> 00:21:58,400 So just in terms of these names, 50S, 30S-- 316 00:21:58,400 --> 00:22:03,600 this is overall the 70S assembled ribosome. 317 00:22:03,600 --> 00:22:05,140 What do these numbers-- 318 00:22:05,140 --> 00:22:06,140 where do they come from? 319 00:22:06,140 --> 00:22:10,050 What does this 50S, 30S, 70S mean? 320 00:22:17,250 --> 00:22:18,886 So what is the S? 321 00:22:18,886 --> 00:22:20,284 AUDIENCE: It's [INAUDIBLE]. 322 00:22:20,284 --> 00:22:24,012 It has to do with the sedimentation [INAUDIBLE].. 323 00:22:24,012 --> 00:22:26,910 ELIZABETH NOLAN: Yeah, it has to do with the sedimentation. 324 00:22:26,910 --> 00:22:30,480 So there's a type of experiment called analytical ultra 325 00:22:30,480 --> 00:22:32,940 centrifugation, and effectively, you 326 00:22:32,940 --> 00:22:37,350 can use this to ask about the sedimentation of a biomolecule. 327 00:22:37,350 --> 00:22:41,610 So effectively, what is the rate at which a biomolecule moves 328 00:22:41,610 --> 00:22:46,260 in response to the centrifugal force in a centrifuge there? 329 00:22:46,260 --> 00:22:50,160 And so you can use optics to monitor the sedimentation 330 00:22:50,160 --> 00:22:53,190 and use mathematics to fit those data to come up 331 00:22:53,190 --> 00:22:55,080 with an S value. 332 00:22:55,080 --> 00:23:00,240 So typically, the larger the S value, the larger the size. 333 00:23:00,240 --> 00:23:02,910 It's not always directly proportional to the mass 334 00:23:02,910 --> 00:23:05,580 because things like shape play a role as well, 335 00:23:05,580 --> 00:23:09,270 but effectively, we see 50S, and that subunit 336 00:23:09,270 --> 00:23:11,250 is larger than the 30S. 337 00:23:11,250 --> 00:23:14,460 And note, when they come together, it's not additive. 338 00:23:14,460 --> 00:23:18,420 It's 70S there, if you're to look at the assembled ribosome 339 00:23:18,420 --> 00:23:20,680 in one of these experiments. 340 00:23:20,680 --> 00:23:25,320 So that's where those values come from here. 341 00:23:25,320 --> 00:23:29,390 So if we take a look from my cartoon depiction 342 00:23:29,390 --> 00:23:33,120 to actual image from cryoelectron microscopy-- 343 00:23:33,120 --> 00:23:37,230 so this is just rotated basically 90 degrees. 344 00:23:37,230 --> 00:23:39,060 What do we see? 345 00:23:39,060 --> 00:23:42,240 We have the 50S here, the catalytic center. 346 00:23:42,240 --> 00:23:43,890 We have the 30S. 347 00:23:43,890 --> 00:23:46,980 Here's the mRNA, and what we're seeing 348 00:23:46,980 --> 00:23:48,930 is that, in this particular structure, 349 00:23:48,930 --> 00:23:51,690 there's some tRNAs bound, and they've indicated also 350 00:23:51,690 --> 00:23:55,260 a ribosomal protein here. 351 00:23:55,260 --> 00:23:57,520 Just as a sense of complexity-- 352 00:23:57,520 --> 00:24:03,390 so in E. coli, the 50S subunit has over 30 proteins 353 00:24:03,390 --> 00:24:05,190 associated with it. 354 00:24:05,190 --> 00:24:06,450 That's a lot-- 355 00:24:06,450 --> 00:24:07,860 30 different proteins. 356 00:24:07,860 --> 00:24:13,230 And the 30S has 21 ribosomal proteins associated with it, 357 00:24:13,230 --> 00:24:16,530 so we need to think about the proteins in addition 358 00:24:16,530 --> 00:24:19,660 to the RNA. 359 00:24:19,660 --> 00:24:24,780 So let's take a look at an image from the crystal 360 00:24:24,780 --> 00:24:27,990 structure reported in 2000, of the 50S 361 00:24:27,990 --> 00:24:32,520 ribosome from a particular prokaryote shown here. 362 00:24:32,520 --> 00:24:36,910 So this is what's described as the crown view, 363 00:24:36,910 --> 00:24:39,870 and in this particular depiction, what we're seeing 364 00:24:39,870 --> 00:24:45,450 is that the ribosomal RNA of the 50S is in gray or white, 365 00:24:45,450 --> 00:24:51,040 and the ribosomal proteins that are bound are in gold. 366 00:24:51,040 --> 00:24:55,140 So taking a look at this, what do we see? 367 00:24:55,140 --> 00:24:59,380 We can ask ourselves some questions from this structure. 368 00:24:59,380 --> 00:25:03,050 So the first question I'll ask is about the RNA. 369 00:25:03,050 --> 00:25:05,970 What does this RNA look like? 370 00:25:05,970 --> 00:25:10,550 So do we see any obvious domains? 371 00:25:10,550 --> 00:25:13,650 If anyone has some experience looking at structures. 372 00:25:18,760 --> 00:25:19,840 I don't see any. 373 00:25:19,840 --> 00:25:23,860 What I see is a compact mass of RNA here. 374 00:25:23,860 --> 00:25:27,460 There's not obvious domains or regions that 375 00:25:27,460 --> 00:25:30,580 are somehow different here. 376 00:25:30,580 --> 00:25:35,620 To me, in this structure, it looks like one big glob of RNA. 377 00:25:35,620 --> 00:25:38,440 But then the question is, is that truly the case, 378 00:25:38,440 --> 00:25:40,120 or is there an organization we're just 379 00:25:40,120 --> 00:25:42,880 not seeing at this level? 