WEBVTT 00:00:00.500 --> 00:00:02.830 The following content is provided under a Creative 00:00:02.830 --> 00:00:04.370 Commons license. 00:00:04.370 --> 00:00:06.670 Your support will help MIT Open Courseware 00:00:06.670 --> 00:00:11.030 continue to offer high quality educational resources for free. 00:00:11.030 --> 00:00:13.660 To make a donation or view additional materials 00:00:13.660 --> 00:00:17.610 from hundreds of MIT courses, visit MIT Open Courseware 00:00:17.610 --> 00:00:18.520 at ocw.mit.edu. 00:00:25.510 --> 00:00:27.010 ELIZABETH NOLAN: What we'll do today 00:00:27.010 --> 00:00:30.730 is have an overview looking at the ribosome structure, 00:00:30.730 --> 00:00:33.190 and also an overview of translation 00:00:33.190 --> 00:00:36.670 to get everyone on the same page for the discussions 00:00:36.670 --> 00:00:39.100 we'll start next week on the elongation 00:00:39.100 --> 00:00:41.290 cycle of translation. 00:00:41.290 --> 00:00:44.380 So I'll post, within lecture notes, 00:00:44.380 --> 00:00:47.110 reading as it applies to a given module 00:00:47.110 --> 00:00:49.610 and information about the problem sets, so you have that 00:00:49.610 --> 00:00:50.110 here. 00:00:53.510 --> 00:00:57.050 So before we get into some more molecular level 00:00:57.050 --> 00:00:59.510 details about ribosome structure, 00:00:59.510 --> 00:01:02.690 it's important to appreciate how we've 00:01:02.690 --> 00:01:06.290 gotten to where we are now in terms of our understanding, 00:01:06.290 --> 00:01:10.130 and so where we'll start is back in some early studies 00:01:10.130 --> 00:01:12.360 of electron microscopy. 00:01:12.360 --> 00:01:17.330 And this is in the '50s, and this researcher, Palade, 00:01:17.330 --> 00:01:20.130 obtained images, that looked like this, 00:01:20.130 --> 00:01:22.460 of rat pancreas tissue. 00:01:22.460 --> 00:01:24.140 And what was seen in these images 00:01:24.140 --> 00:01:26.750 were a lot of dark spheres. 00:01:26.750 --> 00:01:29.480 You can see them throughout, and they were 00:01:29.480 --> 00:01:32.630 called the particles of Palade. 00:01:32.630 --> 00:01:34.580 And one thing the scientists questioned 00:01:34.580 --> 00:01:37.490 is whether these black spheres or dots 00:01:37.490 --> 00:01:41.960 were something real or an artifact from his methods, 00:01:41.960 --> 00:01:43.790 so the perennial and arduous question 00:01:43.790 --> 00:01:46.100 of artifact versus reality. 00:01:46.100 --> 00:01:48.890 So this is something that we all question everyday when 00:01:48.890 --> 00:01:51.720 doing our experiments as well. 00:01:51.720 --> 00:01:54.770 And so he was quite a thorough scientist and experimentalist, 00:01:54.770 --> 00:01:56.510 and he repeated these experiments 00:01:56.510 --> 00:01:59.480 using different procedures to fix the tissues. 00:01:59.480 --> 00:02:02.690 And he observed these types of features 00:02:02.690 --> 00:02:05.090 in many different types of samples, 00:02:05.090 --> 00:02:08.389 and what was determined later on is that these black spheres are 00:02:08.389 --> 00:02:10.430 actually ribosomes. 00:02:10.430 --> 00:02:12.500 So one of the things we're going to look at today 00:02:12.500 --> 00:02:14.570 is how did we get from an image like this, 00:02:14.570 --> 00:02:17.900 just seeing some black dots, to the crystal 00:02:17.900 --> 00:02:20.720 structures we have today and the atomic resolution 00:02:20.720 --> 00:02:22.910 and understanding. 00:02:22.910 --> 00:02:28.390 And so he received the Nobel Prize back in 1974 00:02:28.390 --> 00:02:30.680 for this contribution. 00:02:30.680 --> 00:02:32.690 So just to keep in mind the hypothesis 00:02:32.690 --> 00:02:34.910 of translation, which is easy for us 00:02:34.910 --> 00:02:38.270 to take for granted these days. 00:02:38.270 --> 00:02:41.570 Goes back into the '60s, so there were studies 00:02:41.570 --> 00:02:45.170 during the '60s that resulted in the discovery 00:02:45.170 --> 00:02:48.260 that the 50S subunit of E. coli ribosomes 00:02:48.260 --> 00:02:51.220 catalyzes peptide bond formation. 00:02:51.220 --> 00:02:54.860 And it was discovered that the anticodon of the tRNA 00:02:54.860 --> 00:02:57.290 interacts with the 30S subunit, and that 00:02:57.290 --> 00:03:00.330 was important for translation. 00:03:00.330 --> 00:03:02.180 So this decoding problem-- 00:03:02.180 --> 00:03:05.570 effectively, how do we get from mRNA to protein-- 00:03:05.570 --> 00:03:08.660 was also articulated in the early '60s, 00:03:08.660 --> 00:03:13.160 and this was a puzzle for basically four decades. 00:03:13.160 --> 00:03:15.830 If we think about this from the standpoint of structure 00:03:15.830 --> 00:03:18.950 analysis and crystal structure-- so we'll 00:03:18.950 --> 00:03:22.250 look at images and data from crystal structures 00:03:22.250 --> 00:03:24.560 of the ribosome subunits today. 00:03:24.560 --> 00:03:28.130 If you take a look, what's important to appreciate here 00:03:28.130 --> 00:03:31.580 is that there was huge amounts of effort over many, many years 00:03:31.580 --> 00:03:33.720 to get where we are now. 00:03:33.720 --> 00:03:39.290 So in 1980, first crystals of the ribosome were obtained, 00:03:39.290 --> 00:03:43.670 but these crystals weren't of suitable quality for analysis. 00:03:43.670 --> 00:03:48.290 If we look at 20 years later, in 2000, the first crystal 00:03:48.290 --> 00:03:51.890 structure of the 50S subunit was reported, 00:03:51.890 --> 00:03:54.690 and since then, there's been a flurry of activity. 00:03:54.690 --> 00:03:57.990 So in 2001, first crystal structure of the 30 subunit-- 00:03:57.990 --> 00:04:02.690 30S subunit with mRNA bound, and in this time, 00:04:02.690 --> 00:04:06.290 too, single molecule spectroscopy 00:04:06.290 --> 00:04:08.960 was well on its way, and so there were studies 00:04:08.960 --> 00:04:11.960 beginning of ribosome dynamics. 00:04:11.960 --> 00:04:15.050 And later, 2011, we have a crystal structure 00:04:15.050 --> 00:04:18.950 of a eukaryotic 60S subunit here. 00:04:18.950 --> 00:04:21.170 And so we're going to focus our discussions 00:04:21.170 --> 00:04:24.110 on the prokaryotic ribosome, their similarities 00:04:24.110 --> 00:04:26.570 and differences between prokaryotic ribosomes 00:04:26.570 --> 00:04:28.910 and eukaryotic ribosomes, just to keep 00:04:28.910 --> 00:04:31.430 in mind if you've heard about eukaryotic ribosomes 00:04:31.430 --> 00:04:32.180 in other classes. 00:04:35.100 --> 00:04:39.740 So also to note, in 2009, the Nobel Prize in chemistry 00:04:39.740 --> 00:04:41.930 was awarded for structural studies 00:04:41.930 --> 00:04:46.580 of the ribosome to these three researchers here. 00:04:46.580 --> 00:04:48.560 And their contributions are shown 00:04:48.560 --> 00:04:52.820 ranging from basically the first low quality crystals of the 50S 00:04:52.820 --> 00:04:56.990 ribosomal subunit to understand how important that was, 00:04:56.990 --> 00:04:59.000 to the first crystal structures. 00:04:59.000 --> 00:05:01.320 And something Professor Stubbe and I 00:05:01.320 --> 00:05:04.100 like to remind everyone and keep in mind is, 00:05:04.100 --> 00:05:07.130 with these types of problems and areas, 00:05:07.130 --> 00:05:09.980 there's often many contributors, and they can't all 00:05:09.980 --> 00:05:12.710 be recognized by this prize because it's 00:05:12.710 --> 00:05:15.470 limited to three individuals at maximum. 