WEBVTT 00:00:00.500 --> 00:00:03.479 [CREAKING] 00:00:16.420 --> 00:00:19.100 BARBARA IMPERIALI: So what we are going to talk about today, 00:00:19.100 --> 00:00:23.390 and I'm going to do something a little annoying, as usual, 00:00:23.390 --> 00:00:26.190 I want to take you somewhere here. 00:00:26.190 --> 00:00:29.090 Yeah, sure, back to carbon, that's fine. 00:00:29.090 --> 00:00:31.300 But what I want to show you is the sizes 00:00:31.300 --> 00:00:34.930 of the molecular players in translation 00:00:34.930 --> 00:00:39.220 because we are going here now from the smallest carbon atom. 00:00:39.220 --> 00:00:42.400 And we spent the first four lectures on amino acids, 00:00:42.400 --> 00:00:45.100 nucleotides, sugars, phospholipids. 00:00:45.100 --> 00:00:47.568 But we're entering new territory here, 00:00:47.568 --> 00:00:49.360 and I'm just going to do this in such a way 00:00:49.360 --> 00:00:52.850 that it comes onto the screens. 00:00:52.850 --> 00:00:55.420 What you can see here is, in order 00:00:55.420 --> 00:00:59.300 to make proteins like hemoglobin and antibodies-- 00:00:59.300 --> 00:01:02.260 these are made up of amino acids, the really tiny things 00:01:02.260 --> 00:01:04.269 that are out of view right now-- 00:01:04.269 --> 00:01:08.740 we need large entities that are made up of either nucleic acids 00:01:08.740 --> 00:01:12.140 alone or nucleic acids plus proteins. 00:01:12.140 --> 00:01:14.140 And so the things that will feature today 00:01:14.140 --> 00:01:17.480 are the transfer RNAs and the ribosome. 00:01:17.480 --> 00:01:20.980 And I want you to look at the size of this molecular machine. 00:01:20.980 --> 00:01:23.320 This is we're getting pretty large now. 00:01:23.320 --> 00:01:25.930 We're needing to use large machines 00:01:25.930 --> 00:01:29.950 to make the smaller catalysts that are essential in the cell. 00:01:29.950 --> 00:01:34.000 And, just so sort of maybe size doesn't seem so important, 00:01:34.000 --> 00:01:37.000 but let's just go a little bit further here. 00:01:37.000 --> 00:01:45.090 And what to me is intriguing is that the size of the ribosome 00:01:45.090 --> 00:01:48.450 is pretty similar to the size of the rhinovirus, 00:01:48.450 --> 00:01:51.030 a little smaller than the hepatitis virus, 00:01:51.030 --> 00:01:54.090 but quite a bit smaller than some of these other viruses. 00:01:54.090 --> 00:01:57.960 So the ribosome organelle is a large entity in the cell. 00:01:57.960 --> 00:02:03.930 And, when you do look at electron micrographs of cells, 00:02:03.930 --> 00:02:07.380 you can see these dark dots, which represent ribosomes. 00:02:07.380 --> 00:02:10.770 They're big enough to see, whereas the proteins themselves 00:02:10.770 --> 00:02:12.210 are not big enough to see. 00:02:12.210 --> 00:02:15.450 So that's what you're destined for today. 00:02:19.170 --> 00:02:26.720 All right, OK, so I've started to place on this board 00:02:26.720 --> 00:02:30.200 some of the molecular players, the messenger RNA, the transfer 00:02:30.200 --> 00:02:33.290 RNA, and the ribosomes, which are made up 00:02:33.290 --> 00:02:35.840 of ribosomal RNA plus protein. 00:02:35.840 --> 00:02:39.680 And I want to just remind you about the structure 00:02:39.680 --> 00:02:46.880 of the mature messenger RNA just for a minute 00:02:46.880 --> 00:02:48.680 because, in the last class, we talked 00:02:48.680 --> 00:02:53.750 about a lot of manipulations of that pre-messenger RNA, 00:02:53.750 --> 00:02:56.000 the fresh thing out of transcription. 00:02:56.000 --> 00:02:57.710 And now I just want to remind you 00:02:57.710 --> 00:03:03.230 that the messenger is single-stranded RNA, obviously. 00:03:03.230 --> 00:03:08.690 It has a 5 prime cap, which has got this funky 5 prime, 00:03:08.690 --> 00:03:13.100 5 prime bridge that's resistant to exonucleases. 00:03:13.100 --> 00:03:17.510 Somewhere in that sequence is a start codon. 00:03:17.510 --> 00:03:23.660 It's something that says this is the bit I want to translate. 00:03:23.660 --> 00:03:29.360 Often, there's a lot of stuff here that you don't translate. 00:03:29.360 --> 00:03:36.440 It's part of what's known as the ribosome binding site. 00:03:36.440 --> 00:03:38.750 And there are many features in this part 00:03:38.750 --> 00:03:42.410 of the sequence that are very important for translation. 00:03:42.410 --> 00:03:45.590 They contribute to the efficiency of translation. 00:03:45.590 --> 00:03:48.080 Generically, we'd call it the ribosome binding site, 00:03:48.080 --> 00:03:51.350 but there are funny things called Shine-Dalgarno sequences 00:03:51.350 --> 00:03:52.040 and stuff. 00:03:52.040 --> 00:03:53.910 Don't worry about any of that. 00:03:53.910 --> 00:03:58.190 I just want you to appreciate that, the mature transcript, 00:03:58.190 --> 00:04:00.620 you don't translate the whole thing. 00:04:00.620 --> 00:04:03.230 A lot of this stuff is structural, functional 00:04:03.230 --> 00:04:05.210 for other reasons that contribute 00:04:05.210 --> 00:04:07.160 to the success of translation. 00:04:07.160 --> 00:04:09.560 Once you see one of these, the ribosome 00:04:09.560 --> 00:04:14.060 mows its way through and reads the nucleic acids 00:04:14.060 --> 00:04:15.110 in the message. 00:04:15.110 --> 00:04:18.740 So the message is being conveyed over to the new machinery. 00:04:18.740 --> 00:04:21.640 And then, when you hit one of these three codons, 00:04:21.640 --> 00:04:24.200 and we'll discuss these properly when we get to them, 00:04:24.200 --> 00:04:27.230 it's time to stop and finish translating. 00:04:27.230 --> 00:04:32.000 The other end the message has a poly-adenine tail. 00:04:32.000 --> 00:04:35.330 Remember that, once again, is structural to protect 00:04:35.330 --> 00:04:37.280 the ends of that transcript. 00:04:37.280 --> 00:04:41.720 Even if some of the hundreds of adenine nucleotides 00:04:41.720 --> 00:04:44.810 are nibbled off, you don't get into the part 00:04:44.810 --> 00:04:48.050 of the gene that's critical to be translated. 00:04:48.050 --> 00:04:51.260 At a certain stage, though, you might get in. 00:04:51.260 --> 00:04:53.960 Exonucleases might chew up enough. 00:04:53.960 --> 00:04:56.930 And they may end up chewing up your transcript, 00:04:56.930 --> 00:05:00.140 but that probably suggests that the messenger has been around 00:05:00.140 --> 00:05:04.580 too long, and it's time for it to retire to a better life, OK? 00:05:04.580 --> 00:05:06.840 So remember the poly-A tail. 00:05:06.840 --> 00:05:09.800 And this, once again, plays other functional roles 00:05:09.800 --> 00:05:12.770 with respect to being recognized as a transcript 00:05:12.770 --> 00:05:14.910 and being helped to get out of the nucleus. 00:05:14.910 --> 00:05:18.980 So this was really what we talked about last week. 00:05:18.980 --> 00:05:20.480 There's one more feature in here. 00:05:20.480 --> 00:05:22.460 I'm just going to remind you that 00:05:22.460 --> 00:05:26.030 the final mature transcript has also 00:05:26.030 --> 00:05:31.160 been through splicing with removal of introns 00:05:31.160 --> 00:05:34.520 and the pasting together of exons, which is a wonderful way 00:05:34.520 --> 00:05:37.970 to diversify transcripts of translation 00:05:37.970 --> 00:05:42.590 and give you much more proteins than one gene can encode, OK? 00:05:42.590 --> 00:05:44.090 And we talked about that last time. 00:05:44.090 --> 00:05:44.850 Great. 00:05:44.850 --> 00:05:48.650 So the first thing we have to think about here 00:05:48.650 --> 00:05:54.830 is how do we go from four bases to a language that 00:05:54.830 --> 00:05:56.850 includes 20 letters, right? 00:05:59.370 --> 00:06:01.380 Or it's better really, more precise, 00:06:01.380 --> 00:06:03.180 for me to call them nucleotides. 00:06:07.990 --> 00:06:12.820 It's more precise because the base just 00:06:12.820 --> 00:06:16.660 represents the ring system that's attached to the ribose. 