380 00:25:42,880 --> 00:25:47,170 The next question we can ask is where are the proteins? 381 00:25:47,170 --> 00:25:49,930 So if we look at the proteins and how 382 00:25:49,930 --> 00:25:55,450 they're arranged on this compact mass of RNA, what do we see? 383 00:25:55,450 --> 00:25:56,410 Where are they? 384 00:25:59,637 --> 00:26:00,560 AUDIENCE: The edges? 385 00:26:00,560 --> 00:26:02,060 ELIZABETH NOLAN: On the edges, yeah. 386 00:26:02,060 --> 00:26:04,520 There's many on the edges, like L1 here, 387 00:26:04,520 --> 00:26:08,150 this one on the outside here, over here. 388 00:26:08,150 --> 00:26:11,480 So it looks like these proteins, at least in this view, 389 00:26:11,480 --> 00:26:14,900 are mostly on the outside. 390 00:26:14,900 --> 00:26:18,530 Is there anything unusual or potentially unusual 391 00:26:18,530 --> 00:26:20,795 we can see in addition about these proteins? 392 00:26:25,710 --> 00:26:28,340 Maybe looking at this one here or here. 393 00:26:28,340 --> 00:26:29,280 What's going on? 394 00:26:35,442 --> 00:26:40,220 AUDIENCE: I can't see very well, but I think that there's 395 00:26:40,220 --> 00:26:42,202 not just [INAUDIBLE]. 396 00:26:45,052 --> 00:26:47,510 ELIZABETH NOLAN: It looks like there's some unfolded parts? 397 00:26:47,510 --> 00:26:48,135 AUDIENCE: Yeah. 398 00:26:48,135 --> 00:26:51,020 ELIZABETH NOLAN: Right, so look here. 399 00:26:51,020 --> 00:26:53,240 So it looks like there's some unfolded regions 400 00:26:53,240 --> 00:26:54,005 to these proteins. 401 00:26:56,510 --> 00:26:58,010 And why is that? 402 00:26:58,010 --> 00:27:01,190 And where are these unfolded regions going? 403 00:27:01,190 --> 00:27:02,960 So what we can do is look at the RNA 404 00:27:02,960 --> 00:27:05,750 separately and look at the protein separately now 405 00:27:05,750 --> 00:27:09,320 and see what we learn from these analyses. 406 00:27:09,320 --> 00:27:17,270 So effectively, if we consider the 23S rRNA, 407 00:27:17,270 --> 00:27:19,850 despite that structure we saw before that 408 00:27:19,850 --> 00:27:24,290 looked like a compact mass of RNA, it's structured, 409 00:27:24,290 --> 00:27:27,680 and it consists of six domains. 410 00:27:27,680 --> 00:27:31,340 And these domains have quite complicated shapes, 411 00:27:31,340 --> 00:27:33,170 and they fit together. 412 00:27:33,170 --> 00:27:38,490 And here is just a schematic diagram of this structure. 413 00:27:38,490 --> 00:27:42,110 So if we take a look, we can see that there's domain 1, 414 00:27:42,110 --> 00:27:46,940 domain 2, 3, 4, 5, and 6. 415 00:27:46,940 --> 00:27:49,370 And on the left here, it's indicated 416 00:27:49,370 --> 00:27:53,420 where, in that crown view we just looked at, right here, 417 00:27:53,420 --> 00:27:56,240 these domains are located. 418 00:27:56,240 --> 00:27:59,450 So there is organization, even though in that structure, 419 00:27:59,450 --> 00:28:04,020 it looks like one compact mass of RNA. 420 00:28:04,020 --> 00:28:06,860 So let's think about these proteins a bit more. 421 00:28:06,860 --> 00:28:10,220 And in addition to the crown view and the observations 422 00:28:10,220 --> 00:28:13,640 we had from this particular face of the ribosome, 423 00:28:13,640 --> 00:28:16,370 where it looks like many proteins are on the outside, 424 00:28:16,370 --> 00:28:18,530 and there's some unfolded regions, what 425 00:28:18,530 --> 00:28:20,570 happens if we look elsewhere? 426 00:28:20,570 --> 00:28:25,700 So here, we have rotation, so 180 degrees from here, 427 00:28:25,700 --> 00:28:28,490 effectively looking, we can say, on the backside. 428 00:28:28,490 --> 00:28:30,230 And here, we can look at the view 429 00:28:30,230 --> 00:28:33,350 from the bottom of this subunit. 430 00:28:33,350 --> 00:28:36,800 So what do these images suggest? 431 00:28:36,800 --> 00:28:40,820 Do they support what we were thinking from this one view 432 00:28:40,820 --> 00:28:43,955 here, that proteins are mostly on the outside? 433 00:28:48,350 --> 00:28:51,110 Yeah, I see some shaking heads "yes." 434 00:28:51,110 --> 00:28:53,330 It looks like the surface of this 50S 435 00:28:53,330 --> 00:28:57,710 is covered, effectively, by a protein lattice here. 436 00:28:57,710 --> 00:29:01,860 So what might a role be for these proteins, 437 00:29:01,860 --> 00:29:02,840 an important role? 438 00:29:05,750 --> 00:29:06,720 AUDIENCE: Structural? 439 00:29:06,720 --> 00:29:09,700 ELIZABETH NOLAN: Yeah, so some structural role. 440 00:29:09,700 --> 00:29:13,180 So these proteins can help with stabilizing 441 00:29:13,180 --> 00:29:16,920 this 3D structure of the RNA. 442 00:29:16,920 --> 00:29:20,140 And they have other functions as well, and some of those 443 00:29:20,140 --> 00:29:23,350 will come up as we discuss this elongation cycle. 