00:05:15.470 --> 00:05:18.620 And so other folks like Harry Noller, Peter Moore, 00:05:18.620 --> 00:05:21.320 and Joaquim Frank made really seminal contributions 00:05:21.320 --> 00:05:26.810 to our understanding of this macromolecular machine. 00:05:26.810 --> 00:05:28.700 So what are the questions we're going 00:05:28.700 --> 00:05:31.462 to address in this module? 00:05:31.462 --> 00:05:33.170 And then we'll go over some of the basics 00:05:33.170 --> 00:05:35.370 in ribosome structure. 00:05:35.370 --> 00:05:38.390 So first, one is that we learn from structural studies 00:05:38.390 --> 00:05:40.880 of the ribosome, and really, what 00:05:40.880 --> 00:05:43.730 does ribosome structure at an atomic level 00:05:43.730 --> 00:05:46.160 tell us about its function? 00:05:46.160 --> 00:05:51.260 How does the ribosome recognize, bind, and decode mRNA? 00:05:51.260 --> 00:05:54.620 How are amino acids recognized and delivered, 00:05:54.620 --> 00:05:57.410 and how is the correct amino acid delivered? 00:05:57.410 --> 00:05:59.480 The genetic message needs to be read, 00:05:59.480 --> 00:06:01.910 and it needs to be read properly. 00:06:01.910 --> 00:06:05.330 And what happens if a wrong amino acid is delivered? 00:06:05.330 --> 00:06:06.620 So that's a possibility. 00:06:06.620 --> 00:06:08.660 How does the ribosome cope? 00:06:08.660 --> 00:06:11.270 So this brings up the notion of fidelity. 00:06:11.270 --> 00:06:14.000 How is fidelity of translation maintained? 00:06:14.000 --> 00:06:17.150 And we'll address that next week. 00:06:17.150 --> 00:06:20.180 How is translation initiated? 00:06:20.180 --> 00:06:23.420 How does the ribosome catalyze peptide bond formation? 00:06:23.420 --> 00:06:26.990 So we're interested in that mechanism within the context 00:06:26.990 --> 00:06:28.850 of this course. 00:06:28.850 --> 00:06:31.250 How does the polypeptide leave the ribosome, 00:06:31.250 --> 00:06:34.400 and what happens to that polypeptide after it exits? 00:06:34.400 --> 00:06:36.020 So that will be a transition for us 00:06:36.020 --> 00:06:39.470 into module 2 on protein folding. 00:06:39.470 --> 00:06:42.290 How is translation terminated, and what 00:06:42.290 --> 00:06:44.490 happens to the ribosome after? 00:06:44.490 --> 00:06:47.090 So a given polypeptide chain is made. 00:06:47.090 --> 00:06:49.370 What happens after that? 00:06:49.370 --> 00:06:51.080 And where we'll close this module 00:06:51.080 --> 00:06:54.050 is thinking about how our understanding of the ribosome, 00:06:54.050 --> 00:06:56.810 from all of these basic and fundamental studies, 00:06:56.810 --> 00:06:59.700 allows for the development of new technologies. 00:06:59.700 --> 00:07:01.460 And we'll specifically think about how 00:07:01.460 --> 00:07:03.740 it's possible to use the ribosome 00:07:03.740 --> 00:07:08.170 to incorporate unnatural amino acids into proteins. 00:07:08.170 --> 00:07:09.920 So where we're going to move forward today 00:07:09.920 --> 00:07:14.150 is really structure-- focusing on ribosome structure 00:07:14.150 --> 00:07:16.700 and a general overview of translation, 00:07:16.700 --> 00:07:20.360 basically to have everyone here up to speed for the discussions 00:07:20.360 --> 00:07:22.190 to come next week. 00:07:22.190 --> 00:07:26.360 So first of all, we'll do an overview of key players 00:07:26.360 --> 00:07:29.302 in translation, a brief look at the cycle, 00:07:29.302 --> 00:07:31.010 and then we'll go into structural studies 00:07:31.010 --> 00:07:31.880 of the ribosome. 00:07:46.110 --> 00:07:48.360 And within this set of lecture notes 00:07:48.360 --> 00:07:50.580 are several tables that have lists 00:07:50.580 --> 00:07:53.700 of the players and detailed overall cycle 00:07:53.700 --> 00:07:55.530 that I encourage you to use, just as 00:07:55.530 --> 00:07:58.770 a reference throughout this module for keeping everything 00:07:58.770 --> 00:08:00.150 straight. 00:08:00.150 --> 00:08:03.300 So first, of course, we have the ribosome. 00:08:07.770 --> 00:08:16.785 So the ribosome, as we all know, reads the genetic code 00:08:16.785 --> 00:08:23.880 via the mRNA, and it catalyzes peptide bond formation. 00:08:39.950 --> 00:08:44.449 So in addition to the ribosome, we have the mRNA. 00:08:48.550 --> 00:09:08.070 So this mRNA delivers the genetic code to the ribosome, 00:09:08.070 --> 00:09:09.690 and it provides a template-- 00:09:16.382 --> 00:09:18.310 [AUDIENCE MEMBER SNEEZES] 00:09:18.310 --> 00:09:20.260 Bless you-- for protein synthesis. 00:09:27.380 --> 00:09:34.310 So effectively, we can think about this process 00:09:34.310 --> 00:09:37.280 as a template-driven polymerization. 00:09:44.010 --> 00:09:48.390 So somehow, the amino acids need to get to the ribosome, 00:09:48.390 --> 00:09:50.550 and so we need the help of the tRNAs. 00:09:57.920 --> 00:10:11.130 So these transfer RNAs deliver the amino acid monomers, 00:10:11.130 --> 00:10:25.660 to the ribosome, and they transfer the amino acids 00:10:25.660 --> 00:10:28.690 during synthesis of the polypeptide. 00:10:39.350 --> 00:10:43.730 So in addition to the ribosome, the mRNA, and the tRNAs, 00:10:43.730 --> 00:10:48.740 the ribosome needs some help, so we have translation factors. 00:10:48.740 --> 00:10:51.140 And there's translation factors that 00:10:51.140 --> 00:10:56.030 are involved in each step of the translation cycle. 00:10:56.030 --> 00:10:59.270 So these are proteins that are required at specific points 00:10:59.270 --> 00:11:00.790 during the translation process. 00:11:37.670 --> 00:11:41.150 And so in terms of translation factors, 00:11:41.150 --> 00:11:43.760 we can break the process of translation 00:11:43.760 --> 00:11:45.980 into three or four steps-- 00:11:45.980 --> 00:11:50.970 I prefer three-- which are initiation, elongation, 00:11:50.970 --> 00:11:53.580 and termination. 00:11:53.580 --> 00:11:56.450 Some review articles and papers will divide this 00:11:56.450 --> 00:11:59.390 into four steps, because termination, 00:11:59.390 --> 00:12:01.340 you can think about peptide release 00:12:01.340 --> 00:12:03.650 and then ribosome recycling. 00:12:03.650 --> 00:12:08.090 But regardless to that detail, at each of these stages, 00:12:08.090 --> 00:12:10.400 there are translation factors that help. 00:12:10.400 --> 00:12:18.980 So we have initiation factors that 00:12:18.980 --> 00:12:23.000 help with the process of initiation, and in prokaryotes, 00:12:23.000 --> 00:12:27.080 we have initiation factors 1, 2, and 3, 00:12:27.080 --> 00:12:31.530 so 3 translation factors that help during elongation. 00:12:31.530 --> 00:12:34.820 So the process of making the peptide bond-- 00:12:34.820 --> 00:12:38.820 there are elongation factors. 00:12:38.820 --> 00:12:41.580 EF for Elongation Factor. 00:12:41.580 --> 00:12:43.700 IF for Initiation Factor. 00:12:43.700 --> 00:12:52.310 We have EF-Tu, EF-G, and others, and we'll 00:12:52.310 --> 00:12:56.840 spend quite a bit of time thinking about EF-Tu and EF-G 00:12:56.840 --> 00:12:59.930 over the course of the next week and in recitation 00:12:59.930 --> 00:13:02.090 and in problem sets, thinking about how 00:13:02.090 --> 00:13:05.090 these factors are really facilitating the elongation 00:13:05.090 --> 00:13:07.420 process here. 00:13:07.420 --> 00:13:15.810 And during termination, there are release factors, 00:13:15.810 --> 00:13:21.410 so we have release factors 1, 2, and 3. 