00:06:16.660 --> 00:06:19.930 Nucleotide means the whole thing, including a phosphate. 00:06:19.930 --> 00:06:25.375 So we go from four bases to encoding 20 amino acids. 00:06:29.000 --> 00:06:31.090 Now there are a few organisms-- 00:06:31.090 --> 00:06:35.310 and, in fact, we have one spare one as well, selenocysteine. 00:06:35.310 --> 00:06:37.780 There are a couple of other amino acids 00:06:37.780 --> 00:06:41.860 that might be designated as the so-called 21st, 22nd amino 00:06:41.860 --> 00:06:42.670 acid. 00:06:42.670 --> 00:06:45.910 They're not found globally in all organisms. 00:06:45.910 --> 00:06:49.960 We have selenocysteine in just very, very few proteins. 00:06:49.960 --> 00:06:53.530 So it's something beyond the list of 20 that you saw. 00:06:53.530 --> 00:06:56.200 There are other organisms, for example, 00:06:56.200 --> 00:06:58.090 in archaea, these guys who hang out 00:06:58.090 --> 00:07:01.120 in bubbling hot pots in Yellowstone, 00:07:01.120 --> 00:07:05.200 for example, that have another amino acid known 00:07:05.200 --> 00:07:06.130 as pyrrolysine. 00:07:06.130 --> 00:07:08.650 I'm going to mention that a little bit later on, 00:07:08.650 --> 00:07:10.900 but the ones you really need to think about 00:07:10.900 --> 00:07:15.370 are the ones we're encoding in the global genetic code. 00:07:15.370 --> 00:07:19.270 This is the ones that are common to everybody, all right? 00:07:19.270 --> 00:07:23.950 So, obviously, when you look at the language of bases, 00:07:23.950 --> 00:07:25.690 one base-- 00:07:25.690 --> 00:07:28.960 if the language translated directly one base 00:07:28.960 --> 00:07:36.890 to one amino acid, we could only encode for amino acids. 00:07:36.890 --> 00:07:39.320 So we know it's not one base, one amino acid. 00:07:39.320 --> 00:07:42.770 And I know, at this stage, you know that it's three bases, 00:07:42.770 --> 00:07:46.190 but let's just go through the math or the original questions 00:07:46.190 --> 00:07:48.830 that were sort of circulating. 00:07:48.830 --> 00:07:52.160 Like how do we go from this language to the other language? 00:07:52.160 --> 00:08:02.120 If two bases encoded each amino acid, 00:08:02.120 --> 00:08:05.170 we could only encode eight amino acids. 00:08:05.170 --> 00:08:08.700 That's not enough still. 00:08:08.700 --> 00:08:12.410 16 amino-- wait a minute. 00:08:12.410 --> 00:08:16.760 16 amino acids, sorry, I can never-- 00:08:16.760 --> 00:08:20.390 anyway, that's 4 to the 2. 00:08:20.390 --> 00:08:22.310 It's pretty good if I can't get 4 to the 2 00:08:22.310 --> 00:08:23.510 up at the blackboard. 00:08:23.510 --> 00:08:26.780 This would be 4 to the 1 power and then-- 00:08:26.780 --> 00:08:28.880 so what that came down to realizing 00:08:28.880 --> 00:08:30.560 there were not enough. 00:08:30.560 --> 00:08:33.260 That wouldn't be a sufficient language 00:08:33.260 --> 00:08:35.760 to encode the 20 amino acids. 00:08:35.760 --> 00:08:41.230 So it's finally deduced that three bases 00:08:41.230 --> 00:08:45.400 encoded each amino acid. 00:08:45.400 --> 00:08:52.370 That would give us 64 possible words in the language that 00:08:52.370 --> 00:08:54.310 needs to be translated. 00:08:54.310 --> 00:08:55.820 That's a lot more than we need. 00:08:55.820 --> 00:09:01.150 We only need 20 for the encoded amino acids, 00:09:01.150 --> 00:09:04.630 so 64 possibilities. 00:09:04.630 --> 00:09:07.140 But what else do we need in the language? 00:09:07.140 --> 00:09:08.575 We need a few more things anyway. 00:09:12.420 --> 00:09:14.605 Yeah, up there. 00:09:14.605 --> 00:09:15.980 AUDIENCE: Oh, I don't [INAUDIBLE] 00:09:15.980 --> 00:09:17.680 BARBARA IMPERIALI: Oh, you weren't. 00:09:17.680 --> 00:09:18.695 Up there. 00:09:18.695 --> 00:09:20.778 AUDIENCE: You need to know when to start and stop. 00:09:20.778 --> 00:09:23.510 BARBARA IMPERIALI: So, exactly, so here this isn't necessarily 00:09:23.510 --> 00:09:26.780 one of the ones that uniformly codes amino acid 00:09:26.780 --> 00:09:27.660 through the sequence. 00:09:27.660 --> 00:09:30.800 We need a precise way to say start. 00:09:30.800 --> 00:09:34.190 And, in fact, we need a way to say stop. 00:09:34.190 --> 00:09:37.950 And there are multiple three letter words that say stop. 00:09:37.950 --> 00:09:41.120 So it turns out that the genetic code, which 00:09:41.120 --> 00:09:50.090 forms the basis of this entire concept, 00:09:50.090 --> 00:09:54.500 has some features to it where it does have some degeneracy. 00:09:54.500 --> 00:09:56.240 But we'll go through the degeneracy, 00:09:56.240 --> 00:09:58.040 and we'll take a look at the genetic code 00:09:58.040 --> 00:10:02.555 because it will tell us exactly how the three letter-- 00:10:02.555 --> 00:10:05.570 the words made of three bases encode everything 00:10:05.570 --> 00:10:08.210 we need for translation, all right? 00:10:08.210 --> 00:10:11.120 So let's just go back and take a look at this. 00:10:11.120 --> 00:10:13.160 So we've looked at the messenger. 00:10:13.160 --> 00:10:16.400 I've told you a little bit about the tRNAs and the proteins. 00:10:16.400 --> 00:10:18.650 But then we just sort of give you a little bit 00:10:18.650 --> 00:10:20.150 of the back history. 00:10:20.150 --> 00:10:22.700 And, once the structure of double-stranded DNA 00:10:22.700 --> 00:10:24.800 was deduced, really, everyone was 00:10:24.800 --> 00:10:28.580 moving on to trying to understand how that converted 00:10:28.580 --> 00:10:30.830 to the translation to proteins. 00:10:30.830 --> 00:10:33.800 And there were a lot of workers deeply involved in this. 00:10:33.800 --> 00:10:37.550 Crick and Brenner realized it was three bases to code 00:10:37.550 --> 00:10:42.170 for one amino acid, but Khorana, Nirenberg, and Holley, 00:10:42.170 --> 00:10:46.400 Khorana who was part of our faculty for many, many years, 00:10:46.400 --> 00:10:49.250 actually defined that genetic code 00:10:49.250 --> 00:10:52.100 and got all of the details. 00:10:52.100 --> 00:10:54.950 Brenner and Crick started to have the ideas, 00:10:54.950 --> 00:10:58.760 but, really, the definition by doing a process known 00:10:58.760 --> 00:11:02.720 as cell-free translation where they could very carefully add 00:11:02.720 --> 00:11:08.690 components to understand how the code, the genetic code, 00:11:08.690 --> 00:11:11.630 was formulated where they put in specific messenger 00:11:11.630 --> 00:11:14.330 RNAs and amino acids and transfer 00:11:14.330 --> 00:11:17.570 RNAs and actually made proteins from that. 00:11:17.570 --> 00:11:20.880 So that's the work that Khorana and others did. 00:11:20.880 --> 00:11:23.570 And that was awarded them a Nobel Prize for that work. 00:11:26.450 --> 00:11:30.290 And then, later on, things started to get-- you know, 00:11:30.290 --> 00:11:33.260 these are decades of work I want to point out to you. 00:11:33.260 --> 00:11:34.910 The ribosomes were discovered. 00:11:34.910 --> 00:11:38.240 That was a decade later, the sort 00:11:38.240 --> 00:11:41.390 of details of the structure, but not the structure itself. 00:11:41.390 --> 00:11:44.450 And it was really exciting in the 2000s 00:11:44.450 --> 00:11:48.080 when Ramakrishnan, Steitz, and Yonath solved the structure 00:11:48.080 --> 00:11:50.150 of the prokaryotic ribosome. 00:11:50.150 --> 00:11:53.670 So each of these things has taken a decade to happen, 00:11:53.670 --> 00:11:56.300 but they are fundamental, major, important things 00:11:56.300 --> 00:12:00.780 that we can act on and move forward to understand more. 00:12:00.780 --> 00:12:06.220 All right, so let's move to the transfer RNAs. 00:12:06.220 --> 00:12:09.040 And I've flashed up this slide a couple of times, 00:12:09.040 --> 00:12:12.940 but I actually now have the movie of the structure 00:12:12.940 --> 00:12:14.870 of a transfer RNA. 00:12:14.870 --> 00:12:19.600 So the transfer RNA is a linear segment of RNA, 00:12:19.600 --> 00:12:25.030 but it's folded up the way ribonucleic acids are 00:12:25.030 --> 00:12:29.260 with three, four stretches of double strand coming together 00:12:29.260 --> 00:12:31.340 and loops in between them. 00:12:31.340 --> 00:12:33.400 And the one end, this is the 3 prime end. 00:12:33.400 --> 00:12:35.200 I always try to draw it on the left 00:12:35.200 --> 00:12:37.270 because, otherwise, things get confusing. 