444 00:29:23,350 --> 00:29:28,030 But one function is certainly structural. 445 00:29:28,030 --> 00:29:30,490 If we just think about the distribution 446 00:29:30,490 --> 00:29:34,030 of the proteins along the surface of this 50S, 447 00:29:34,030 --> 00:29:36,010 it looks more or less uniform. 448 00:29:36,010 --> 00:29:38,648 There aren't patches where there's no protein or patches 449 00:29:38,648 --> 00:29:39,940 where there's a lot of protein. 450 00:29:39,940 --> 00:29:43,360 They're pretty much evenly distributed here. 451 00:29:43,360 --> 00:29:47,410 So as it turns out, most of the segments of the 23S 452 00:29:47,410 --> 00:29:51,370 do interact with protein, and if we look at these proteins 453 00:29:51,370 --> 00:29:54,280 more closely, we're going to follow up on the observation 454 00:29:54,280 --> 00:29:58,010 that it looks like they have some unfolded regions. 455 00:29:58,010 --> 00:29:59,920 So what we're looking at here are just 456 00:29:59,920 --> 00:30:05,410 a selection of the 50S proteins in the absence of the RNA. 457 00:30:05,410 --> 00:30:07,750 So these structures have been taken out 458 00:30:07,750 --> 00:30:09,610 of that total structure. 459 00:30:09,610 --> 00:30:13,030 In terms of nomenclature, l means large 460 00:30:13,030 --> 00:30:15,250 and s means small, in terms of thinking 461 00:30:15,250 --> 00:30:17,440 about ribosomal proteins. 462 00:30:17,440 --> 00:30:20,470 And so what's found in the 50S is 463 00:30:20,470 --> 00:30:26,740 that we can categorize 17 of the proteins as globular or folded 464 00:30:26,740 --> 00:30:31,130 and 13 of the proteins as cases where 465 00:30:31,130 --> 00:30:34,360 there's extensions that are non-globular 466 00:30:34,360 --> 00:30:36,220 or have no clear structure. 467 00:30:36,220 --> 00:30:39,340 And that's color coded in these examples, where 468 00:30:39,340 --> 00:30:44,080 we have folded regions in green and then unfolded regions 469 00:30:44,080 --> 00:30:46,360 in red. 470 00:30:46,360 --> 00:30:51,850 So why is this, and where are these red extensions going? 471 00:30:51,850 --> 00:30:55,510 So what's seen is that these non-globular extensions work 472 00:30:55,510 --> 00:30:59,350 their way into the interior of the ribosome, 473 00:30:59,350 --> 00:31:01,780 so we can think about them kind of like tentacles, 474 00:31:01,780 --> 00:31:05,200 for instance, going into the interior. 475 00:31:05,200 --> 00:31:07,150 So how might they interact with the RNA? 476 00:31:14,120 --> 00:31:15,560 So I'll give you a hint. 477 00:31:15,560 --> 00:31:19,460 In these regions in red, there are quite a number 478 00:31:19,460 --> 00:31:23,165 of arginine and lysine residues compared to other regions. 479 00:31:26,540 --> 00:31:29,945 So what properties of arginine or lysine would be important? 480 00:31:29,945 --> 00:31:30,820 AUDIENCE: [INAUDIBLE] 481 00:31:30,820 --> 00:31:32,195 ELIZABETH NOLAN: Positive charge. 482 00:31:32,195 --> 00:31:34,600 Right, we have positively charged amino acids. 483 00:31:34,600 --> 00:31:36,010 What about PKAs? 484 00:31:36,010 --> 00:31:39,280 So who votes for arginine having a higher PKA than lysine? 485 00:31:42,190 --> 00:31:43,645 The opposite? 486 00:31:47,050 --> 00:31:48,820 So that's a point for review. 487 00:31:48,820 --> 00:31:50,440 Lysine around 10.5. 488 00:31:50,440 --> 00:31:51,850 arginine around 12.5. 489 00:31:51,850 --> 00:31:55,030 arginine's higher here. 490 00:31:55,030 --> 00:31:58,270 So if we have a bunch of positively charged residues 491 00:31:58,270 --> 00:31:59,680 in these extensions, how are they 492 00:31:59,680 --> 00:32:02,800 going to interact with the rRNA? 493 00:32:02,800 --> 00:32:05,620 What are the molecular features there that are important? 494 00:32:05,620 --> 00:32:06,920 AUDIENCE: [INAUDIBLE] 495 00:32:06,920 --> 00:32:07,920 ELIZABETH NOLAN: Pardon? 496 00:32:07,920 --> 00:32:08,090 AUDIENCE: Phosphates? 497 00:32:08,090 --> 00:32:09,880 ELIZABETH NOLAN: Yeah, the phosphate backbones. 498 00:32:09,880 --> 00:32:12,280 So we have the negatively charged phosphates, positively 499 00:32:12,280 --> 00:32:13,480 charged amino acids-- 500 00:32:13,480 --> 00:32:18,380 effectively formation of salt bridges here. 501 00:32:18,380 --> 00:32:23,230 AUDIENCE: So I know structure for a lot of these, well, 502 00:32:23,230 --> 00:32:24,460 non-globular regions. 503 00:32:24,460 --> 00:32:27,010 Does it mean that they're more disordered, 504 00:32:27,010 --> 00:32:31,820 or do they still have relatively similar B factor 505 00:32:31,820 --> 00:32:34,390 compared to the rest of the globular region? 