00:13:21.410 --> 00:13:22.370 And we can also-- 00:13:22.370 --> 00:13:25.250 these are involved in release of the polypeptide that's 00:13:25.250 --> 00:13:27.800 been synthesized from the ribosome, 00:13:27.800 --> 00:13:29.390 and there's other players as well 00:13:29.390 --> 00:13:33.560 that I'll list here, including ribosome recycling 00:13:33.560 --> 00:13:40.070 factor, so the subunits get recycled, as we'll see. 00:13:40.070 --> 00:13:43.730 And we can also include a protein called trigger factor 00:13:43.730 --> 00:13:45.140 here. 00:13:45.140 --> 00:13:48.140 That is involved in folding of nascent polypeptide 00:13:48.140 --> 00:13:51.770 chains or the polypeptide chain as it's 00:13:51.770 --> 00:13:53.120 coming off the ribosome. 00:13:58.220 --> 00:14:02.270 And then just to summarize in terms 00:14:02.270 --> 00:14:10.180 of three stages of translation as I'll present them 00:14:10.180 --> 00:14:18.430 to you within this course, we have the initiation process; 00:14:18.430 --> 00:14:27.430 two, elongation; and three, termination. 00:14:30.800 --> 00:14:34.580 And where we'll be focusing the lectures next week, and really, 00:14:34.580 --> 00:14:41.340 this whole module, is here on elongation. 00:14:41.340 --> 00:14:43.730 And I'll just note that the elongation 00:14:43.730 --> 00:14:47.030 cycle is highly conserved. 00:14:47.030 --> 00:14:50.750 Termination and initiation vary quite a bit between prokaryotic 00:14:50.750 --> 00:14:54.830 and eukaryotes there in terms of the processes 00:14:54.830 --> 00:14:57.890 and involved players. 00:14:57.890 --> 00:15:01.070 So where are we going? 00:15:01.070 --> 00:15:03.710 Just as a brief overview of the cycle, 00:15:03.710 --> 00:15:07.020 and we'll come back to this later within today's lecture, 00:15:07.020 --> 00:15:08.750 or if not, on Monday. 00:15:08.750 --> 00:15:10.910 So we start with initiation, and we're 00:15:10.910 --> 00:15:13.310 going to have to ask ourselves, how 00:15:13.310 --> 00:15:18.450 is it that this 70S prokaryotic ribosome or initiation complex 00:15:18.450 --> 00:15:19.730 is assembled? 00:15:19.730 --> 00:15:23.510 And so there's a special tRNA involved, the initiator tRNA 00:15:23.510 --> 00:15:25.430 that we see binds here, and we'll 00:15:25.430 --> 00:15:29.150 talk more about these E, P, and A-sites in a moment. 00:15:29.150 --> 00:15:32.360 So we see the ribosome is assembled, the mRNA is bound, 00:15:32.360 --> 00:15:35.630 and there's an initiator tRNA bound. 00:15:35.630 --> 00:15:40.730 In order for the elongation cycle to be entered, 00:15:40.730 --> 00:15:43.170 an amino acid needs to be delivered, 00:15:43.170 --> 00:15:46.670 and that's delivered by an aminoacyl tRNA. 00:15:46.670 --> 00:15:50.730 That's in a ternary complex, so three components. 00:15:50.730 --> 00:15:56.270 We have the tRNA, the elongation factor Tu, and GTP. 00:15:56.270 --> 00:16:01.040 So this complex here somehow delivers an aminoacyl tRNA 00:16:01.040 --> 00:16:03.350 to the ribosome, and we're going to look 00:16:03.350 --> 00:16:05.800 at this process in detail next week. 00:16:05.800 --> 00:16:07.460 So this will be one of our case studies 00:16:07.460 --> 00:16:10.910 thinking about experiments and how experiments have supported 00:16:10.910 --> 00:16:14.090 a specific kinetic model here. 00:16:14.090 --> 00:16:19.400 So here, we have a complex where the tRNA 00:16:19.400 --> 00:16:21.680 is ready to occupy the A-site. 00:16:21.680 --> 00:16:25.310 What happens here-- we see that there's a GTP hydrolysis event. 00:16:25.310 --> 00:16:27.650 We'll talk about more as we go forward. 00:16:27.650 --> 00:16:30.320 Peptide transfer reaction-- so we 00:16:30.320 --> 00:16:32.600 have formation of a peptide bond, 00:16:32.600 --> 00:16:35.330 and then this elongation factor G 00:16:35.330 --> 00:16:38.930 comes in to facilitate the elongation cycle. 00:16:38.930 --> 00:16:42.950 And then this cycle will continue until some point that 00:16:42.950 --> 00:16:45.590 signals to stop synthesis, so a stop codon 00:16:45.590 --> 00:16:47.340 will enter the A-site. 00:16:47.340 --> 00:16:50.840 And there's a termination process, ribosome recycling, 00:16:50.840 --> 00:16:54.920 and you can imagine this whole cycle happening again. 00:16:54.920 --> 00:16:58.100 So how do we get to this cartoon to some more detailed 00:16:58.100 --> 00:16:59.060 understanding? 00:16:59.060 --> 00:17:00.390 That's where we're going. 00:17:00.390 --> 00:17:03.710 So come back to this cartoon at various stages 00:17:03.710 --> 00:17:05.940 throughout the course. 00:17:05.940 --> 00:17:14.420 So first, we'll do a cartoon overview of the prokaryotic 70S 00:17:14.420 --> 00:17:16.500 ribosome, and then we're going to look 00:17:16.500 --> 00:17:20.010 at some of the data from crystallography studies here. 00:17:34.660 --> 00:17:37.690 So as I think we all know the ribosome is comprised 00:17:37.690 --> 00:17:57.310 of RNA and proteins, and by mass, it's about 66% 00:17:57.310 --> 00:18:01.390 RNA and about 34% protein. 00:18:05.360 --> 00:18:09.310 And it's comprised of two subunits, 00:18:09.310 --> 00:18:12.655 and those are indicated in the cartoon by different colors. 00:18:23.150 --> 00:18:28.995 So in prokaryotes, we have the 50S, 00:18:28.995 --> 00:18:30.120 which is the large subunit. 00:18:33.710 --> 00:18:41.120 This is made up of 23S ribosomal RNA, a piece of 5S 00:18:41.120 --> 00:18:44.375 ribosomal RNA, and proteins. 00:18:48.200 --> 00:18:51.290 In terms of size this is huge, so it's 00:18:51.290 --> 00:18:53.580 approximately 1.5 megadaltons. 00:19:00.300 --> 00:19:02.820 And what we find within this subunit 00:19:02.820 --> 00:19:06.030 is the catalytic center, or peptidyl transferase center. 00:19:06.030 --> 00:19:08.085 This is sometimes abbreviated as PTC. 00:19:18.690 --> 00:19:22.380 And what we also find in the 50S subunit 00:19:22.380 --> 00:19:24.480 are three sites for tRNA binding. 00:19:55.500 --> 00:19:59.135 And so the other subunit in prokaryotes is the 30S. 00:20:04.620 --> 00:20:05.715 This is a small subunit. 00:20:11.590 --> 00:20:21.480 It's comprised of 16S rRNA and proteins, 00:20:21.480 --> 00:20:22.930 and it's also quite large. 00:20:22.930 --> 00:20:31.000 Just smaller than the 50S, so on the order of 0.8 megadaltons. 00:20:31.000 --> 00:20:34.750 And in terms of function, what we have in the 30S 00:20:34.750 --> 00:20:41.065 is the decoding center, so for decoding the mRNA, 00:20:41.065 --> 00:20:43.315 and the site of mRNA binding. 00:20:47.520 --> 00:20:51.590 So if we draw this in cartoon form-- 00:20:51.590 --> 00:20:55.220 and this is something I really encourage you all 00:20:55.220 --> 00:20:58.710 to do when thinking about the experiments and the problem 00:20:58.710 --> 00:21:00.710 sets because that's going to help you understand 00:21:00.710 --> 00:21:04.580 the experimental design and what actually happened. 00:21:04.580 --> 00:21:10.940 Here, what we have on top is the 50S subunit. 00:21:10.940 --> 00:21:14.090 On bottom, the 30S subunit. 