00:12:37.270 --> 00:12:40.090 It's much easier to sort of see where everything ends up 00:12:40.090 --> 00:12:43.470 if you put the long arm on the 3 prime side 00:12:43.470 --> 00:12:45.303 to the left of your picture. 00:12:45.303 --> 00:12:46.720 And what I think is cool, when you 00:12:46.720 --> 00:12:50.260 look at the structure of RNA, we think of messenger RNA 00:12:50.260 --> 00:12:52.880 as being a sort of rather floppy entity, 00:12:52.880 --> 00:12:55.300 but it actually really likes to form 00:12:55.300 --> 00:12:57.430 short segments of double helix. 00:12:57.430 --> 00:12:59.500 It just doesn't do well with the really long 00:12:59.500 --> 00:13:02.980 double-stranded structure the same way that DNA does. 00:13:02.980 --> 00:13:05.950 But this folded up structure is very important. 00:13:05.950 --> 00:13:09.300 And it was on the observation of these folded structures 00:13:09.300 --> 00:13:13.090 that the ribosome hypothesis was formulated. 00:13:13.090 --> 00:13:16.540 But the two things that you want to remember about the ribosome 00:13:16.540 --> 00:13:18.940 are that the 3 prime hydroxyl group 00:13:18.940 --> 00:13:23.620 of the last ribose within this ribosomal sequence 00:13:23.620 --> 00:13:26.920 is where the amino acid that's going to be loaded 00:13:26.920 --> 00:13:29.230 into your protein is attached. 00:13:29.230 --> 00:13:30.740 So that's one point. 00:13:30.740 --> 00:13:33.790 And then there's another landmark on this structure. 00:13:33.790 --> 00:13:35.410 And that's what's called-- 00:13:35.410 --> 00:13:37.760 one of the loops has a special name. 00:13:37.760 --> 00:13:40.300 It's called the anticodon loop. 00:13:40.300 --> 00:13:43.900 It comprises three nucleotides that 00:13:43.900 --> 00:13:48.970 are complementary to the nucleotides in your messenger 00:13:48.970 --> 00:13:50.050 sequence. 00:13:50.050 --> 00:13:52.900 So this really is a decoder because, at one end, 00:13:52.900 --> 00:13:55.150 it's carrying an amino acid, but it's 00:13:55.150 --> 00:13:58.600 carrying the amino acid that corresponds 00:13:58.600 --> 00:14:02.830 to the code that's in the messenger via that anticodon 00:14:02.830 --> 00:14:03.460 loop. 00:14:03.460 --> 00:14:06.340 So it's a large structure, but don't mistake it 00:14:06.340 --> 00:14:08.110 for being something that's just sort 00:14:08.110 --> 00:14:10.300 of amino acid and anticodon. 00:14:10.300 --> 00:14:12.520 A lot goes on with the rest of the structure. 00:14:12.520 --> 00:14:16.600 It's a very important structure in the mechanisms of protein 00:14:16.600 --> 00:14:19.360 translation and synthesis. 00:14:19.360 --> 00:14:24.190 OK, so here I've got to sum that up with a couple more 00:14:24.190 --> 00:14:27.670 of the ways that you would see the transfer RNAs. 00:14:27.670 --> 00:14:29.890 You might see it in this globular form. 00:14:29.890 --> 00:14:32.920 And I pointed out the anticodon loop. 00:14:32.920 --> 00:14:35.590 The place where the amino acid gets linked 00:14:35.590 --> 00:14:38.110 is also called the acceptor stem. 00:14:38.110 --> 00:14:41.020 And, up here, I show you that linkage. 00:14:41.020 --> 00:14:42.380 And you should-- yes? 00:14:42.380 --> 00:14:43.797 AUDIENCE: I was just going to ask, 00:14:43.797 --> 00:14:46.885 I see how the anticodons are specialized. 00:14:46.885 --> 00:14:50.443 How does the 3 prime end of the tRNA 00:14:50.443 --> 00:14:51.610 know which protein is bound? 00:14:51.610 --> 00:14:54.160 BARBARA IMPERIALI: Yeah, and, in a moment, not quite yet, 00:14:54.160 --> 00:14:56.710 I will show you structures of tRNAs 00:14:56.710 --> 00:14:59.530 bound to their synthetases, which 00:14:59.530 --> 00:15:01.160 are the enzymes that load them. 00:15:01.160 --> 00:15:04.000 So there is specificity throughout that whole thing. 00:15:04.000 --> 00:15:06.890 It's not just bystander stuffing. 00:15:06.890 --> 00:15:08.450 It's really involved. 00:15:08.450 --> 00:15:10.720 And it's a great question, and I hope 00:15:10.720 --> 00:15:13.150 you'll get an answer that's reasonably satisfied 00:15:13.150 --> 00:15:15.290 from the structure perspective. 00:15:15.290 --> 00:15:19.120 And so, if you look up here at the acceptor stem, 00:15:19.120 --> 00:15:21.910 the amino acid is joined by an ester bond 00:15:21.910 --> 00:15:26.590 to the 3 prime end of the transfer RNA, 00:15:26.590 --> 00:15:28.120 but, hopefully, you can see in here. 00:15:28.120 --> 00:15:30.040 There's the carboxyl. 00:15:30.040 --> 00:15:31.270 There's the amine. 00:15:31.270 --> 00:15:34.900 And CHR designates the amino acid 00:15:34.900 --> 00:15:37.990 where R would be the side chain of your amino acid. 00:15:37.990 --> 00:15:40.660 So that's what that looks like at that end of the transfer 00:15:40.660 --> 00:15:41.590 RNA. 00:15:41.590 --> 00:15:44.560 And, coming down to the anticodon loop, 00:15:44.560 --> 00:15:49.420 you're going to read the messenger 5 prime to 3 prime. 00:15:49.420 --> 00:15:52.390 And the anticodon loop, when you draw 00:15:52.390 --> 00:15:54.670 this in this configuration, actually 00:15:54.670 --> 00:15:57.880 shows you that the anticodon loop 00:15:57.880 --> 00:16:00.730 is antiparallel to the codon loop 00:16:00.730 --> 00:16:03.650 to make that good hydrogen-bonding network. 00:16:03.650 --> 00:16:06.790 So that's why I like to be consistent in the way I 00:16:06.790 --> 00:16:08.230 render this structure. 00:16:08.230 --> 00:16:13.195 So the anticodon loop of the RNA complements that triplet codon 00:16:13.195 --> 00:16:15.550 in the messenger RNA, all right? 00:16:15.550 --> 00:16:16.406 Yes? 00:16:16.406 --> 00:16:17.894 AUDIENCE: [INAUDIBLE]. 00:16:17.894 --> 00:16:19.393 What's between the G and T? 00:16:19.393 --> 00:16:21.310 BARBARA IMPERIALI: What's between the G and T? 00:16:21.310 --> 00:16:21.810 Hold on. 00:16:21.810 --> 00:16:25.282 I'm going to-- what's between the G and the-- 00:16:25.282 --> 00:16:26.529 AUDIENCE: On the loop there. 00:16:26.529 --> 00:16:27.737 BARBARA IMPERIALI: Over here? 00:16:27.737 --> 00:16:28.930 AUDIENCE: No, above that on the left. 00:16:28.930 --> 00:16:30.222 BARBARA IMPERIALI: On the left. 00:16:30.222 --> 00:16:32.740 Oh, this guy? 00:16:32.740 --> 00:16:34.180 So cool you ask that. 00:16:34.180 --> 00:16:38.620 So this guy is what's known as a funny base. 00:16:38.620 --> 00:16:42.882 It's actually-- that's the symbol for it 00:16:42.882 --> 00:16:43.840 that you've picked out. 00:16:43.840 --> 00:16:46.210 And it's a base known as pseudouridine. 00:16:50.920 --> 00:16:56.020 And it turns out that these unusual bases show up 00:16:56.020 --> 00:16:58.210 in RNA sequences. 00:16:58.210 --> 00:17:01.030 Pseudouridine has an interesting structure 00:17:01.030 --> 00:17:04.480 where the bond to the ribose ring 00:17:04.480 --> 00:17:06.940 is not a carbon-nitrogen bond, but, rather, 00:17:06.940 --> 00:17:07.960 a carbon-carbon bond. 00:17:07.960 --> 00:17:09.460 And it's a bit more stable. 00:17:09.460 --> 00:17:13.690 So, in RNA, there's some of these other unusual bases. 00:17:13.690 --> 00:17:17.500 And pseudouridine is the most common of the unusual ones. 00:17:17.500 --> 00:17:19.630 It can still hydrogen bond, but it 00:17:19.630 --> 00:17:22.869 tends to show up in these sort of different loops 00:17:22.869 --> 00:17:24.527 and turn-type places, OK? 00:17:24.527 --> 00:17:25.569 Thanks for noticing that. 00:17:25.569 --> 00:17:26.227 Yeah? 00:17:26.227 --> 00:17:28.060 AUDIENCE: So do you get the G in parentheses 00:17:28.060 --> 00:17:29.640 on the yellow part [INAUDIBLE]? 