506 00:32:34,390 --> 00:32:37,173 It's just that they don't fall under [INAUDIBLE]---- 507 00:32:37,173 --> 00:32:39,340 ELIZABETH NOLAN: I don't know what the B factors are 508 00:32:39,340 --> 00:32:41,470 for the different regions of these proteins, 509 00:32:41,470 --> 00:32:43,660 and for the case of discussion here, I 510 00:32:43,660 --> 00:32:46,390 would have it fall under a lack of secondary structure. 511 00:32:46,390 --> 00:32:49,240 And keep in mind, the ribosome is quite dynamic, 512 00:32:49,240 --> 00:32:52,942 and in isolation, are all the proteins there 513 00:32:52,942 --> 00:32:55,150 and in their native way or not is just something else 514 00:32:55,150 --> 00:32:55,880 to keep in mind. 515 00:32:55,880 --> 00:32:58,920 But these are certainly lacking a fold 516 00:32:58,920 --> 00:33:03,100 and going into the interior and working from salt bridges here. 517 00:33:06,660 --> 00:33:12,530 Here's just an example of the 50S with tRNAs bound. 518 00:33:12,530 --> 00:33:14,970 So we have the 50S. 519 00:33:14,970 --> 00:33:20,160 We see tRNA in the E-site, the P-site, and the A-site. 520 00:33:20,160 --> 00:33:23,490 And so what are these three sites? 521 00:33:23,490 --> 00:33:27,970 Effectively, their names indicate what they bind 522 00:33:27,970 --> 00:33:31,080 or what they do in terms of these letters. 523 00:33:31,080 --> 00:33:37,140 The A-site binds aminoacyl tRNAs with the exception of initiator 524 00:33:37,140 --> 00:33:41,310 tRNA, which cannot bind to the A-site. 525 00:33:41,310 --> 00:33:44,340 The P-site binds the initiator tRNA 526 00:33:44,340 --> 00:33:47,460 during the initiation process of translation, 527 00:33:47,460 --> 00:33:51,750 and then it also binds peptydil tRNAs, so effectively 528 00:33:51,750 --> 00:33:55,890 the tRNA that has the growing peptide chain attached. 529 00:33:55,890 --> 00:34:00,720 And then the E-site binds the DA slated tRNA, 530 00:34:00,720 --> 00:34:03,510 and this is called the E-site because it's the exit site. 531 00:34:03,510 --> 00:34:07,800 And eventually, this tRNA that has lost its amino acid 532 00:34:07,800 --> 00:34:10,039 needs to get kicked out of the ribosome. 533 00:34:12,739 --> 00:34:18,260 So one more point-- just going back about these proteins 534 00:34:18,260 --> 00:34:21,440 to highlight. 535 00:34:21,440 --> 00:34:24,860 We stated that these proteins are mostly on the exterior, 536 00:34:24,860 --> 00:34:27,320 and there's just these extensions that go in. 537 00:34:27,320 --> 00:34:29,870 One thing I didn't explicitly say 538 00:34:29,870 --> 00:34:31,909 is that this peptidyl transferase 539 00:34:31,909 --> 00:34:34,730 center is devoid of protein. 540 00:34:34,730 --> 00:34:37,040 So in this catalytic center that's 541 00:34:37,040 --> 00:34:42,050 responsible for peptide bond formation, there's no protein. 542 00:34:42,050 --> 00:34:45,080 So based on all of the structural evidence, 543 00:34:45,080 --> 00:34:49,100 the nearest protein is 18 Angstroms away. 544 00:34:49,100 --> 00:34:53,239 That's quite far when thinking about making a peptide bond 545 00:34:53,239 --> 00:34:55,370 in a catalytic center. 546 00:34:55,370 --> 00:34:58,340 And also we'll learn that magnesium ions 547 00:34:58,340 --> 00:35:00,620 are important for ribosome assembly. 548 00:35:00,620 --> 00:35:03,230 I'll just point out that the closest magnesium 549 00:35:03,230 --> 00:35:06,320 ion is 8 Angstroms away. 550 00:35:06,320 --> 00:35:10,760 So if there's no protein in this catalytic center that's 551 00:35:10,760 --> 00:35:13,910 responsible for formation of peptide bonds in this growing 552 00:35:13,910 --> 00:35:16,040 polypeptide chain, what does that 553 00:35:16,040 --> 00:35:20,650 tell us right off the bat about the ribosome and catalysis? 554 00:35:25,033 --> 00:35:26,440 AUDIENCE: [INAUDIBLE] 555 00:35:26,440 --> 00:35:27,440 ELIZABETH NOLAN: Pardon? 556 00:35:27,440 --> 00:35:29,273 AUDIENCE: You have many functional component 557 00:35:29,273 --> 00:35:32,030 of [INAUDIBLE] ribozymes. 558 00:35:32,030 --> 00:35:33,680 ELIZABETH NOLAN: Yeah, so the ribo-- 559 00:35:33,680 --> 00:35:36,100 the ribosome is a ribozyme, yes. 560 00:35:36,100 --> 00:35:38,590 So there's many functional components, 561 00:35:38,590 --> 00:35:41,320 but in terms of peptide bond formation, 562 00:35:41,320 --> 00:35:44,890 it's the RNA that's catalyzing that reaction. 563 00:35:44,890 --> 00:35:49,510 So it's a ribozyme, or an RNA based catalyst. 564 00:35:49,510 --> 00:35:52,630 And so this is something many of us may take for granted 565 00:35:52,630 --> 00:35:58,630 right now, but it was a big surprise to see this here. 566 00:35:58,630 --> 00:36:01,950 And to the best of my knowledge, the ribosome 567 00:36:01,950 --> 00:36:06,610 is the only natural ribozyme that has a polymerase activity. 