WEBVTT 00:00:01.000 --> 00:00:08.000 Good morning. Good morning. 00:00:08.000 --> 00:00:14.000 So, I'd like to pick up where we left off last time and just finish 00:00:14.000 --> 00:00:20.000 off translation and then step back and look at how this central dogma 00:00:20.000 --> 00:00:26.000 of DNA is replicated into DNA, is read into RNA, and is translated 00:00:26.000 --> 00:00:30.000 into protein. Or, actually, as Francis Crick 00:00:30.000 --> 00:00:33.000 really put it, all information flow from nucleic 00:00:33.000 --> 00:00:37.000 acid to protein. How that varies amongst organisms. 00:00:37.000 --> 00:00:40.000 Because first we're going through it and looking at the absolutely 00:00:40.000 --> 00:00:43.000 common features, DNA replication, so it's five prime 00:00:43.000 --> 00:00:46.000 to three prime, et cetera, et cetera, 00:00:46.000 --> 00:00:50.000 transcription, translation. But in a moment I'd like to turn to 00:00:50.000 --> 00:00:53.000 the variations between different kinds of organisms. 00:00:53.000 --> 00:00:56.000 But let me briefly finish up, if I may, the bit about translation 00:00:56.000 --> 00:01:00.000 in general so we can look at its variation. 00:01:00.000 --> 00:01:06.000 As we talked about last time, we have a messenger RNA that has 00:01:06.000 --> 00:01:13.000 been transcribed from a specific region of the chromosome starting at 00:01:13.000 --> 00:01:20.000 a promoter and going to some stop of transcription. 00:01:20.000 --> 00:01:27.000 And that messenger RNA will include some particular sequence, 00:01:27.000 --> 00:01:34.000 and I'll copy one here, A-U-A-C-G-A-U-G-A-A-G-A-G-G-C-C-C, 00:01:34.000 --> 00:01:41.000 et cetera, et cetera, et cetera, out to a UAG. 00:01:41.000 --> 00:01:45.000 And this is the direction five prime to three prime. 00:01:45.000 --> 00:01:49.000 We'll remember that all nucleic acid polymerization goes five prime 00:01:49.000 --> 00:01:53.000 to three prime. So, what happens is the cell begins 00:01:53.000 --> 00:01:57.000 scanning this message. And it does that by this message 00:01:57.000 --> 00:02:01.000 being exported into the cytoplasm of the cell. The ribosome coming along 00:02:01.000 --> 00:02:05.000 and glomming onto this message and scanning on for the 00:02:05.000 --> 00:02:09.000 place to start. It looks, it looks, 00:02:09.000 --> 00:02:13.000 it looks, it looks, and it finds the first AUG. Footnote, 00:02:13.000 --> 00:02:18.000 this isn't 100% true. There are occasional messages that start their 00:02:18.000 --> 00:02:22.000 translation not at an AUG, and there are even occasional, 00:02:22.000 --> 00:02:27.000 there are even more messages that don't quite start at the first AUG 00:02:27.000 --> 00:02:31.000 because the ribosome is really is looking for something a little bit 00:02:31.000 --> 00:02:36.000 special, but to a first order approximation. 00:02:36.000 --> 00:02:40.000 Good enough for the textbooks. It goes along to the first AUG. 00:02:40.000 --> 00:02:44.000 In reality it's a little more subtle than that. 00:02:44.000 --> 00:02:48.000 But it starts at the first AUG. And what it does is it builds a 00:02:48.000 --> 00:02:52.000 protein that corresponds to it according to a three letter genetic 00:02:52.000 --> 00:02:56.000 code. And you all know the lookup table. It's in your book. 00:02:56.000 --> 00:03:00.000 AUG, always the first amino acid put in. A methionine. 00:03:00.000 --> 00:03:04.000 Then AAG. Lysine, I think. Then arginine. 00:03:04.000 --> 00:03:08.000 Then a proline. Now, I mean this is this particular sequence. 00:03:08.000 --> 00:03:12.000 Any other sequence would be different. Et cetera. 