00:17:29.640 --> 00:17:31.960 BARBARA IMPERIALI: The G, which is in parentheses, 00:17:31.960 --> 00:17:34.960 designates that even those sort of bulges 00:17:34.960 --> 00:17:37.810 between the real loops can vary in length. 00:17:37.810 --> 00:17:39.490 So it could be more than one. 00:17:39.490 --> 00:17:42.120 So that addresses-- comes back to that other question. 00:17:42.120 --> 00:17:44.920 But that stuff in between has variables. 00:17:44.920 --> 00:17:50.920 It has variable bulges and variable shapes associated 00:17:50.920 --> 00:17:54.010 with the synthetase enzymes that I'll introduce in a minute 00:17:54.010 --> 00:17:56.680 that it recognizes, all great questions. 00:17:56.680 --> 00:18:01.160 OK, all right, cool. 00:18:01.160 --> 00:18:06.290 OK, so [HUMMING] we've got all of that. 00:18:06.290 --> 00:18:10.300 So now let's move ourselves so we've looked at-- 00:18:10.300 --> 00:18:12.250 we know the messenger well. 00:18:12.250 --> 00:18:14.740 We're starting to understand the tRNAs. 00:18:14.740 --> 00:18:16.960 What we need to move on to now is 00:18:16.960 --> 00:18:19.600 sort of the most important part of the game, which 00:18:19.600 --> 00:18:23.360 is really taking a look at the genetic code. 00:18:23.360 --> 00:18:30.478 So this table is but one rendition of the genetic code. 00:18:30.478 --> 00:18:32.770 Sometimes, you'll see it in different shapes and sizes. 00:18:32.770 --> 00:18:35.560 In a second, I'll show you one of the other renditions. 00:18:35.560 --> 00:18:37.930 But it is the absolute-- 00:18:37.930 --> 00:18:50.940 the sort of Rosetta Stone for translating 00:18:50.940 --> 00:18:59.460 messenger RNA to amino acid sequence using codons. 00:18:59.460 --> 00:19:03.120 So this sort of-- whoa. 00:19:03.120 --> 00:19:04.890 Getting a little-- I love translation. 00:19:04.890 --> 00:19:05.130 I'm sorry. 00:19:05.130 --> 00:19:07.255 I'm getting a little bit excited about translation. 00:19:07.255 --> 00:19:11.700 OK, so there are a few features of this genetic code. 00:19:11.700 --> 00:19:13.770 Number one, you won't have to remember it. 00:19:13.770 --> 00:19:15.960 It would be foolish for us to think you could, 00:19:15.960 --> 00:19:18.960 but there are characteristics about the genetic code that 00:19:18.960 --> 00:19:19.840 are very important. 00:19:19.840 --> 00:19:22.110 But, first of all, let me sort of calibrate you. 00:19:22.110 --> 00:19:26.310 This would be the first of the three letters in the codon. 00:19:26.310 --> 00:19:28.530 And, by the way, the genetic code 00:19:28.530 --> 00:19:31.440 gives you the identities of what are 00:19:31.440 --> 00:19:37.950 known as the codons, which is how we designate 00:19:37.950 --> 00:19:40.290 the triplet of nucleotides. 00:19:44.720 --> 00:19:47.680 So you'll hear a lot about codons and anticodons 00:19:47.680 --> 00:19:48.730 of course. 00:19:48.730 --> 00:19:53.530 So the way you read this table is you read the first letter. 00:19:53.530 --> 00:19:57.160 So all of these begin with U. All of these begin with C 00:19:57.160 --> 00:19:58.240 and so on. 00:19:58.240 --> 00:20:02.710 Then you read the second letter, and there's those designations. 00:20:02.710 --> 00:20:07.420 So, if I'm going here, here, it's going to be starting UU. 00:20:07.420 --> 00:20:09.310 And then, within each block, there 00:20:09.310 --> 00:20:13.690 are the four alternatives for the other four bases. 00:20:13.690 --> 00:20:16.120 And then the third one, basically, 00:20:16.120 --> 00:20:17.860 just designates those. 00:20:17.860 --> 00:20:22.450 So you can read, for each amino acid, what three-letter codon 00:20:22.450 --> 00:20:24.170 would correspond to it. 00:20:24.170 --> 00:20:25.850 Some people quite like-- 00:20:25.850 --> 00:20:27.110 oops. 00:20:27.110 --> 00:20:28.600 I got rid of it too fast. 00:20:28.600 --> 00:20:31.420 Some people quite like this other rendition 00:20:31.420 --> 00:20:35.160 where the first of the three letters is in the center. 00:20:35.160 --> 00:20:40.120 So it's either G, U, A, or C. Then the second one comes out. 00:20:40.120 --> 00:20:42.460 It's in the brown circle. 00:20:42.460 --> 00:20:47.650 And then the third one is the third letter in the codon. 00:20:47.650 --> 00:20:51.640 And then that tells you what amino acid that corresponds to. 00:20:51.640 --> 00:20:56.390 And we'll generally tend to just stick with this one table 00:20:56.390 --> 00:20:57.590 and stick with it. 00:20:57.590 --> 00:21:01.120 So you might as well get used to that particular table. 00:21:01.120 --> 00:21:04.750 Now there's a couple of characteristics in the table. 00:21:04.750 --> 00:21:08.530 The first thing to notice is that, within that table, 00:21:08.530 --> 00:21:12.130 there is a codon to start. 00:21:12.130 --> 00:21:13.510 And it's AUG. 00:21:13.510 --> 00:21:16.720 And the one thing that's sort of sent to drive you crazy 00:21:16.720 --> 00:21:28.960 is that AUG codon equals start, but it also equals methionine. 00:21:28.960 --> 00:21:33.640 And, in bacteria, it equals a modified version of methionine. 00:21:33.640 --> 00:21:37.690 So, if the ribosome is reading the messenger RNA, 00:21:37.690 --> 00:21:42.100 it will look for the first one of these and start reading. 00:21:42.100 --> 00:21:44.470 But, once you find another one of these further 00:21:44.470 --> 00:21:47.260 into the sequence, it will put in methionine. 00:21:47.260 --> 00:21:48.640 Methionine is fairly rare. 00:21:48.640 --> 00:21:51.240 There may only be one or two more in the protein. 00:21:51.240 --> 00:21:54.790 And I hate the illogic of that, but, nevertheless, it 00:21:54.790 --> 00:21:56.020 is the case. 00:21:56.020 --> 00:21:59.120 In some organisms, you start with different amino acids, 00:21:59.120 --> 00:22:02.800 but the most common start is the codon for methionine 00:22:02.800 --> 00:22:03.770 is the start. 00:22:03.770 --> 00:22:07.270 So what that means is that every protein you translate 00:22:07.270 --> 00:22:09.370 has a methionine at one of the termini. 00:22:09.370 --> 00:22:11.180 Which terminus would it be at? 00:22:11.180 --> 00:22:12.160 AUDIENCE: [INAUDIBLE] 00:22:12.160 --> 00:22:20.050 BARBARA IMPERIALI: Yeah, OK, so that's 00:22:20.050 --> 00:22:23.650 the details with methionine and the start codon. 00:22:23.650 --> 00:22:26.500 Then there are several stop codons. 00:22:26.500 --> 00:22:29.080 And I've shown you the three of them there. 00:22:29.080 --> 00:22:32.050 The stop codons tend to be used variously. 00:22:32.050 --> 00:22:35.620 Some are more predominant in some organisms than other. 00:22:35.620 --> 00:22:37.960 And some of them respond differently 00:22:37.960 --> 00:22:40.090 when the process of stopping occurs, which 00:22:40.090 --> 00:22:41.980 we'll talk about in a second. 00:22:41.980 --> 00:22:47.930 But the next important thing to notice about the genetic code 00:22:47.930 --> 00:22:49.960 is that it's what's called degenerate. 00:22:56.323 --> 00:22:57.990 Now, when you call something degenerate, 00:22:57.990 --> 00:23:00.323 it seems like sort of a really nasty thing to call them. 00:23:00.323 --> 00:23:02.460 And it doesn't mean, oh, it's so bad. 00:23:02.460 --> 00:23:03.510 It's just a degenerate. 00:23:03.510 --> 00:23:08.627 It just means it is specific, but it's not-- 00:23:08.627 --> 00:23:09.210 wait a minute. 00:23:09.210 --> 00:23:11.210 I've got the right wording here because I always 00:23:11.210 --> 00:23:12.390 get this sort of-- 00:23:12.390 --> 00:23:18.390 degenerate, ah, it's not ambiguous. 00:23:18.390 --> 00:23:19.813 It's degenerate. 00:23:19.813 --> 00:23:20.730 That's the best thing. 