568 00:36:06,610 --> 00:36:09,250 So many of these natural ribozymes 569 00:36:09,250 --> 00:36:14,290 are involved in RNA maturation here, so for those of you 570 00:36:14,290 --> 00:36:19,900 interested in evolution and hypotheses about RNA world, 571 00:36:19,900 --> 00:36:23,110 this observation that there's no protein in the catalytic center 572 00:36:23,110 --> 00:36:27,730 of the ribosome supports an RNA world hypothesis, 573 00:36:27,730 --> 00:36:32,170 the idea that the RNA, which stores genetic information, 574 00:36:32,170 --> 00:36:37,870 can perform chemical catalysis predates DNA and proteins. 575 00:36:37,870 --> 00:36:39,550 One thing I'll just, though, point out 576 00:36:39,550 --> 00:36:42,700 is that, prior to this structural study, roughly two 577 00:36:42,700 --> 00:36:46,480 years before, there was some experimental work done just 578 00:36:46,480 --> 00:36:51,730 looking at isolated 50S rRNA with no proteins. 579 00:36:51,730 --> 00:36:56,230 And it was found that isolated 50S rRNA could catalyze 580 00:36:56,230 --> 00:37:00,520 peptide bond formation, and that, specifically, domain 5 581 00:37:00,520 --> 00:37:04,353 was important for that reaction here. 582 00:37:04,353 --> 00:37:05,770 So if you're curious about that, I 583 00:37:05,770 --> 00:37:10,030 can point you in the direction of a paper. 584 00:37:10,030 --> 00:37:14,500 One last observation about the 50S subunit 585 00:37:14,500 --> 00:37:17,620 involves a peptide exit tunnel. 586 00:37:17,620 --> 00:37:21,460 And so somehow, the growing polypeptide chain 587 00:37:21,460 --> 00:37:24,840 needs to get out of this macromolecular machine, 588 00:37:24,840 --> 00:37:27,070 and in order for that to happen, there's 589 00:37:27,070 --> 00:37:30,190 an exit tunnel in the 50S subunit. 590 00:37:30,190 --> 00:37:34,360 So here, if we go back to that cryo-EM image, what's 591 00:37:34,360 --> 00:37:36,280 shown in this particular depiction 592 00:37:36,280 --> 00:37:41,260 is a polypeptide chain emerging from the 50S here. 593 00:37:41,260 --> 00:37:44,440 If we look at this view, a top or bottom view, what we see 594 00:37:44,440 --> 00:37:49,750 is that there's a hole here, and that hole is this exit tunnel. 595 00:37:49,750 --> 00:37:54,370 This is just another view of the same thing rotated, 596 00:37:54,370 --> 00:37:56,830 and a macrolide is a type of antibiotic 597 00:37:56,830 --> 00:38:00,280 that can bind in the region and is thought to block 598 00:38:00,280 --> 00:38:02,830 exit of the polypeptide. 599 00:38:02,830 --> 00:38:05,290 So there's some features about this exit tunnel 600 00:38:05,290 --> 00:38:10,070 that are interesting and that we need to consider. 601 00:38:10,070 --> 00:38:15,130 First of all, it's long, so approximately 100 Angstroms. 602 00:38:15,130 --> 00:38:18,940 And the diameter is relatively small, 603 00:38:18,940 --> 00:38:23,710 so the diameter is on the order of 15 Angstroms. 604 00:38:23,710 --> 00:38:27,820 So what we need to think about, from the perspective 605 00:38:27,820 --> 00:38:32,350 of this diameter, is what can fit, 606 00:38:32,350 --> 00:38:35,620 and so this week in recitation, you're 607 00:38:35,620 --> 00:38:41,060 looking at using PyMOL and ubiquitin as an example. 608 00:38:41,060 --> 00:38:43,170 If you just ask yourself, would ubiquitin, 609 00:38:43,170 --> 00:38:49,370 folded ubiquitin, fit in this exit tunnel based on its size? 610 00:38:49,370 --> 00:38:53,560 And so where does protein folding occur? 611 00:38:53,560 --> 00:38:57,580 We think about this as primarily and predominantly happening 612 00:38:57,580 --> 00:39:00,670 after the polypeptide comes out of the ribosome 613 00:39:00,670 --> 00:39:03,010 because there just isn't room in this exit 614 00:39:03,010 --> 00:39:07,990 tunnel for some folded structure to exist here. 615 00:39:07,990 --> 00:39:12,280 Also, the exit tunnel not shown in these images 616 00:39:12,280 --> 00:39:15,940 is lined with hydrophobic residues, just as 617 00:39:15,940 --> 00:39:17,650 another feature. 618 00:39:17,650 --> 00:39:20,695 So it's narrow, and it cannot accommodate folded proteins. 619 00:39:23,490 --> 00:39:30,210 So briefly on the 30S, similar to the 50S as said before, 620 00:39:30,210 --> 00:39:35,460 this 30S is comprised of RNA and proteins. 621 00:39:35,460 --> 00:39:39,600 It has the sites of mRNA binding and decoding. 622 00:39:39,600 --> 00:39:43,770 Here's just a structural overview of the 30S 623 00:39:43,770 --> 00:39:47,850 with different regions named, and similar to what 624 00:39:47,850 --> 00:39:54,720 we saw for the 23S rRNA of the 50S subunit, 625 00:39:54,720 --> 00:39:58,410 the 16S rRNA also has structure. 