00:21:14.090 --> 00:21:19.400 We have the mRNA, and note the directionality, so 5 prime end, 00:21:19.400 --> 00:21:22.820 5 prime end of the ribose, 3 prime end here. 00:21:22.820 --> 00:21:25.580 And then within this 50S, we can think 00:21:25.580 --> 00:21:31.640 about these sites for tRNA binding ordered as such, 00:21:31.640 --> 00:21:38.540 so E, P, and A here, so this is the catalytic center 00:21:38.540 --> 00:21:40.470 or peptidyl transferase center here. 00:21:43.250 --> 00:21:47.360 So overall, this assembled ribosome 00:21:47.360 --> 00:21:50.240 is on the order of 2.3 megadaltons 00:21:50.240 --> 00:21:53.780 and is about 200 Angstroms in diameter. 00:21:53.780 --> 00:21:58.400 So just in terms of these names, 50S, 30S-- 00:21:58.400 --> 00:22:03.600 this is overall the 70S assembled ribosome. 00:22:03.600 --> 00:22:05.140 What do these numbers-- 00:22:05.140 --> 00:22:06.140 where do they come from? 00:22:06.140 --> 00:22:10.050 What does this 50S, 30S, 70S mean? 00:22:17.250 --> 00:22:18.886 So what is the S? 00:22:18.886 --> 00:22:20.284 AUDIENCE: It's [INAUDIBLE]. 00:22:20.284 --> 00:22:24.012 It has to do with the sedimentation [INAUDIBLE].. 00:22:24.012 --> 00:22:26.910 ELIZABETH NOLAN: Yeah, it has to do with the sedimentation. 00:22:26.910 --> 00:22:30.480 So there's a type of experiment called analytical ultra 00:22:30.480 --> 00:22:32.940 centrifugation, and effectively, you 00:22:32.940 --> 00:22:37.350 can use this to ask about the sedimentation of a biomolecule. 00:22:37.350 --> 00:22:41.610 So effectively, what is the rate at which a biomolecule moves 00:22:41.610 --> 00:22:46.260 in response to the centrifugal force in a centrifuge there? 00:22:46.260 --> 00:22:50.160 And so you can use optics to monitor the sedimentation 00:22:50.160 --> 00:22:53.190 and use mathematics to fit those data to come up 00:22:53.190 --> 00:22:55.080 with an S value. 00:22:55.080 --> 00:23:00.240 So typically, the larger the S value, the larger the size. 00:23:00.240 --> 00:23:02.910 It's not always directly proportional to the mass 00:23:02.910 --> 00:23:05.580 because things like shape play a role as well, 00:23:05.580 --> 00:23:09.270 but effectively, we see 50S, and that subunit 00:23:09.270 --> 00:23:11.250 is larger than the 30S. 00:23:11.250 --> 00:23:14.460 And note, when they come together, it's not additive. 00:23:14.460 --> 00:23:18.420 It's 70S there, if you're to look at the assembled ribosome 00:23:18.420 --> 00:23:20.680 in one of these experiments. 00:23:20.680 --> 00:23:25.320 So that's where those values come from here. 00:23:25.320 --> 00:23:29.390 So if we take a look from my cartoon depiction 00:23:29.390 --> 00:23:33.120 to actual image from cryoelectron microscopy-- 00:23:33.120 --> 00:23:37.230 so this is just rotated basically 90 degrees. 00:23:37.230 --> 00:23:39.060 What do we see? 00:23:39.060 --> 00:23:42.240 We have the 50S here, the catalytic center. 00:23:42.240 --> 00:23:43.890 We have the 30S. 00:23:43.890 --> 00:23:46.980 Here's the mRNA, and what we're seeing 00:23:46.980 --> 00:23:48.930 is that, in this particular structure, 00:23:48.930 --> 00:23:51.690 there's some tRNAs bound, and they've indicated also 00:23:51.690 --> 00:23:55.260 a ribosomal protein here. 00:23:55.260 --> 00:23:57.520 Just as a sense of complexity-- 00:23:57.520 --> 00:24:03.390 so in E. coli, the 50S subunit has over 30 proteins 00:24:03.390 --> 00:24:05.190 associated with it. 00:24:05.190 --> 00:24:06.450 That's a lot-- 00:24:06.450 --> 00:24:07.860 30 different proteins. 00:24:07.860 --> 00:24:13.230 And the 30S has 21 ribosomal proteins associated with it, 00:24:13.230 --> 00:24:16.530 so we need to think about the proteins in addition 00:24:16.530 --> 00:24:19.660 to the RNA. 00:24:19.660 --> 00:24:24.780 So let's take a look at an image from the crystal 00:24:24.780 --> 00:24:27.990 structure reported in 2000, of the 50S 00:24:27.990 --> 00:24:32.520 ribosome from a particular prokaryote shown here. 00:24:32.520 --> 00:24:36.910 So this is what's described as the crown view, 00:24:36.910 --> 00:24:39.870 and in this particular depiction, what we're seeing 00:24:39.870 --> 00:24:45.450 is that the ribosomal RNA of the 50S is in gray or white, 00:24:45.450 --> 00:24:51.040 and the ribosomal proteins that are bound are in gold. 00:24:51.040 --> 00:24:55.140 So taking a look at this, what do we see? 00:24:55.140 --> 00:24:59.380 We can ask ourselves some questions from this structure. 00:24:59.380 --> 00:25:03.050 So the first question I'll ask is about the RNA. 00:25:03.050 --> 00:25:05.970 What does this RNA look like? 00:25:05.970 --> 00:25:10.550 So do we see any obvious domains? 00:25:10.550 --> 00:25:13.650 If anyone has some experience looking at structures. 00:25:18.760 --> 00:25:19.840 I don't see any. 00:25:19.840 --> 00:25:23.860 What I see is a compact mass of RNA here. 00:25:23.860 --> 00:25:27.460 There's not obvious domains or regions that 00:25:27.460 --> 00:25:30.580 are somehow different here. 00:25:30.580 --> 00:25:35.620 To me, in this structure, it looks like one big glob of RNA. 00:25:35.620 --> 00:25:38.440 But then the question is, is that truly the case, 00:25:38.440 --> 00:25:40.120 or is there an organization we're just 00:25:40.120 --> 00:25:42.880 not seeing at this level? 00:25:42.880 --> 00:25:47.170 The next question we can ask is where are the proteins? 00:25:47.170 --> 00:25:49.930 So if we look at the proteins and how 00:25:49.930 --> 00:25:55.450 they're arranged on this compact mass of RNA, what do we see? 00:25:55.450 --> 00:25:56.410 Where are they? 00:25:59.637 --> 00:26:00.560 AUDIENCE: The edges? 00:26:00.560 --> 00:26:02.060 ELIZABETH NOLAN: On the edges, yeah. 00:26:02.060 --> 00:26:04.520 There's many on the edges, like L1 here, 00:26:04.520 --> 00:26:08.150 this one on the outside here, over here. 00:26:08.150 --> 00:26:11.480 So it looks like these proteins, at least in this view, 00:26:11.480 --> 00:26:14.900 are mostly on the outside. 00:26:14.900 --> 00:26:18.530 Is there anything unusual or potentially unusual 00:26:18.530 --> 00:26:20.795 we can see in addition about these proteins? 00:26:25.710 --> 00:26:28.340 Maybe looking at this one here or here. 00:26:28.340 --> 00:26:29.280 What's going on? 00:26:35.442 --> 00:26:40.220 AUDIENCE: I can't see very well, but I think that there's 00:26:40.220 --> 00:26:42.202 not just [INAUDIBLE]. 00:26:45.052 --> 00:26:47.510 ELIZABETH NOLAN: It looks like there's some unfolded parts? 00:26:47.510 --> 00:26:48.135 AUDIENCE: Yeah. 00:26:48.135 --> 00:26:51.020 ELIZABETH NOLAN: Right, so look here. 00:26:51.020 --> 00:26:53.240 So it looks like there's some unfolded regions 00:26:53.240 --> 00:26:54.005 to these proteins. 00:26:56.510 --> 00:26:58.010 And why is that? 00:26:58.010 --> 00:27:01.190 And where are these unfolded regions going? 00:27:01.190 --> 00:27:02.960 So what we can do is look at the RNA 00:27:02.960 --> 00:27:05.750 separately and look at the protein separately now 00:27:05.750 --> 00:27:09.320 and see what we learn from these analyses. 00:27:09.320 --> 00:27:17.270 So effectively, if we consider the 23S rRNA, 00:27:17.270 --> 00:27:19.850 despite that structure we saw before that 00:27:19.850 --> 00:27:24.290 looked like a compact mass of RNA, it's structured, 00:27:24.290 --> 00:27:27.680 and it consists of six domains. 