00:23:20.730 --> 00:23:23.680 But it's not ambiguous. 00:23:26.570 --> 00:23:28.760 What this means is there are all, 00:23:28.760 --> 00:23:33.110 for several amino acids, more than one codon that specify it. 00:23:38.900 --> 00:23:41.700 And let's take a look at some of the examples 00:23:41.700 --> 00:23:43.360 of that degeneracy. 00:23:43.360 --> 00:23:46.530 It's at its most extreme with residues 00:23:46.530 --> 00:23:50.490 such as leucine where there are six codons that specify 00:23:50.490 --> 00:23:52.200 leucine. 00:23:52.200 --> 00:23:55.080 Alanine has four different codons. 00:23:55.080 --> 00:23:57.630 They all specify alanine. 00:23:57.630 --> 00:23:59.760 Lysine has two codons. 00:23:59.760 --> 00:24:02.850 And it's generally appreciated that the residues that 00:24:02.850 --> 00:24:07.350 have more codons tend to be the more common in your messenger 00:24:07.350 --> 00:24:11.310 RNAs, in your final protein sequence, because you'll find, 00:24:11.310 --> 00:24:13.930 in your protein, a lot of leucines. 00:24:13.930 --> 00:24:17.550 So we need a few more codons to stack the deck in favor 00:24:17.550 --> 00:24:19.720 of putting in more leucines. 00:24:19.720 --> 00:24:21.610 But so it's ambiguous-- 00:24:21.610 --> 00:24:23.610 it's degenerate. 00:24:23.610 --> 00:24:26.010 But, what I mean by it's not ambiguous, 00:24:26.010 --> 00:24:28.680 you know what it's going to code for. 00:24:28.680 --> 00:24:33.690 The other place where degenerate codons can become important 00:24:33.690 --> 00:24:35.910 is that there is species specificity. 00:24:39.582 --> 00:24:42.040 So I'm going to write that in here and explain what I mean. 00:24:47.510 --> 00:24:51.680 And what that means is that some organisms might prefer 00:24:51.680 --> 00:24:54.320 two or three of the degenerate codons, 00:24:54.320 --> 00:24:57.110 and others may prefer a couple of the others. 00:24:57.110 --> 00:24:59.480 And what that means, once in the laboratory, 00:24:59.480 --> 00:25:01.490 is it's really annoying if we want 00:25:01.490 --> 00:25:05.650 to express a protein in a really convenient bacterial system 00:25:05.650 --> 00:25:08.030 that we've taken from a mammalian system. 00:25:08.030 --> 00:25:11.210 Our codon mix may not be the same. 00:25:11.210 --> 00:25:13.490 And so there are companies now that 00:25:13.490 --> 00:25:16.670 actually will fix the codons in a gene for you 00:25:16.670 --> 00:25:19.550 to make them compatible with a different organism 00:25:19.550 --> 00:25:20.450 for expression. 00:25:20.450 --> 00:25:22.580 So it has huge practical implications 00:25:22.580 --> 00:25:23.990 to be quite honest. 00:25:23.990 --> 00:25:27.930 And it can be very annoying in the laboratory. 00:25:27.930 --> 00:25:34.700 OK, and then so codon usage varies amongst organisms. 00:25:34.700 --> 00:25:37.850 All right, so now, one last thing, so we've 00:25:37.850 --> 00:25:41.530 talked about the genetic code. 00:25:41.530 --> 00:25:45.220 It's the code that's going to be embedded within the messenger 00:25:45.220 --> 00:25:47.440 RNA. 00:25:47.440 --> 00:25:50.470 The last thing I want to do is, basically, explain to you 00:25:50.470 --> 00:25:52.330 one more time, when you're looking 00:25:52.330 --> 00:25:54.940 to read what your amino acids that get put in 00:25:54.940 --> 00:25:57.520 may be, you're going to look at the codon. 00:25:57.520 --> 00:26:01.050 And it will tell you exactly the amino acid. 00:26:01.050 --> 00:26:03.340 A long time ago, I used to be confused 00:26:03.340 --> 00:26:05.650 because I thought I should be looking at the anticodon. 00:26:05.650 --> 00:26:07.422 And I was trying to translate everything, 00:26:07.422 --> 00:26:08.380 and it was a real mess. 00:26:08.380 --> 00:26:10.690 But it's the genetic code in that box 00:26:10.690 --> 00:26:12.760 that I just showed you is written down 00:26:12.760 --> 00:26:16.480 for maximum clarity and ease of use. 00:26:16.480 --> 00:26:20.320 So, whenever you see a particular three-letter code 00:26:20.320 --> 00:26:22.690 on the messenger, you will then be 00:26:22.690 --> 00:26:25.750 able to know what amino acid it would code. 00:26:25.750 --> 00:26:27.320 So this is kind of interesting. 00:26:27.320 --> 00:26:31.800 It just reinforces to you that the codon and the anticodon 00:26:31.800 --> 00:26:33.920 are antiparallel. 00:26:33.920 --> 00:26:37.490 And so what I want to do is, basically, in this diagram, 00:26:37.490 --> 00:26:41.620 you would be reading from the 5 prime to the 3 prime end, 00:26:41.620 --> 00:26:45.820 as the transfer RNAs attach to the messenger RNA. 00:26:45.820 --> 00:26:51.310 And that would give you a codon here that would be AUC. 00:26:51.310 --> 00:26:53.260 If you'd written this the wrong way round, 00:26:53.260 --> 00:26:54.670 it would look like CUA. 00:26:54.670 --> 00:26:57.580 So you really want to be reading 5 prime to 3 prime 00:26:57.580 --> 00:26:58.700 in the codon. 00:26:58.700 --> 00:27:01.990 And then you can go to your favorite genetic code map 00:27:01.990 --> 00:27:05.080 and say this thing is read 5 prime to 3 prime. 00:27:05.080 --> 00:27:10.690 And the amino acid that gets put in is isoleucine, OK? 00:27:10.690 --> 00:27:15.250 So you'll need to be able to do that quite readily. 00:27:15.250 --> 00:27:19.780 All right, next portion, those monster-- 00:27:19.780 --> 00:27:21.460 loading the amino acid. 00:27:21.460 --> 00:27:24.920 OK, so, as I said, this is a many building block, many parts 00:27:24.920 --> 00:27:25.420 thing. 00:27:25.420 --> 00:27:26.980 So we've got the transfer RNAs. 00:27:26.980 --> 00:27:31.540 We know where we load them onto the amino acids. 00:27:31.540 --> 00:27:35.620 We know where we load the amino acids onto the transfer RNA. 00:27:35.620 --> 00:27:38.020 But we do not know how that is done. 00:27:38.020 --> 00:27:40.840 So I want to show you briefly how that occurs. 00:27:40.840 --> 00:27:45.730 So let me just start by drawing transfer RNAs the way 00:27:45.730 --> 00:27:54.210 I usually draw them in a very sort of cartoon form. 00:27:54.210 --> 00:28:00.450 And, to attach an amino acid to the 3 prime end of the transfer 00:28:00.450 --> 00:28:04.380 RNA, you have an amino acid residue-- 00:28:04.380 --> 00:28:07.355 we're just going to go R here-- 00:28:07.355 --> 00:28:10.830 carboxylic acid, amine. 00:28:10.830 --> 00:28:14.130 Actually, I'm going to draw them in their appropriate charged 00:28:14.130 --> 00:28:15.230 states. 00:28:15.230 --> 00:28:17.970 And what I need to be able to do is faithfully 00:28:17.970 --> 00:28:25.640 fix this amino acid to the 3 prime OH of the transfer RNA. 00:28:25.640 --> 00:28:28.250 And what we do is we need adenosine 00:28:28.250 --> 00:28:34.070 triphosphate to activate this chemistry. 00:28:34.070 --> 00:28:39.768 And then the OH at the 3 prime end reacts with the amino acid. 00:28:39.768 --> 00:28:41.060 So I'm just going to draw that. 00:28:41.060 --> 00:28:44.990 I'm leaving out steps because, otherwise, it's too many, 00:28:44.990 --> 00:28:46.400 and you'll be cross with me. 00:28:59.080 --> 00:29:02.920 So you attach through an ester to the amino acid 00:29:02.920 --> 00:29:06.610 from the 3 prime end of these transfer RNA. 00:29:06.610 --> 00:29:07.690 That's what's done. 00:29:07.690 --> 00:29:10.510 The ATP makes this chemistry feasible, 00:29:10.510 --> 00:29:12.570 but there's one more player here. 00:29:12.570 --> 00:29:15.560 And that's the enzyme that brings them all together, 00:29:15.560 --> 00:29:31.600 which is known as an aminoacyl-tRNA synthetase, 00:29:31.600 --> 00:29:36.880 meaning it's an enzyme that makes an aminoacyl-tRNA. 00:29:36.880 --> 00:29:38.270 And it synthetases it. 00:29:38.270 --> 00:29:41.690 So that's how its name gets subtracted. 