626 00:39:58,410 --> 00:40:03,000 And I just show you the domain organization here, 627 00:40:03,000 --> 00:40:06,870 so we see that there are four domains, 628 00:40:06,870 --> 00:40:10,380 and they're color coded in green, yellow, blue, and red 629 00:40:10,380 --> 00:40:12,210 here. 630 00:40:12,210 --> 00:40:20,340 And so another point just to make in passing about 16S-- 631 00:40:23,040 --> 00:40:27,300 16S rRNA is highly conserved amongst species, 632 00:40:27,300 --> 00:40:30,450 so sequencing the 16S is commonly 633 00:40:30,450 --> 00:40:33,210 done in studies of, say, the microbiome 634 00:40:33,210 --> 00:40:36,090 to figure out something about the distribution 635 00:40:36,090 --> 00:40:39,600 of different types of prokaryotic organisms 636 00:40:39,600 --> 00:40:43,050 there for that. 637 00:40:43,050 --> 00:40:47,730 So why spend so much time on the individual subunits? 638 00:40:47,730 --> 00:40:51,090 What we find is that the structures 639 00:40:51,090 --> 00:40:54,120 are very similar when the ribosome is assembled. 640 00:40:54,120 --> 00:40:56,520 So we can think of the 30S and the 50S 641 00:40:56,520 --> 00:40:59,550 as coming together to give the 70S, 642 00:40:59,550 --> 00:41:01,830 and these subunits basically look the same 643 00:41:01,830 --> 00:41:03,840 as they do in isolation. 644 00:41:03,840 --> 00:41:08,640 And that's depicted here, in just another example. 645 00:41:08,640 --> 00:41:11,700 So if we're looking at this structure based 646 00:41:11,700 --> 00:41:14,640 on the cartoon and our discussions, 647 00:41:14,640 --> 00:41:18,570 you should be able to identify the different components. 648 00:41:18,570 --> 00:41:21,338 So here, what do we have? 649 00:41:21,338 --> 00:41:22,230 AUDIENCE: 50S. 650 00:41:22,230 --> 00:41:23,613 ELIZABETH NOLAN: Yeah, and here? 651 00:41:23,613 --> 00:41:24,540 AUDIENCE: 30S. 652 00:41:24,540 --> 00:41:25,707 ELIZABETH NOLAN: 30S, right. 653 00:41:25,707 --> 00:41:28,177 What's this? 654 00:41:28,177 --> 00:41:29,052 AUDIENCE: [INAUDIBLE] 655 00:41:29,052 --> 00:41:30,543 AUDIENCE: [INAUDIBLE] 656 00:41:30,543 --> 00:41:34,130 ELIZABETH NOLAN: Yeah, we have a tRNA bound here. 657 00:41:34,130 --> 00:41:36,920 Here, a protein. 658 00:41:36,920 --> 00:41:41,720 So bring yourself back to this cartoon and its simplicity 659 00:41:41,720 --> 00:41:45,050 as we work through problems next week. 660 00:41:45,050 --> 00:41:48,290 So another point to make, just to think about, 661 00:41:48,290 --> 00:41:51,710 is how is it that these subunits actually come together, 662 00:41:51,710 --> 00:41:56,100 and what mediates that interaction there? 663 00:41:56,100 --> 00:42:01,250 And so these subunits basically come into contact 664 00:42:01,250 --> 00:42:05,120 at about 12 positions, and magnesium ions 665 00:42:05,120 --> 00:42:08,420 are really important for mediating the interaction 666 00:42:08,420 --> 00:42:11,630 between the 30S and the 50S. 667 00:42:11,630 --> 00:42:15,320 So there's bound magnesium ions that mediate interactions 668 00:42:15,320 --> 00:42:17,930 between these subunits here. 669 00:42:17,930 --> 00:42:20,510 And so in week four recitation, we're 670 00:42:20,510 --> 00:42:23,990 going to think about how to purify ribosomes. 671 00:42:23,990 --> 00:42:26,360 And if you're interested in purifying ribosomes, how 672 00:42:26,360 --> 00:42:31,370 do you get an assembled 70S prokaryotic ribosome? 673 00:42:31,370 --> 00:42:35,120 And based on the need for magnesium ions here, 674 00:42:35,120 --> 00:42:37,820 we'll see how that's an important variable 675 00:42:37,820 --> 00:42:41,340 in these procedures. 676 00:42:41,340 --> 00:42:45,230 So we'll close just with some overview 677 00:42:45,230 --> 00:42:49,340 points about the translation process as a whole. 678 00:42:49,340 --> 00:42:53,990 So during translation, mRNA is read from the five prime 679 00:42:53,990 --> 00:42:56,780 to the three prime end. 680 00:42:56,780 --> 00:42:59,660 Polypeptides are synthesized from the N terminus 681 00:42:59,660 --> 00:43:02,810 to the C terminus, so there's directionality. 682 00:43:02,810 --> 00:43:05,390 As I said earlier, translation factors 683 00:43:05,390 --> 00:43:08,000 are required at each stage-- 684 00:43:08,000 --> 00:43:12,050 initiation, elongation, and termination. 685 00:43:12,050 --> 00:43:14,030 Something that I haven't highlighted yet 686 00:43:14,030 --> 00:43:16,320 is the importance of GTP. 687 00:43:16,320 --> 00:43:18,740 So in that initial overview of the cycle, 688 00:43:18,740 --> 00:43:22,340 we saw that there were some instances of GTP hydrolysis 689 00:43:22,340 --> 00:43:26,120 by certain translation factors, and in this translation 690 00:43:26,120 --> 00:43:31,310 process, GTP hydrolysis provides a means to convert chemical 691 00:43:31,310 --> 00:43:33,500 energy into mechanical energy. 