00:27:27.680 --> 00:27:31.340 And these domains have quite complicated shapes, 00:27:31.340 --> 00:27:33.170 and they fit together. 00:27:33.170 --> 00:27:38.490 And here is just a schematic diagram of this structure. 00:27:38.490 --> 00:27:42.110 So if we take a look, we can see that there's domain 1, 00:27:42.110 --> 00:27:46.940 domain 2, 3, 4, 5, and 6. 00:27:46.940 --> 00:27:49.370 And on the left here, it's indicated 00:27:49.370 --> 00:27:53.420 where, in that crown view we just looked at, right here, 00:27:53.420 --> 00:27:56.240 these domains are located. 00:27:56.240 --> 00:27:59.450 So there is organization, even though in that structure, 00:27:59.450 --> 00:28:04.020 it looks like one compact mass of RNA. 00:28:04.020 --> 00:28:06.860 So let's think about these proteins a bit more. 00:28:06.860 --> 00:28:10.220 And in addition to the crown view and the observations 00:28:10.220 --> 00:28:13.640 we had from this particular face of the ribosome, 00:28:13.640 --> 00:28:16.370 where it looks like many proteins are on the outside, 00:28:16.370 --> 00:28:18.530 and there's some unfolded regions, what 00:28:18.530 --> 00:28:20.570 happens if we look elsewhere? 00:28:20.570 --> 00:28:25.700 So here, we have rotation, so 180 degrees from here, 00:28:25.700 --> 00:28:28.490 effectively looking, we can say, on the backside. 00:28:28.490 --> 00:28:30.230 And here, we can look at the view 00:28:30.230 --> 00:28:33.350 from the bottom of this subunit. 00:28:33.350 --> 00:28:36.800 So what do these images suggest? 00:28:36.800 --> 00:28:40.820 Do they support what we were thinking from this one view 00:28:40.820 --> 00:28:43.955 here, that proteins are mostly on the outside? 00:28:48.350 --> 00:28:51.110 Yeah, I see some shaking heads "yes." 00:28:51.110 --> 00:28:53.330 It looks like the surface of this 50S 00:28:53.330 --> 00:28:57.710 is covered, effectively, by a protein lattice here. 00:28:57.710 --> 00:29:01.860 So what might a role be for these proteins, 00:29:01.860 --> 00:29:02.840 an important role? 00:29:05.750 --> 00:29:06.720 AUDIENCE: Structural? 00:29:06.720 --> 00:29:09.700 ELIZABETH NOLAN: Yeah, so some structural role. 00:29:09.700 --> 00:29:13.180 So these proteins can help with stabilizing 00:29:13.180 --> 00:29:16.920 this 3D structure of the RNA. 00:29:16.920 --> 00:29:20.140 And they have other functions as well, and some of those 00:29:20.140 --> 00:29:23.350 will come up as we discuss this elongation cycle. 00:29:23.350 --> 00:29:28.030 But one function is certainly structural. 00:29:28.030 --> 00:29:30.490 If we just think about the distribution 00:29:30.490 --> 00:29:34.030 of the proteins along the surface of this 50S, 00:29:34.030 --> 00:29:36.010 it looks more or less uniform. 00:29:36.010 --> 00:29:38.648 There aren't patches where there's no protein or patches 00:29:38.648 --> 00:29:39.940 where there's a lot of protein. 00:29:39.940 --> 00:29:43.360 They're pretty much evenly distributed here. 00:29:43.360 --> 00:29:47.410 So as it turns out, most of the segments of the 23S 00:29:47.410 --> 00:29:51.370 do interact with protein, and if we look at these proteins 00:29:51.370 --> 00:29:54.280 more closely, we're going to follow up on the observation 00:29:54.280 --> 00:29:58.010 that it looks like they have some unfolded regions. 00:29:58.010 --> 00:29:59.920 So what we're looking at here are just 00:29:59.920 --> 00:30:05.410 a selection of the 50S proteins in the absence of the RNA. 00:30:05.410 --> 00:30:07.750 So these structures have been taken out 00:30:07.750 --> 00:30:09.610 of that total structure. 00:30:09.610 --> 00:30:13.030 In terms of nomenclature, l means large 00:30:13.030 --> 00:30:15.250 and s means small, in terms of thinking 00:30:15.250 --> 00:30:17.440 about ribosomal proteins. 00:30:17.440 --> 00:30:20.470 And so what's found in the 50S is 00:30:20.470 --> 00:30:26.740 that we can categorize 17 of the proteins as globular or folded 00:30:26.740 --> 00:30:31.130 and 13 of the proteins as cases where 00:30:31.130 --> 00:30:34.360 there's extensions that are non-globular 00:30:34.360 --> 00:30:36.220 or have no clear structure. 00:30:36.220 --> 00:30:39.340 And that's color coded in these examples, where 00:30:39.340 --> 00:30:44.080 we have folded regions in green and then unfolded regions 00:30:44.080 --> 00:30:46.360 in red. 00:30:46.360 --> 00:30:51.850 So why is this, and where are these red extensions going? 00:30:51.850 --> 00:30:55.510 So what's seen is that these non-globular extensions work 00:30:55.510 --> 00:30:59.350 their way into the interior of the ribosome, 00:30:59.350 --> 00:31:01.780 so we can think about them kind of like tentacles, 00:31:01.780 --> 00:31:05.200 for instance, going into the interior. 00:31:05.200 --> 00:31:07.150 So how might they interact with the RNA? 00:31:14.120 --> 00:31:15.560 So I'll give you a hint. 00:31:15.560 --> 00:31:19.460 In these regions in red, there are quite a number 00:31:19.460 --> 00:31:23.165 of arginine and lysine residues compared to other regions. 00:31:26.540 --> 00:31:29.945 So what properties of arginine or lysine would be important? 00:31:29.945 --> 00:31:30.820 AUDIENCE: [INAUDIBLE] 00:31:30.820 --> 00:31:32.195 ELIZABETH NOLAN: Positive charge. 00:31:32.195 --> 00:31:34.600 Right, we have positively charged amino acids. 00:31:34.600 --> 00:31:36.010 What about PKAs? 00:31:36.010 --> 00:31:39.280 So who votes for arginine having a higher PKA than lysine? 00:31:42.190 --> 00:31:43.645 The opposite? 00:31:47.050 --> 00:31:48.820 So that's a point for review. 00:31:48.820 --> 00:31:50.440 Lysine around 10.5. 00:31:50.440 --> 00:31:51.850 arginine around 12.5. 00:31:51.850 --> 00:31:55.030 arginine's higher here. 00:31:55.030 --> 00:31:58.270 So if we have a bunch of positively charged residues 00:31:58.270 --> 00:31:59.680 in these extensions, how are they 00:31:59.680 --> 00:32:02.800 going to interact with the rRNA? 00:32:02.800 --> 00:32:05.620 What are the molecular features there that are important? 00:32:05.620 --> 00:32:06.920 AUDIENCE: [INAUDIBLE] 00:32:06.920 --> 00:32:07.920 ELIZABETH NOLAN: Pardon? 00:32:07.920 --> 00:32:08.090 AUDIENCE: Phosphates? 00:32:08.090 --> 00:32:09.880 ELIZABETH NOLAN: Yeah, the phosphate backbones. 00:32:09.880 --> 00:32:12.280 So we have the negatively charged phosphates, positively 00:32:12.280 --> 00:32:13.480 charged amino acids-- 00:32:13.480 --> 00:32:18.380 effectively formation of salt bridges here. 00:32:18.380 --> 00:32:23.230 AUDIENCE: So I know structure for a lot of these, well, 00:32:23.230 --> 00:32:24.460 non-globular regions. 00:32:24.460 --> 00:32:27.010 Does it mean that they're more disordered, 00:32:27.010 --> 00:32:31.820 or do they still have relatively similar B factor 00:32:31.820 --> 00:32:34.390 compared to the rest of the globular region? 00:32:34.390 --> 00:32:37.173 It's just that they don't fall under [INAUDIBLE]---- 00:32:37.173 --> 00:32:39.340 ELIZABETH NOLAN: I don't know what the B factors are 00:32:39.340 --> 00:32:41.470 for the different regions of these proteins, 00:32:41.470 --> 00:32:43.660 and for the case of discussion here, I 00:32:43.660 --> 00:32:46.390 would have it fall under a lack of secondary structure. 