00:29:41.690 --> 00:29:45.370 So, with reference to an earlier question, what I'm showing 00:29:45.370 --> 00:29:48.010 you here are different synthetases 00:29:48.010 --> 00:29:50.680 for different amino acids that show you 00:29:50.680 --> 00:29:54.130 that there's a recognition not just for the amino acid 00:29:54.130 --> 00:29:55.600 that's being loaded, but, rather, 00:29:55.600 --> 00:29:58.100 for the entire transfer RNA. 00:29:58.100 --> 00:30:00.280 So some of these look quite different. 00:30:00.280 --> 00:30:04.450 The isoleucine one interacts in one way. 00:30:04.450 --> 00:30:06.580 The valine one is a little different. 00:30:06.580 --> 00:30:08.860 And the glutamine one is different again. 00:30:08.860 --> 00:30:13.630 So they vary in the way they interact with the transfer 00:30:13.630 --> 00:30:14.180 RNAs. 00:30:14.180 --> 00:30:16.750 So the transfer RNAs are specific 00:30:16.750 --> 00:30:19.660 for the amino acid that is loaded onto them, 00:30:19.660 --> 00:30:22.840 but also for the synthetase that does the loading. 00:30:22.840 --> 00:30:24.940 That's how you get the specificity. 00:30:24.940 --> 00:30:27.940 Does that address your question from earlier? 00:30:27.940 --> 00:30:28.480 Hello? 00:30:28.480 --> 00:30:29.840 OK. 00:30:29.840 --> 00:30:30.840 That makes sense, right? 00:30:30.840 --> 00:30:33.760 So they fall into a lot of different families, 00:30:33.760 --> 00:30:35.470 but they're quite varied when you 00:30:35.470 --> 00:30:38.770 look at specific interactions between the synthetase 00:30:38.770 --> 00:30:40.110 and the amino acid. 00:30:40.110 --> 00:30:42.820 And, on those synthetases, there will also 00:30:42.820 --> 00:30:46.420 be specificity for the amino acid side 00:30:46.420 --> 00:30:50.380 chain of the amino acid that gets loaded. 00:30:50.380 --> 00:30:52.660 Now let's look at the ribosome components 00:30:52.660 --> 00:30:55.390 because they're the last big monsters. 00:30:55.390 --> 00:31:00.580 Ribosomes have a small and a large-- 00:31:00.580 --> 00:31:03.340 that's the large and a small subunit. 00:31:03.340 --> 00:31:06.880 They are made up, as I mentioned over here, 00:31:06.880 --> 00:31:17.700 of RNA and protein, so small and large subunits. 00:31:17.700 --> 00:31:21.220 And I've shown them there in two different colors. 00:31:21.220 --> 00:31:24.010 The prokaryotic ribosomes are pretty 00:31:24.010 --> 00:31:26.170 different from the eukaryotic ones. 00:31:26.170 --> 00:31:29.170 There's a higher proportion of RNA 00:31:29.170 --> 00:31:32.710 in the prokaryotic ones than the eukaryotic ones, which 00:31:32.710 --> 00:31:34.730 is kind of interesting. 00:31:34.730 --> 00:31:36.970 And these complexes are so big, and they're 00:31:36.970 --> 00:31:39.970 made up of so many protein and RNA 00:31:39.970 --> 00:31:42.550 strands, that we don't so much measure them 00:31:42.550 --> 00:31:44.860 by the number of those components, 00:31:44.860 --> 00:31:47.470 but, rather, by what's known as their sedimentation 00:31:47.470 --> 00:31:50.590 coefficient, which gives us a sense of the weight 00:31:50.590 --> 00:31:53.120 of the module or the complex. 00:31:53.120 --> 00:31:54.455 So the small subunit-- 00:31:58.260 --> 00:32:01.850 oh, he's back [INAUDIBLE]. 00:32:01.850 --> 00:32:07.380 The small subunit would have a 30S sedimentation coefficient. 00:32:07.380 --> 00:32:10.010 The S stands for Svedberg units. 00:32:10.010 --> 00:32:13.490 It's how fast its sediments in an ultra centrifuge. 00:32:13.490 --> 00:32:17.840 And the large subunit would have a 50S sedimentation 00:32:17.840 --> 00:32:18.920 coefficient. 00:32:18.920 --> 00:32:21.800 And those correspond to a certain number of daltons. 00:32:21.800 --> 00:32:25.668 If you see that S term after a number, 00:32:25.668 --> 00:32:26.960 that's what it's talking about. 00:32:26.960 --> 00:32:29.002 It's talking about the sedimentation coefficient, 00:32:29.002 --> 00:32:31.775 which gives us a sense of the size of the complex. 00:32:35.140 --> 00:32:38.950 And, when you start to bring all the pieces together now, 00:32:38.950 --> 00:32:42.820 what we can see on this slide is the messenger, the transfer 00:32:42.820 --> 00:32:48.280 RNAs, and the ribosomal all to scale in such a way 00:32:48.280 --> 00:32:50.110 that it can really explain it. 00:32:50.110 --> 00:32:54.580 So what I show you here is the small and large subunit. 00:32:54.580 --> 00:32:58.270 In orange-- well, that's kind of a burnt orange-- 00:32:58.270 --> 00:33:01.030 is a sneaky little bit of the messenger. 00:33:01.030 --> 00:33:03.640 In yellow are the transfer RNAs. 00:33:03.640 --> 00:33:05.410 And there's one more unit on here 00:33:05.410 --> 00:33:07.330 that I won't describe too much. 00:33:07.330 --> 00:33:11.230 It's a protein factor that helps all the processes occur. 00:33:11.230 --> 00:33:15.610 Generally, it's thought to help the loaded tRNA come 00:33:15.610 --> 00:33:18.790 to the ribosome, get it in place, and then go away. 00:33:18.790 --> 00:33:20.380 So it's some of these extra helper 00:33:20.380 --> 00:33:22.330 proteins that are involved. 00:33:22.330 --> 00:33:24.070 OK, so let's build a protein because I 00:33:24.070 --> 00:33:26.200 know it's the moment we've all been waiting for, 00:33:26.200 --> 00:33:29.770 and we're going to walk through how those pieces come together. 00:33:29.770 --> 00:33:32.860 It's a very clunky animation, but I'm very proud of myself 00:33:32.860 --> 00:33:34.340 because I did it myself. 00:33:34.340 --> 00:33:36.700 So I'm just going to show you how these things happen, 00:33:36.700 --> 00:33:40.120 as you assemble a chunk of polypeptide chain 00:33:40.120 --> 00:33:41.310 from a messenger. 00:33:41.310 --> 00:33:43.450 And so here's the messenger. 00:33:43.450 --> 00:33:45.970 It's being read 5 prime to 3 prime. 00:33:45.970 --> 00:33:49.720 What happens, first of all, is that the small subunit 00:33:49.720 --> 00:33:52.480 kind of floats along, looking for the place that 00:33:52.480 --> 00:33:56.350 would be the ribosome binding site that I mentioned here, 00:33:56.350 --> 00:33:59.920 and then sliding its way to position the start 00:33:59.920 --> 00:34:03.520 codon in the right place to start the synthesis. 00:34:03.520 --> 00:34:06.820 Once that happens, the methionine 00:34:06.820 --> 00:34:11.710 that's on its tRNA, the start one, gets into place. 00:34:11.710 --> 00:34:14.370 And, at the same time, the large subunit, 00:34:14.370 --> 00:34:17.469 completing the ribosome complex, comes together. 00:34:17.469 --> 00:34:22.120 So you now have large and small ribosomal complexes 00:34:22.120 --> 00:34:25.600 stuck onto that messenger, ready to carry on. 00:34:25.600 --> 00:34:27.850 And, in each case, you're translocating 00:34:27.850 --> 00:34:30.469 through the messenger RNA. 00:34:30.469 --> 00:34:34.000 And, in each step, you're bringing in a tRNA 00:34:34.000 --> 00:34:38.840 that's loaded with an amino acid where the anticodon 00:34:38.840 --> 00:34:41.350 of the amino acid-- and I've got those little letters shown 00:34:41.350 --> 00:34:42.610 on the bottom here-- 00:34:42.610 --> 00:34:45.460 is complementary to the codon that's 00:34:45.460 --> 00:34:47.420 within the messenger RNA. 00:34:47.420 --> 00:34:50.530 So we can start building this protein. 00:34:50.530 --> 00:34:52.150 AUG is that start codon. 00:34:52.150 --> 00:34:54.190 Methionine is the first amino acid. 00:34:54.190 --> 00:34:56.409 And it's always at the N-terminus. 00:34:56.409 --> 00:34:59.080 Then there's another amino acid comes in. 00:34:59.080 --> 00:35:03.550 The codon was UUU, and that corresponds to phenylalanine. 00:35:03.550 --> 00:35:05.410 And then the next thing that happens 00:35:05.410 --> 00:35:09.040 is there's a movement such that a new bond gets-- 00:35:09.040 --> 00:35:12.880 a new amide bond is formed between the methionine 00:35:12.880 --> 00:35:14.020 and the phenylalanine. 