692 00:43:33,500 --> 00:43:38,150 And so we're going to think a lot about how GTP hydrolysis 693 00:43:38,150 --> 00:43:41,060 plays a role next week. 694 00:43:41,060 --> 00:43:43,640 And although we're going to look at many structures, 695 00:43:43,640 --> 00:43:46,430 keep in mind that conformational changes are 696 00:43:46,430 --> 00:43:49,670 essential for catalysis by the ribosome, 697 00:43:49,670 --> 00:43:53,570 and that this is a very dynamic system here. 698 00:43:56,120 --> 00:43:59,060 So just some additional facts-- 699 00:43:59,060 --> 00:44:04,070 so ribosomes will synthesize six to 20 peptide bonds per second. 700 00:44:04,070 --> 00:44:09,100 The error rate is less than 1 in 1,000, which brings up fidelity 701 00:44:09,100 --> 00:44:09,600 again. 702 00:44:09,600 --> 00:44:12,470 How does the ribosome maintain this? 703 00:44:12,470 --> 00:44:14,810 And the rate accelerations are on the order of 10 704 00:44:14,810 --> 00:44:19,670 to the 7-fold, so less than many enzymes, but quite good. 705 00:44:19,670 --> 00:44:22,370 And in all living organisms, these ribosomes 706 00:44:22,370 --> 00:44:25,760 carry out protein synthesis, so all ribosomes 707 00:44:25,760 --> 00:44:29,480 contain two subunits that reversibly associate 708 00:44:29,480 --> 00:44:32,060 during the translation cycle. 709 00:44:32,060 --> 00:44:34,400 Protein synthesis occurs through the binding 710 00:44:34,400 --> 00:44:38,300 of the aminoacyl tRNAs to the 70S ribosome 711 00:44:38,300 --> 00:44:41,150 in an order dictated by the mRNA. 712 00:44:41,150 --> 00:44:43,670 And next week, we're going to dissect how this actually 713 00:44:43,670 --> 00:44:47,240 occurs, and we think this will be quite new for all of you, 714 00:44:47,240 --> 00:44:51,710 even if you've learned about the ribosome in other courses. 715 00:44:51,710 --> 00:44:54,710 These tRNAs move sequentially through these three 716 00:44:54,710 --> 00:44:58,914 ribosome binding sites, as we saw before here. 717 00:45:01,890 --> 00:45:08,290 So we can return to our overview cycle here, that we saw before. 718 00:45:08,290 --> 00:45:12,180 And so we'll briefly address how initiation occurs. 719 00:45:12,180 --> 00:45:16,200 So how is this 70S ribosome assembled? 720 00:45:16,200 --> 00:45:19,490 We'll have a detailed case study of EF-Tu 721 00:45:19,490 --> 00:45:21,840 and then look through this elongation 722 00:45:21,840 --> 00:45:22,755 cycle in more detail. 723 00:45:25,760 --> 00:45:28,440 In terms of the players and the outcomes-- 724 00:45:28,440 --> 00:45:31,670 so this is a reference slide for all of you, where 725 00:45:31,670 --> 00:45:36,740 the stages are listed, that all of the players are listed, 726 00:45:36,740 --> 00:45:42,470 so some more detail than what's up here, and then the outcome. 727 00:45:42,470 --> 00:45:45,590 So what you can see from this overview, 728 00:45:45,590 --> 00:45:48,920 and go back and study it outside of lecture, 729 00:45:48,920 --> 00:45:52,410 is that, in each case, we see GTP, 730 00:45:52,410 --> 00:45:56,300 which means GTP hydrolysis occurs at each step. 731 00:45:56,300 --> 00:46:00,110 In initiation, our outcome is assembly of the 70S 732 00:46:00,110 --> 00:46:05,360 with mRNA bound and with an initiator tRNA in the P-site. 733 00:46:05,360 --> 00:46:08,270 The outcome of elongation is synthesis 734 00:46:08,270 --> 00:46:12,200 of this nascent, or new, polypeptide chain, 735 00:46:12,200 --> 00:46:15,410 and termination is the hydrolytic release 736 00:46:15,410 --> 00:46:18,290 of the peptide, release of the tRNAs 737 00:46:18,290 --> 00:46:21,680 and mRNAs and dissociation of the 70S 738 00:46:21,680 --> 00:46:23,810 and, ultimately, recycling. 739 00:46:23,810 --> 00:46:27,880 So there's many factors that need to be taken into account 740 00:46:27,880 --> 00:46:32,570 and dealt with it at every stage here. 741 00:46:32,570 --> 00:46:35,240 This is just another reference table. 742 00:46:35,240 --> 00:46:39,110 It has some additional players, like EF-Ts, 743 00:46:39,110 --> 00:46:43,280 and this is a nucleotide exchange factor for EF-Tu. 744 00:46:43,280 --> 00:46:47,510 So EF-Tu is a GTP-ase that we'll hear more about in lecture 745 00:46:47,510 --> 00:46:51,860 next week, and in recitation next week. 746 00:46:51,860 --> 00:46:56,180 Briefly, some topics for review-- 747 00:46:56,180 --> 00:46:59,690 if you need to review the genetic code, please do. 748 00:46:59,690 --> 00:47:04,070 We're not going to spend much time on it here. 749 00:47:04,070 --> 00:47:06,620 But in brief, I think we all know this genetic code is 750 00:47:06,620 --> 00:47:08,340 based on codons. 