00:32:46.390 --> 00:32:49.240 And keep in mind, the ribosome is quite dynamic, 00:32:49.240 --> 00:32:52.942 and in isolation, are all the proteins there 00:32:52.942 --> 00:32:55.150 and in their native way or not is just something else 00:32:55.150 --> 00:32:55.880 to keep in mind. 00:32:55.880 --> 00:32:58.920 But these are certainly lacking a fold 00:32:58.920 --> 00:33:03.100 and going into the interior and working from salt bridges here. 00:33:06.660 --> 00:33:12.530 Here's just an example of the 50S with tRNAs bound. 00:33:12.530 --> 00:33:14.970 So we have the 50S. 00:33:14.970 --> 00:33:20.160 We see tRNA in the E-site, the P-site, and the A-site. 00:33:20.160 --> 00:33:23.490 And so what are these three sites? 00:33:23.490 --> 00:33:27.970 Effectively, their names indicate what they bind 00:33:27.970 --> 00:33:31.080 or what they do in terms of these letters. 00:33:31.080 --> 00:33:37.140 The A-site binds aminoacyl tRNAs with the exception of initiator 00:33:37.140 --> 00:33:41.310 tRNA, which cannot bind to the A-site. 00:33:41.310 --> 00:33:44.340 The P-site binds the initiator tRNA 00:33:44.340 --> 00:33:47.460 during the initiation process of translation, 00:33:47.460 --> 00:33:51.750 and then it also binds peptydil tRNAs, so effectively 00:33:51.750 --> 00:33:55.890 the tRNA that has the growing peptide chain attached. 00:33:55.890 --> 00:34:00.720 And then the E-site binds the DA slated tRNA, 00:34:00.720 --> 00:34:03.510 and this is called the E-site because it's the exit site. 00:34:03.510 --> 00:34:07.800 And eventually, this tRNA that has lost its amino acid 00:34:07.800 --> 00:34:10.039 needs to get kicked out of the ribosome. 00:34:12.739 --> 00:34:18.260 So one more point-- just going back about these proteins 00:34:18.260 --> 00:34:21.440 to highlight. 00:34:21.440 --> 00:34:24.860 We stated that these proteins are mostly on the exterior, 00:34:24.860 --> 00:34:27.320 and there's just these extensions that go in. 00:34:27.320 --> 00:34:29.870 One thing I didn't explicitly say 00:34:29.870 --> 00:34:31.909 is that this peptidyl transferase 00:34:31.909 --> 00:34:34.730 center is devoid of protein. 00:34:34.730 --> 00:34:37.040 So in this catalytic center that's 00:34:37.040 --> 00:34:42.050 responsible for peptide bond formation, there's no protein. 00:34:42.050 --> 00:34:45.080 So based on all of the structural evidence, 00:34:45.080 --> 00:34:49.100 the nearest protein is 18 Angstroms away. 00:34:49.100 --> 00:34:53.239 That's quite far when thinking about making a peptide bond 00:34:53.239 --> 00:34:55.370 in a catalytic center. 00:34:55.370 --> 00:34:58.340 And also we'll learn that magnesium ions 00:34:58.340 --> 00:35:00.620 are important for ribosome assembly. 00:35:00.620 --> 00:35:03.230 I'll just point out that the closest magnesium 00:35:03.230 --> 00:35:06.320 ion is 8 Angstroms away. 00:35:06.320 --> 00:35:10.760 So if there's no protein in this catalytic center that's 00:35:10.760 --> 00:35:13.910 responsible for formation of peptide bonds in this growing 00:35:13.910 --> 00:35:16.040 polypeptide chain, what does that 00:35:16.040 --> 00:35:20.650 tell us right off the bat about the ribosome and catalysis? 00:35:25.033 --> 00:35:26.440 AUDIENCE: [INAUDIBLE] 00:35:26.440 --> 00:35:27.440 ELIZABETH NOLAN: Pardon? 00:35:27.440 --> 00:35:29.273 AUDIENCE: You have many functional component 00:35:29.273 --> 00:35:32.030 of [INAUDIBLE] ribozymes. 00:35:32.030 --> 00:35:33.680 ELIZABETH NOLAN: Yeah, so the ribo-- 00:35:33.680 --> 00:35:36.100 the ribosome is a ribozyme, yes. 00:35:36.100 --> 00:35:38.590 So there's many functional components, 00:35:38.590 --> 00:35:41.320 but in terms of peptide bond formation, 00:35:41.320 --> 00:35:44.890 it's the RNA that's catalyzing that reaction. 00:35:44.890 --> 00:35:49.510 So it's a ribozyme, or an RNA based catalyst. 00:35:49.510 --> 00:35:52.630 And so this is something many of us may take for granted 00:35:52.630 --> 00:35:58.630 right now, but it was a big surprise to see this here. 00:35:58.630 --> 00:36:01.950 And to the best of my knowledge, the ribosome 00:36:01.950 --> 00:36:06.610 is the only natural ribozyme that has a polymerase activity. 00:36:06.610 --> 00:36:09.250 So many of these natural ribozymes 00:36:09.250 --> 00:36:14.290 are involved in RNA maturation here, so for those of you 00:36:14.290 --> 00:36:19.900 interested in evolution and hypotheses about RNA world, 00:36:19.900 --> 00:36:23.110 this observation that there's no protein in the catalytic center 00:36:23.110 --> 00:36:27.730 of the ribosome supports an RNA world hypothesis, 00:36:27.730 --> 00:36:32.170 the idea that the RNA, which stores genetic information, 00:36:32.170 --> 00:36:37.870 can perform chemical catalysis predates DNA and proteins. 00:36:37.870 --> 00:36:39.550 One thing I'll just, though, point out 00:36:39.550 --> 00:36:42.700 is that, prior to this structural study, roughly two 00:36:42.700 --> 00:36:46.480 years before, there was some experimental work done just 00:36:46.480 --> 00:36:51.730 looking at isolated 50S rRNA with no proteins. 00:36:51.730 --> 00:36:56.230 And it was found that isolated 50S rRNA could catalyze 00:36:56.230 --> 00:37:00.520 peptide bond formation, and that, specifically, domain 5 00:37:00.520 --> 00:37:04.353 was important for that reaction here. 00:37:04.353 --> 00:37:05.770 So if you're curious about that, I 00:37:05.770 --> 00:37:10.030 can point you in the direction of a paper. 00:37:10.030 --> 00:37:14.500 One last observation about the 50S subunit 00:37:14.500 --> 00:37:17.620 involves a peptide exit tunnel. 00:37:17.620 --> 00:37:21.460 And so somehow, the growing polypeptide chain 00:37:21.460 --> 00:37:24.840 needs to get out of this macromolecular machine, 00:37:24.840 --> 00:37:27.070 and in order for that to happen, there's 00:37:27.070 --> 00:37:30.190 an exit tunnel in the 50S subunit. 00:37:30.190 --> 00:37:34.360 So here, if we go back to that cryo-EM image, what's 00:37:34.360 --> 00:37:36.280 shown in this particular depiction 00:37:36.280 --> 00:37:41.260 is a polypeptide chain emerging from the 50S here. 00:37:41.260 --> 00:37:44.440 If we look at this view, a top or bottom view, what we see 00:37:44.440 --> 00:37:49.750 is that there's a hole here, and that hole is this exit tunnel. 00:37:49.750 --> 00:37:54.370 This is just another view of the same thing rotated, 00:37:54.370 --> 00:37:56.830 and a macrolide is a type of antibiotic 00:37:56.830 --> 00:38:00.280 that can bind in the region and is thought to block 00:38:00.280 --> 00:38:02.830 exit of the polypeptide. 00:38:02.830 --> 00:38:05.290 So there's some features about this exit tunnel 00:38:05.290 --> 00:38:10.070 that are interesting and that we need to consider. 00:38:10.070 --> 00:38:15.130 First of all, it's long, so approximately 100 Angstroms. 00:38:15.130 --> 00:38:18.940 And the diameter is relatively small, 00:38:18.940 --> 00:38:23.710 so the diameter is on the order of 15 Angstroms. 00:38:23.710 --> 00:38:27.820 So what we need to think about, from the perspective 00:38:27.820 --> 00:38:32.350 of this diameter, is what can fit, 00:38:32.350 --> 00:38:35.620 and so this week in recitation, you're 00:38:35.620 --> 00:38:41.060 looking at using PyMOL and ubiquitin as an example. 00:38:41.060 --> 00:38:43.170 If you just ask yourself, would ubiquitin, 00:38:43.170 --> 00:38:49.370 folded ubiquitin, fit in this exit tunnel based on its size? 00:38:49.370 --> 00:38:53.560 And so where does protein folding occur? 