00:35:16.960 --> 00:35:18.920 And that new bond is an amide bond. 00:35:18.920 --> 00:35:20.240 It's not any of the others. 00:35:20.240 --> 00:35:25.360 So you're literally intercepting this complex on the transfer 00:35:25.360 --> 00:35:28.488 RNA with the amine of a new amino acid. 00:35:28.488 --> 00:35:29.780 That's how that comes together. 00:35:29.780 --> 00:35:33.160 I'm not going to worry you too much with the chemical details, 00:35:33.160 --> 00:35:36.310 but a lot of them have been illuminated by having 00:35:36.310 --> 00:35:38.270 the structure of the ribosome. 00:35:38.270 --> 00:35:41.980 And it shows you, in fact, that it's not proteins that 00:35:41.980 --> 00:35:43.570 are catalyzing that reaction. 00:35:43.570 --> 00:35:45.170 It's nucleic acids. 00:35:45.170 --> 00:35:49.150 So now I'm just going to move you on through the synthesis, 00:35:49.150 --> 00:35:53.560 in each case, building that polypeptide chain 00:35:53.560 --> 00:35:56.770 and then moving, translocating. 00:35:56.770 --> 00:35:57.640 This guy leaves. 00:35:57.640 --> 00:35:59.080 The ribosome moves along. 00:36:02.770 --> 00:36:05.980 This was a good Saturday afternoon's work. 00:36:05.980 --> 00:36:07.923 We've got to use up all those tRNAs. 00:36:07.923 --> 00:36:10.090 It doesn't get interesting until you get to the end. 00:36:10.090 --> 00:36:14.440 So we're using them, but now we come and hit a stop codon. 00:36:14.440 --> 00:36:15.910 We have UAG. 00:36:15.910 --> 00:36:17.410 So what happens here? 00:36:17.410 --> 00:36:21.460 The whole process slows down because you can either 00:36:21.460 --> 00:36:25.630 load what's known as a suppressor tRNA-- 00:36:25.630 --> 00:36:28.300 that's the one, the magenta one, that went up there-- 00:36:28.300 --> 00:36:32.710 or a protein release factor can come in and bind. 00:36:32.710 --> 00:36:36.250 But, in either case, there's no new amino acid to come in, 00:36:36.250 --> 00:36:39.850 and translation finishes and releases 00:36:39.850 --> 00:36:44.170 the protein in its complete form. 00:36:44.170 --> 00:36:46.780 Now the reason I want to differentiate 00:36:46.780 --> 00:36:51.160 between the release factor and an RNA that 00:36:51.160 --> 00:36:54.070 has the complementary stop codon is 00:36:54.070 --> 00:36:58.240 because the RNAs that are known as the suppressor RNAs 00:36:58.240 --> 00:37:00.670 have now been completely hijacked 00:37:00.670 --> 00:37:03.550 to make an enhanced genetic code where 00:37:03.550 --> 00:37:06.970 we can load lots of different amino acids using 00:37:06.970 --> 00:37:08.650 the suppressor RNAs. 00:37:08.650 --> 00:37:11.050 And, if anyone would like to chat to me about this, 00:37:11.050 --> 00:37:13.990 I'd love to because I think it's a fascinating field, 00:37:13.990 --> 00:37:16.110 but it's a bit beyond the scope of. 00:37:16.110 --> 00:37:20.290 OK, so this entire process took the following 00:37:20.290 --> 00:37:24.010 where you've got small and large subunits, all the elongation 00:37:24.010 --> 00:37:26.950 factors and initiation and release. 00:37:26.950 --> 00:37:28.630 And then, in each case, the energy 00:37:28.630 --> 00:37:30.850 is actually not provided by ATP. 00:37:30.850 --> 00:37:32.890 It's provided by GTP. 00:37:32.890 --> 00:37:37.120 But where ATP is important is in loading the amino acids 00:37:37.120 --> 00:37:39.290 onto the transfer RNAs. 00:37:39.290 --> 00:37:43.780 This occurs at about a rate of 20 amino acids per second, 00:37:43.780 --> 00:37:49.210 meaning you're reading about 60 bases per second, which 00:37:49.210 --> 00:37:52.750 is pretty consistent with the rate of transcription, 00:37:52.750 --> 00:37:54.250 not the rate of replication. 00:37:54.250 --> 00:37:56.380 That's far faster. 00:37:56.380 --> 00:38:00.400 OK, sometimes, when you're making a lot of a protein, 00:38:00.400 --> 00:38:05.920 you will see that ribosomes line up on the messenger RNA. 00:38:05.920 --> 00:38:07.600 And you'll have many proteins being 00:38:07.600 --> 00:38:10.600 made at once and at different stages in the game. 00:38:10.600 --> 00:38:13.810 And this is a lovely electron micrograph that actually 00:38:13.810 --> 00:38:15.970 shows this process in action. 00:38:15.970 --> 00:38:19.150 And, when you have a lot of ribosomes on one messenger, 00:38:19.150 --> 00:38:20.560 we call them polysomes. 00:38:20.560 --> 00:38:21.760 They have that name. 00:38:21.760 --> 00:38:26.980 OK, good, all right, so what I want to do now-- so 00:38:26.980 --> 00:38:28.390 does everyone feel good about how 00:38:28.390 --> 00:38:32.530 you translate a messenger into a protein and the various moving 00:38:32.530 --> 00:38:33.490 parts? 00:38:33.490 --> 00:38:36.070 You would always know the amino acid structures, 00:38:36.070 --> 00:38:37.630 have the genetic code. 00:38:37.630 --> 00:38:39.850 Just be familiar with reading it and making 00:38:39.850 --> 00:38:43.030 sure you could pick out which amino acid might 00:38:43.030 --> 00:38:47.590 be incorporated in response to which particular codon. 00:38:47.590 --> 00:38:50.020 So, when proteins are made on the ribosome, 00:38:50.020 --> 00:38:51.500 they have a bit of a choice. 00:38:51.500 --> 00:38:53.920 They can get made and fold beautifully 00:38:53.920 --> 00:38:55.930 into active proteins. 00:38:55.930 --> 00:38:57.570 Those proteins could be modified. 00:38:57.570 --> 00:39:00.520 They could go to different places in the cell. 00:39:00.520 --> 00:39:03.010 Occasionally, proteins misfold. 00:39:03.010 --> 00:39:05.380 Maybe the rate of synthesis is too fast, 00:39:05.380 --> 00:39:07.240 or the environment isn't right. 00:39:07.240 --> 00:39:10.750 So there will need to be mechanisms whereby proteins 00:39:10.750 --> 00:39:13.660 get degraded if they're not folded properly, 00:39:13.660 --> 00:39:15.610 but that's the story for another day. 00:39:15.610 --> 00:39:18.880 It's not always perfect, but what is known now 00:39:18.880 --> 00:39:21.940 is that, as proteins are emerging from the ribosome, 00:39:21.940 --> 00:39:25.000 they're starting to fold almost immediately 00:39:25.000 --> 00:39:27.790 from that N-terminus to, ultimately, 00:39:27.790 --> 00:39:29.800 attain their compact shape. 00:39:29.800 --> 00:39:31.300 The last thing I want to talk to you 00:39:31.300 --> 00:39:34.090 about today is what happens-- what 00:39:34.090 --> 00:39:37.640 are the types of errors we get in translation. 00:39:37.640 --> 00:39:41.170 Now translation, like transcription and regulation, 00:39:41.170 --> 00:39:44.470 has some editing mechanisms to fix errors, 00:39:44.470 --> 00:39:47.590 but, occasionally, there are errors in the DNA that 00:39:47.590 --> 00:39:49.750 put different amino acids. 00:39:49.750 --> 00:39:54.190 And the editing at that stage, by the way, to fix errors 00:39:54.190 --> 00:39:57.010 is editing when you've loaded the wrong amino acid 00:39:57.010 --> 00:39:58.690 onto a tRNA. 00:39:58.690 --> 00:40:03.190 But what happens when the DNA message, the DNA starting 00:40:03.190 --> 00:40:06.550 point, is wrong, which means the messenger is wrong, 00:40:06.550 --> 00:40:11.320 which then means we get a different translated proteins? 00:40:11.320 --> 00:40:14.800 So I'm just going to give you a couple of terms here. 00:40:14.800 --> 00:40:17.740 When we have here-- and, on all these slides, 00:40:17.740 --> 00:40:20.320 I'm going to show you the double-stranded DNA. 00:40:20.320 --> 00:40:22.900 I'm going to show you the messenger that gets made 00:40:22.900 --> 00:40:24.460 and the protein that comes forward. 00:40:24.460 --> 00:40:28.090 And they're all lined up so you can follow them really nicely, 00:40:28.090 --> 00:40:31.270 always writing 5 prime to 3 prime, except when 00:40:31.270 --> 00:40:32.950 we have double-stranded because we 00:40:32.950 --> 00:40:36.830 have to put the bottom strand in a different order. 