751 00:47:08,340 --> 00:47:11,900 They're read sequentially from a fixed starting point, 752 00:47:11,900 --> 00:47:13,700 and the code, which is a triplet code, 753 00:47:13,700 --> 00:47:16,730 is degenerate and non-overlapping. 754 00:47:16,730 --> 00:47:18,470 So why do we have a triplet code? 755 00:47:25,890 --> 00:47:27,050 We have four bases. 756 00:47:27,050 --> 00:47:29,678 AUDIENCE: We need enough combinations to [INAUDIBLE].. 757 00:47:29,678 --> 00:47:31,220 ELIZABETH NOLAN: Exactly, there needs 758 00:47:31,220 --> 00:47:34,730 to be enough combinations for all the amino acids. 759 00:47:34,730 --> 00:47:37,370 So we have 20 proteinogenic amino acids, 760 00:47:37,370 --> 00:47:38,690 and what else do we have? 761 00:47:38,690 --> 00:47:40,160 We have selenocysteine. 762 00:47:40,160 --> 00:47:41,690 We have pyrrolysine. 763 00:47:41,690 --> 00:47:44,960 So a triplet code with four bases 764 00:47:44,960 --> 00:47:48,530 covers everything we need here. 765 00:47:48,530 --> 00:47:52,100 We have start codons and stop codons we have to keep in mind, 766 00:47:52,100 --> 00:47:54,530 listed here. 767 00:47:54,530 --> 00:47:59,510 And as a reminder, in translation, the amino acids 768 00:47:59,510 --> 00:48:02,630 are delivered by the aminoacyl tRNAs. 769 00:48:02,630 --> 00:48:06,500 So the mRNA does not recognize these amino acids directly. 770 00:48:06,500 --> 00:48:12,470 We need the tRNAs that allows this reading to occur. 771 00:48:12,470 --> 00:48:14,630 Throughout this course, we're going to refer-- 772 00:48:14,630 --> 00:48:17,120 well, throughout this section with the ribosome, 773 00:48:17,120 --> 00:48:22,640 we'll be referring to nucleotides, et cetera, 774 00:48:22,640 --> 00:48:24,890 by the letter abbreviations. 775 00:48:24,890 --> 00:48:27,890 There are structures, chemical structures, 776 00:48:27,890 --> 00:48:29,780 associated with these abbreviations, 777 00:48:29,780 --> 00:48:32,660 and it's important to know those and be thinking about those 778 00:48:32,660 --> 00:48:34,460 as you work problems. 779 00:48:34,460 --> 00:48:37,640 So just as review, we have the DNA bases, 780 00:48:37,640 --> 00:48:43,160 C, G, A, and T. In RNA, we have uracil instead of thymine. 781 00:48:43,160 --> 00:48:45,590 The purines, A and G, have two rings, 782 00:48:45,590 --> 00:48:47,930 and the Pyrimidines, one ring. 783 00:48:47,930 --> 00:48:52,850 For nomenclature, nucleoside versus nucleotide-- 784 00:48:52,850 --> 00:48:56,950 so the nucleoside is a base plus a sugar, 785 00:48:56,950 --> 00:49:00,260 so there's this glycosidic bond here between the base 786 00:49:00,260 --> 00:49:04,850 and the carbon here of the ribose. 787 00:49:04,850 --> 00:49:08,090 And then the nucleotide is this nucleoside 788 00:49:08,090 --> 00:49:11,690 with one or more phosphate groups attached 789 00:49:11,690 --> 00:49:13,100 at the five prime carbon. 790 00:49:13,100 --> 00:49:15,440 So we go one prime, two prime, three prime, 791 00:49:15,440 --> 00:49:19,250 four prime, five prime for the numbering of the ribose. 792 00:49:19,250 --> 00:49:23,510 And keep in mind, from 5.07 or 7.05-- 793 00:49:23,510 --> 00:49:26,270 I think this should be known, but these phosphates, 794 00:49:26,270 --> 00:49:29,540 we have alpha, beta, and gamma phosphates. 795 00:49:29,540 --> 00:49:34,190 And depending on whether your ATP or some other nucleotide 796 00:49:34,190 --> 00:49:37,430 is being hydrolyzed to, say, an AMP or and ADP, 797 00:49:37,430 --> 00:49:40,440 you're going to have attack at different positions. 798 00:49:40,440 --> 00:49:44,900 So if you need to review, visit your basic biochemistry 799 00:49:44,900 --> 00:49:47,240 textbook for these details. 800 00:49:47,240 --> 00:49:49,340 Also just to keep in mind, the Watson-Crick 801 00:49:49,340 --> 00:49:53,660 based pairing, so G and C. We have three hydrogen bonds 802 00:49:53,660 --> 00:49:57,380 here, A and T, two hydrogen bonds. 803 00:49:57,380 --> 00:49:59,840 And after spring break, Professor Stubbe 804 00:49:59,840 --> 00:50:04,070 will be presenting a module on nucleotide metabolism, where 805 00:50:04,070 --> 00:50:07,410 we'll be thinking about these things in some more detail. 806 00:50:07,410 --> 00:50:11,900 So where we'll begin on Monday is briefly 807 00:50:11,900 --> 00:50:14,480 looking at an overview of initiation, 808 00:50:14,480 --> 00:50:16,700 and then we're going to begin to ask 809 00:50:16,700 --> 00:50:20,090 how did these amino acids get attached to tRNAs, 810 00:50:20,090 --> 00:50:22,970 and how did those aminoacyl tRNAs 811 00:50:22,970 --> 00:50:25,760 get to the A-site of the ribosome. 812 00:50:25,760 --> 00:50:28,510 So we'll see you then.