00:38:53.560 --> 00:38:57.580 We think about this as primarily and predominantly happening 00:38:57.580 --> 00:39:00.670 after the polypeptide comes out of the ribosome 00:39:00.670 --> 00:39:03.010 because there just isn't room in this exit 00:39:03.010 --> 00:39:07.990 tunnel for some folded structure to exist here. 00:39:07.990 --> 00:39:12.280 Also, the exit tunnel not shown in these images 00:39:12.280 --> 00:39:15.940 is lined with hydrophobic residues, just as 00:39:15.940 --> 00:39:17.650 another feature. 00:39:17.650 --> 00:39:20.695 So it's narrow, and it cannot accommodate folded proteins. 00:39:23.490 --> 00:39:30.210 So briefly on the 30S, similar to the 50S as said before, 00:39:30.210 --> 00:39:35.460 this 30S is comprised of RNA and proteins. 00:39:35.460 --> 00:39:39.600 It has the sites of mRNA binding and decoding. 00:39:39.600 --> 00:39:43.770 Here's just a structural overview of the 30S 00:39:43.770 --> 00:39:47.850 with different regions named, and similar to what 00:39:47.850 --> 00:39:54.720 we saw for the 23S rRNA of the 50S subunit, 00:39:54.720 --> 00:39:58.410 the 16S rRNA also has structure. 00:39:58.410 --> 00:40:03.000 And I just show you the domain organization here, 00:40:03.000 --> 00:40:06.870 so we see that there are four domains, 00:40:06.870 --> 00:40:10.380 and they're color coded in green, yellow, blue, and red 00:40:10.380 --> 00:40:12.210 here. 00:40:12.210 --> 00:40:20.340 And so another point just to make in passing about 16S-- 00:40:23.040 --> 00:40:27.300 16S rRNA is highly conserved amongst species, 00:40:27.300 --> 00:40:30.450 so sequencing the 16S is commonly 00:40:30.450 --> 00:40:33.210 done in studies of, say, the microbiome 00:40:33.210 --> 00:40:36.090 to figure out something about the distribution 00:40:36.090 --> 00:40:39.600 of different types of prokaryotic organisms 00:40:39.600 --> 00:40:43.050 there for that. 00:40:43.050 --> 00:40:47.730 So why spend so much time on the individual subunits? 00:40:47.730 --> 00:40:51.090 What we find is that the structures 00:40:51.090 --> 00:40:54.120 are very similar when the ribosome is assembled. 00:40:54.120 --> 00:40:56.520 So we can think of the 30S and the 50S 00:40:56.520 --> 00:40:59.550 as coming together to give the 70S, 00:40:59.550 --> 00:41:01.830 and these subunits basically look the same 00:41:01.830 --> 00:41:03.840 as they do in isolation. 00:41:03.840 --> 00:41:08.640 And that's depicted here, in just another example. 00:41:08.640 --> 00:41:11.700 So if we're looking at this structure based 00:41:11.700 --> 00:41:14.640 on the cartoon and our discussions, 00:41:14.640 --> 00:41:18.570 you should be able to identify the different components. 00:41:18.570 --> 00:41:21.338 So here, what do we have? 00:41:21.338 --> 00:41:22.230 AUDIENCE: 50S. 00:41:22.230 --> 00:41:23.613 ELIZABETH NOLAN: Yeah, and here? 00:41:23.613 --> 00:41:24.540 AUDIENCE: 30S. 00:41:24.540 --> 00:41:25.707 ELIZABETH NOLAN: 30S, right. 00:41:25.707 --> 00:41:28.177 What's this? 00:41:28.177 --> 00:41:29.052 AUDIENCE: [INAUDIBLE] 00:41:29.052 --> 00:41:30.543 AUDIENCE: [INAUDIBLE] 00:41:30.543 --> 00:41:34.130 ELIZABETH NOLAN: Yeah, we have a tRNA bound here. 00:41:34.130 --> 00:41:36.920 Here, a protein. 00:41:36.920 --> 00:41:41.720 So bring yourself back to this cartoon and its simplicity 00:41:41.720 --> 00:41:45.050 as we work through problems next week. 00:41:45.050 --> 00:41:48.290 So another point to make, just to think about, 00:41:48.290 --> 00:41:51.710 is how is it that these subunits actually come together, 00:41:51.710 --> 00:41:56.100 and what mediates that interaction there? 00:41:56.100 --> 00:42:01.250 And so these subunits basically come into contact 00:42:01.250 --> 00:42:05.120 at about 12 positions, and magnesium ions 00:42:05.120 --> 00:42:08.420 are really important for mediating the interaction 00:42:08.420 --> 00:42:11.630 between the 30S and the 50S. 00:42:11.630 --> 00:42:15.320 So there's bound magnesium ions that mediate interactions 00:42:15.320 --> 00:42:17.930 between these subunits here. 00:42:17.930 --> 00:42:20.510 And so in week four recitation, we're 00:42:20.510 --> 00:42:23.990 going to think about how to purify ribosomes. 00:42:23.990 --> 00:42:26.360 And if you're interested in purifying ribosomes, how 00:42:26.360 --> 00:42:31.370 do you get an assembled 70S prokaryotic ribosome? 00:42:31.370 --> 00:42:35.120 And based on the need for magnesium ions here, 00:42:35.120 --> 00:42:37.820 we'll see how that's an important variable 00:42:37.820 --> 00:42:41.340 in these procedures. 00:42:41.340 --> 00:42:45.230 So we'll close just with some overview 00:42:45.230 --> 00:42:49.340 points about the translation process as a whole. 00:42:49.340 --> 00:42:53.990 So during translation, mRNA is read from the five prime 00:42:53.990 --> 00:42:56.780 to the three prime end. 00:42:56.780 --> 00:42:59.660 Polypeptides are synthesized from the N terminus 00:42:59.660 --> 00:43:02.810 to the C terminus, so there's directionality. 00:43:02.810 --> 00:43:05.390 As I said earlier, translation factors 00:43:05.390 --> 00:43:08.000 are required at each stage-- 00:43:08.000 --> 00:43:12.050 initiation, elongation, and termination. 00:43:12.050 --> 00:43:14.030 Something that I haven't highlighted yet 00:43:14.030 --> 00:43:16.320 is the importance of GTP. 00:43:16.320 --> 00:43:18.740 So in that initial overview of the cycle, 00:43:18.740 --> 00:43:22.340 we saw that there were some instances of GTP hydrolysis 00:43:22.340 --> 00:43:26.120 by certain translation factors, and in this translation 00:43:26.120 --> 00:43:31.310 process, GTP hydrolysis provides a means to convert chemical 00:43:31.310 --> 00:43:33.500 energy into mechanical energy. 00:43:33.500 --> 00:43:38.150 And so we're going to think a lot about how GTP hydrolysis 00:43:38.150 --> 00:43:41.060 plays a role next week. 00:43:41.060 --> 00:43:43.640 And although we're going to look at many structures, 00:43:43.640 --> 00:43:46.430 keep in mind that conformational changes are 00:43:46.430 --> 00:43:49.670 essential for catalysis by the ribosome, 00:43:49.670 --> 00:43:53.570 and that this is a very dynamic system here. 00:43:56.120 --> 00:43:59.060 So just some additional facts-- 00:43:59.060 --> 00:44:04.070 so ribosomes will synthesize six to 20 peptide bonds per second. 00:44:04.070 --> 00:44:09.100 The error rate is less than 1 in 1,000, which brings up fidelity 00:44:09.100 --> 00:44:09.600 again. 00:44:09.600 --> 00:44:12.470 How does the ribosome maintain this? 00:44:12.470 --> 00:44:14.810 And the rate accelerations are on the order of 10 00:44:14.810 --> 00:44:19.670 to the 7-fold, so less than many enzymes, but quite good. 00:44:19.670 --> 00:44:22.370 And in all living organisms, these ribosomes 00:44:22.370 --> 00:44:25.760 carry out protein synthesis, so all ribosomes 00:44:25.760 --> 00:44:29.480 contain two subunits that reversibly associate 00:44:29.480 --> 00:44:32.060 during the translation cycle. 00:44:32.060 --> 00:44:34.400 Protein synthesis occurs through the binding 00:44:34.400 --> 00:44:38.300 of the aminoacyl tRNAs to the 70S ribosome 00:44:38.300 --> 00:44:41.150 in an order dictated by the mRNA. 00:44:41.150 --> 00:44:43.670 And next week, we're going to dissect how this actually 00:44:43.670 --> 00:44:47.240 occurs, and we think this will be quite new for all of you, 00:44:47.240 --> 00:44:51.710 even if you've learned about the ribosome in other courses. 