00:40:36.830 --> 00:40:39.220 First of all, this entire chunk would 00:40:39.220 --> 00:40:41.840 be called the reading frame. 00:40:41.840 --> 00:40:44.350 It's the portion of DNA that's going 00:40:44.350 --> 00:40:48.115 to be read and transcribed into the messenger RNA. 00:40:50.930 --> 00:40:52.733 And, when you look at the two strands, 00:40:52.733 --> 00:40:54.400 you're going to have to figure out which 00:40:54.400 --> 00:40:56.560 one is the template strand. 00:40:56.560 --> 00:41:00.580 And you would figure that out by knowing that you read 3 prime 00:41:00.580 --> 00:41:05.720 to 5 prime, but that you transcribe 5 prime to 3 prime, 00:41:05.720 --> 00:41:06.220 right? 00:41:06.220 --> 00:41:09.150 So you could recognize which of the two strands 00:41:09.150 --> 00:41:11.470 you're going to make the messenger out of. 00:41:11.470 --> 00:41:13.780 Once you get the messenger, you come straight 00:41:13.780 --> 00:41:16.750 to the genetic code because you can use the genetic code 00:41:16.750 --> 00:41:18.340 to translate each of these. 00:41:18.340 --> 00:41:19.810 Does that make sense? 00:41:19.810 --> 00:41:21.250 So this would be a situation where 00:41:21.250 --> 00:41:22.990 you have a wild-type enzyme. 00:41:22.990 --> 00:41:27.520 Everything is transcribed and translated properly. 00:41:27.520 --> 00:41:29.620 Occasionally, though, there are errors 00:41:29.620 --> 00:41:33.530 that will introduce defects into the ultimate protein. 00:41:33.530 --> 00:41:37.480 The first type of error is a nonsense mutation, 00:41:37.480 --> 00:41:41.530 which might be leaving out a base pair, 00:41:41.530 --> 00:41:43.870 inserting one, substituting one. 00:41:43.870 --> 00:41:47.650 And this would, ultimately, cause an error 00:41:47.650 --> 00:41:52.760 in the DNA that then causes an error in the messenger RNA. 00:41:52.760 --> 00:41:56.620 So let's say we delete by mistake, 00:41:56.620 --> 00:41:59.720 or we're missing the C-G base pair. 00:41:59.720 --> 00:42:02.520 Then the messenger RNA is now different. 00:42:02.520 --> 00:42:06.430 And a lot of the base pairs-- the bases slide up 00:42:06.430 --> 00:42:07.700 to fill the gap. 00:42:07.700 --> 00:42:11.560 So we have what's known as a frame-shift mutation. 00:42:11.560 --> 00:42:13.260 We've shifted things. 00:42:13.260 --> 00:42:17.890 And what we've done by doing that is introduce wrong codons. 00:42:17.890 --> 00:42:21.670 So the first codon was read properly. 00:42:21.670 --> 00:42:25.210 The next one was read properly, even though there 00:42:25.210 --> 00:42:29.050 was a mistake, but then, all of a sudden, we get in this mess 00:42:29.050 --> 00:42:32.410 where one of the later codons carries a mistake 00:42:32.410 --> 00:42:34.580 and becomes a stop codon. 00:42:34.580 --> 00:42:36.220 OK, does everybody see that? 00:42:36.220 --> 00:42:39.730 So that would be called a frame-shift mutation that 00:42:39.730 --> 00:42:44.260 introduces a nonsense mutation and puts a stop codon 00:42:44.260 --> 00:42:46.510 into your messenger RNA, OK? 00:42:49.690 --> 00:42:52.270 The next types of mistakes are ones 00:42:52.270 --> 00:42:54.880 that are what are called silent mutations. 00:42:54.880 --> 00:42:58.900 It doesn't matter, for example, if you made an error 00:42:58.900 --> 00:43:03.790 in the DNA, which ended up with an error in the messenger RNA, 00:43:03.790 --> 00:43:07.420 if you still code the same amino acid, right? 00:43:07.420 --> 00:43:08.620 I go to the genetic code. 00:43:08.620 --> 00:43:10.690 I'm like, oh my goodness, I've got a mutation. 00:43:10.690 --> 00:43:11.800 Oh, it's fine. 00:43:11.800 --> 00:43:16.430 CCC codes for proline, but CCA also codes for proline. 00:43:16.430 --> 00:43:19.970 So that's called a silent mutation, OK? 00:43:19.970 --> 00:43:24.080 Then the last ones are the ones where 00:43:24.080 --> 00:43:27.350 we start to encounter errors in DNA that 00:43:27.350 --> 00:43:30.530 result in errors in proteins that may cause 00:43:30.530 --> 00:43:32.580 genetically inherited diseases. 00:43:32.580 --> 00:43:34.040 So let's take a look at that. 00:43:34.040 --> 00:43:38.090 Here it's a missense mutation where 00:43:38.090 --> 00:43:40.220 we've got an error in the DNA, which 00:43:40.220 --> 00:43:44.130 has resulted in an error in the messenger RNA. 00:43:44.130 --> 00:43:45.800 So, instead of valine-- 00:43:45.800 --> 00:43:48.410 instead of leucine, we've put in valine. 00:43:48.410 --> 00:43:51.380 That's not so bad because it's quite what 00:43:51.380 --> 00:43:53.420 we would call conservative. 00:43:53.420 --> 00:43:55.650 They're sort of similar amino acids. 00:43:55.650 --> 00:43:57.620 They have similar personalities. 00:43:57.620 --> 00:44:01.340 So this is a missense mutation, but it 00:44:01.340 --> 00:44:05.810 doesn't cause any dramatic changes, probably, in the DNA. 00:44:05.810 --> 00:44:07.370 And then the last one I want to show 00:44:07.370 --> 00:44:11.840 you is a missense mutation that is non-conservative 00:44:11.840 --> 00:44:14.070 and causes a serious defect. 00:44:14.070 --> 00:44:17.150 And this takes you back to the beginning of the class 00:44:17.150 --> 00:44:19.940 where we've put a mistake in the sequence 00:44:19.940 --> 00:44:23.045 where we've changed a glycine to an arginine. 00:44:23.045 --> 00:44:24.620 And that's a big change. 00:44:24.620 --> 00:44:28.210 And I just want to remind you of the situation in hemoglobin 00:44:28.210 --> 00:44:33.140 when we had a missense mutation, and we incorporated a valine 00:44:33.140 --> 00:44:36.560 instead of a glutamic acid, just through one change 00:44:36.560 --> 00:44:39.920 in the DNA, which made one change in the messenger, which 00:44:39.920 --> 00:44:43.340 put a drastic change in the protein that caused sickle cell 00:44:43.340 --> 00:44:44.570 anemia. 00:44:44.570 --> 00:44:46.070 So missense mutations are where you 00:44:46.070 --> 00:44:48.050 put in the wrong amino acids. 00:44:48.050 --> 00:44:51.410 And those are the ones where you end up, in a lot of cases, 00:44:51.410 --> 00:44:53.520 with inherited diseases. 00:44:53.520 --> 00:44:55.550 Nonsense mutations are not so bad 00:44:55.550 --> 00:44:58.040 because you probably just truncate the protein. 00:44:58.040 --> 00:45:02.640 You don't-- nonsense mutations aren't so bad because you end 00:45:02.640 --> 00:45:06.150 up with a truncated protein, which would be degraded. 00:45:06.150 --> 00:45:09.317 The missense mutations are the more serious ones 00:45:09.317 --> 00:45:11.400 because you end up with a full length protein that 00:45:11.400 --> 00:45:13.110 might have a mistake in it. 00:45:13.110 --> 00:45:15.570 And then that would affect the function. 00:45:15.570 --> 00:45:17.620 Am I being clear enough to everyone? 00:45:17.620 --> 00:45:18.120 Yeah? 00:45:18.120 --> 00:45:18.620 Good. 00:45:18.620 --> 00:45:22.440 OK, I am going to tell you that I'm handing over the baton 00:45:22.440 --> 00:45:25.350 to my colleague, Professor Martin. 00:45:25.350 --> 00:45:27.270 He'll take over on Monday. 00:45:27.270 --> 00:45:28.680 Mouse is pretty happy. 00:45:28.680 --> 00:45:30.630 He's pretty excited about genetics. 00:45:30.630 --> 00:45:32.770 And these will be the lectures that will occur. 00:45:32.770 --> 00:45:33.270 What? 00:45:33.270 --> 00:45:34.830 You haven't seen him, have you? 00:45:34.830 --> 00:45:36.962 He's keen on genetics, yeah. 00:45:36.962 --> 00:45:37.848 AUDIENCE: Great. 00:45:37.848 --> 00:45:39.390 BARBARA IMPERIALI: OK, and that's it. 00:45:39.390 --> 00:45:43.350 Don't forget my office hours on Monday if you need them. 00:45:43.350 --> 00:45:44.850 I think this field is fascinating. 00:45:44.850 --> 00:45:47.160 Once you get used to the mechanics of it, 00:45:47.160 --> 00:45:50.160 it's really cool to think of how you go from DNA 00:45:50.160 --> 00:45:53.390 to RNA to folded proteins.