00:44:51.710 --> 00:44:54.710 These tRNAs move sequentially through these three 00:44:54.710 --> 00:44:58.914 ribosome binding sites, as we saw before here. 00:45:01.890 --> 00:45:08.290 So we can return to our overview cycle here, that we saw before. 00:45:08.290 --> 00:45:12.180 And so we'll briefly address how initiation occurs. 00:45:12.180 --> 00:45:16.200 So how is this 70S ribosome assembled? 00:45:16.200 --> 00:45:19.490 We'll have a detailed case study of EF-Tu 00:45:19.490 --> 00:45:21.840 and then look through this elongation 00:45:21.840 --> 00:45:22.755 cycle in more detail. 00:45:25.760 --> 00:45:28.440 In terms of the players and the outcomes-- 00:45:28.440 --> 00:45:31.670 so this is a reference slide for all of you, where 00:45:31.670 --> 00:45:36.740 the stages are listed, that all of the players are listed, 00:45:36.740 --> 00:45:42.470 so some more detail than what's up here, and then the outcome. 00:45:42.470 --> 00:45:45.590 So what you can see from this overview, 00:45:45.590 --> 00:45:48.920 and go back and study it outside of lecture, 00:45:48.920 --> 00:45:52.410 is that, in each case, we see GTP, 00:45:52.410 --> 00:45:56.300 which means GTP hydrolysis occurs at each step. 00:45:56.300 --> 00:46:00.110 In initiation, our outcome is assembly of the 70S 00:46:00.110 --> 00:46:05.360 with mRNA bound and with an initiator tRNA in the P-site. 00:46:05.360 --> 00:46:08.270 The outcome of elongation is synthesis 00:46:08.270 --> 00:46:12.200 of this nascent, or new, polypeptide chain, 00:46:12.200 --> 00:46:15.410 and termination is the hydrolytic release 00:46:15.410 --> 00:46:18.290 of the peptide, release of the tRNAs 00:46:18.290 --> 00:46:21.680 and mRNAs and dissociation of the 70S 00:46:21.680 --> 00:46:23.810 and, ultimately, recycling. 00:46:23.810 --> 00:46:27.880 So there's many factors that need to be taken into account 00:46:27.880 --> 00:46:32.570 and dealt with it at every stage here. 00:46:32.570 --> 00:46:35.240 This is just another reference table. 00:46:35.240 --> 00:46:39.110 It has some additional players, like EF-Ts, 00:46:39.110 --> 00:46:43.280 and this is a nucleotide exchange factor for EF-Tu. 00:46:43.280 --> 00:46:47.510 So EF-Tu is a GTP-ase that we'll hear more about in lecture 00:46:47.510 --> 00:46:51.860 next week, and in recitation next week. 00:46:51.860 --> 00:46:56.180 Briefly, some topics for review-- 00:46:56.180 --> 00:46:59.690 if you need to review the genetic code, please do. 00:46:59.690 --> 00:47:04.070 We're not going to spend much time on it here. 00:47:04.070 --> 00:47:06.620 But in brief, I think we all know this genetic code is 00:47:06.620 --> 00:47:08.340 based on codons. 00:47:08.340 --> 00:47:11.900 They're read sequentially from a fixed starting point, 00:47:11.900 --> 00:47:13.700 and the code, which is a triplet code, 00:47:13.700 --> 00:47:16.730 is degenerate and non-overlapping. 00:47:16.730 --> 00:47:18.470 So why do we have a triplet code? 00:47:25.890 --> 00:47:27.050 We have four bases. 00:47:27.050 --> 00:47:29.678 AUDIENCE: We need enough combinations to [INAUDIBLE].. 00:47:29.678 --> 00:47:31.220 ELIZABETH NOLAN: Exactly, there needs 00:47:31.220 --> 00:47:34.730 to be enough combinations for all the amino acids. 00:47:34.730 --> 00:47:37.370 So we have 20 proteinogenic amino acids, 00:47:37.370 --> 00:47:38.690 and what else do we have? 00:47:38.690 --> 00:47:40.160 We have selenocysteine. 00:47:40.160 --> 00:47:41.690 We have pyrrolysine. 00:47:41.690 --> 00:47:44.960 So a triplet code with four bases 00:47:44.960 --> 00:47:48.530 covers everything we need here. 00:47:48.530 --> 00:47:52.100 We have start codons and stop codons we have to keep in mind, 00:47:52.100 --> 00:47:54.530 listed here. 00:47:54.530 --> 00:47:59.510 And as a reminder, in translation, the amino acids 00:47:59.510 --> 00:48:02.630 are delivered by the aminoacyl tRNAs. 00:48:02.630 --> 00:48:06.500 So the mRNA does not recognize these amino acids directly. 00:48:06.500 --> 00:48:12.470 We need the tRNAs that allows this reading to occur. 00:48:12.470 --> 00:48:14.630 Throughout this course, we're going to refer-- 00:48:14.630 --> 00:48:17.120 well, throughout this section with the ribosome, 00:48:17.120 --> 00:48:22.640 we'll be referring to nucleotides, et cetera, 00:48:22.640 --> 00:48:24.890 by the letter abbreviations. 00:48:24.890 --> 00:48:27.890 There are structures, chemical structures, 00:48:27.890 --> 00:48:29.780 associated with these abbreviations, 00:48:29.780 --> 00:48:32.660 and it's important to know those and be thinking about those 00:48:32.660 --> 00:48:34.460 as you work problems. 00:48:34.460 --> 00:48:37.640 So just as review, we have the DNA bases, 00:48:37.640 --> 00:48:43.160 C, G, A, and T. In RNA, we have uracil instead of thymine. 00:48:43.160 --> 00:48:45.590 The purines, A and G, have two rings, 00:48:45.590 --> 00:48:47.930 and the Pyrimidines, one ring. 00:48:47.930 --> 00:48:52.850 For nomenclature, nucleoside versus nucleotide-- 00:48:52.850 --> 00:48:56.950 so the nucleoside is a base plus a sugar, 00:48:56.950 --> 00:49:00.260 so there's this glycosidic bond here between the base 00:49:00.260 --> 00:49:04.850 and the carbon here of the ribose. 00:49:04.850 --> 00:49:08.090 And then the nucleotide is this nucleoside 00:49:08.090 --> 00:49:11.690 with one or more phosphate groups attached 00:49:11.690 --> 00:49:13.100 at the five prime carbon. 00:49:13.100 --> 00:49:15.440 So we go one prime, two prime, three prime, 00:49:15.440 --> 00:49:19.250 four prime, five prime for the numbering of the ribose. 00:49:19.250 --> 00:49:23.510 And keep in mind, from 5.07 or 7.05-- 00:49:23.510 --> 00:49:26.270 I think this should be known, but these phosphates, 00:49:26.270 --> 00:49:29.540 we have alpha, beta, and gamma phosphates. 00:49:29.540 --> 00:49:34.190 And depending on whether your ATP or some other nucleotide 00:49:34.190 --> 00:49:37.430 is being hydrolyzed to, say, an AMP or and ADP, 00:49:37.430 --> 00:49:40.440 you're going to have attack at different positions. 00:49:40.440 --> 00:49:44.900 So if you need to review, visit your basic biochemistry 00:49:44.900 --> 00:49:47.240 textbook for these details. 00:49:47.240 --> 00:49:49.340 Also just to keep in mind, the Watson-Crick 00:49:49.340 --> 00:49:53.660 based pairing, so G and C. We have three hydrogen bonds 00:49:53.660 --> 00:49:57.380 here, A and T, two hydrogen bonds. 00:49:57.380 --> 00:49:59.840 And after spring break, Professor Stubbe 00:49:59.840 --> 00:50:04.070 will be presenting a module on nucleotide metabolism, where 00:50:04.070 --> 00:50:07.410 we'll be thinking about these things in some more detail. 00:50:07.410 --> 00:50:11.900 So where we'll begin on Monday is briefly 00:50:11.900 --> 00:50:14.480 looking at an overview of initiation, 00:50:14.480 --> 00:50:16.700 and then we're going to begin to ask 00:50:16.700 --> 00:50:20.090 how did these amino acids get attached to tRNAs, 00:50:20.090 --> 00:50:22.970 and how did those aminoacyl tRNAs 00:50:22.970 --> 00:50:25.760 get to the A-site of the ribosome. 00:50:25.760 --> 00:50:28.510 So we'll see you then.