00:03:12.000 --> 00:03:17.000 How does it accomplish this matching between three letters of 00:03:17.000 --> 00:03:21.000 the genetic code? Oh, and when it gets to AUG, 00:03:21.000 --> 00:03:25.000 that is one of the three singles for stop, don't put in any more amino 00:03:25.000 --> 00:03:30.000 acids. There are three such stop signals. 00:03:30.000 --> 00:03:37.000 AUG, sorry, UAG, UGG and U, oops, what did I just do 00:03:37.000 --> 00:03:44.000 here? Let's get that right. UAG, UGG and UGA. Those are the 00:03:44.000 --> 00:03:51.000 three stop codons. So, how many total codons are there? 00:03:51.000 --> 00:03:59.000 64 codons. Three of them spell stop. 61 of them spell 00:03:59.000 --> 00:04:05.000 specific amino acids. And how many amino acids are there? 00:04:05.000 --> 00:04:11.000 20. So, the average redundancy is three. Some are specified by 00:04:11.000 --> 00:04:16.000 multiple codons. The most extreme is some amino 00:04:16.000 --> 00:04:21.000 acids are specified by as many as six codons. Did I, 00:04:21.000 --> 00:04:27.000 oh, thank you. Come back down. Of course. U-A, so it's UAG, right? 00:04:27.000 --> 00:04:32.000 Sorry, UAA and UGA and UAG. 00:04:32.000 --> 00:04:37.000 Thank you. Very good. All right. So, now, how does it accomplish 00:04:37.000 --> 00:04:42.000 this feat of taking amino acids, of taking nucleotide sequence, RNA 00:04:42.000 --> 00:04:47.000 sequence and converting it into the sequence of amino acids? 00:04:47.000 --> 00:04:52.000 As I mentioned last time, there was lots of original somewhat 00:04:52.000 --> 00:04:57.000 nutty thinking about some looping codes that would make the RNA fold 00:04:57.000 --> 00:05:03.000 up in such a way to bind the amino acids and all that. 00:05:03.000 --> 00:05:12.000 But, as Francis Crick thought up, there had to be some kind of an 00:05:12.000 --> 00:05:21.000 adapter molecule that would take the RNA sequence and would somehow 00:05:21.000 --> 00:05:30.000 connect it up to the correct amino acid, and that was UAC. 00:05:30.000 --> 00:05:35.000 A particular transfer RNA molecule. And the tRNA molecule is an adapter 00:05:35.000 --> 00:05:40.000 sequence that has three nucleotides here that match up to the three 00:05:40.000 --> 00:05:45.000 nucleotides of the codon that we're trying to translate, 00:05:45.000 --> 00:05:50.000 and it has the appropriate amino acid that's been stuck on the end of 00:05:50.000 --> 00:05:55.000 it. And how does it get there? How does the right tRNA, the tRNA 00:05:55.000 --> 00:06:00.000 to match this codon have the right amino acid put on it? 00:06:00.000 --> 00:06:03.000 There's a dedicated enzyme that recognizes that tRNA and puts on 00:06:03.000 --> 00:06:07.000 that amino acid. It's aminoacyl-tRNA synthetase. 00:06:07.000 --> 00:06:11.000 It sticks the right amino on the right transfer RNA. 00:06:11.000 --> 00:06:15.000 So, that's how it accomplishes the physical recognition of these three 00:06:15.000 --> 00:06:18.000 bases and has the right amino acid attached to it. 00:06:18.000 --> 00:06:22.000 There's an enzymatic machinery that has all of these tRNAs floating 00:06:22.000 --> 00:06:26.000 around in the cell which can be used for this translation here. 00:06:26.000 --> 00:06:30.000 How does this actually happen physically? 00:06:30.000 --> 00:06:35.000 It happens in this vast machine called the ribosome. 00:06:35.000 --> 00:06:41.000 In the ribosome, if we have, say, our codon here and we have a 00:06:41.000 --> 00:06:47.000 tRNA that, well, we'll put that actually in the 00:06:47.000 --> 00:06:53.000 ribosome that, say, has the first amino acid here, 00:06:53.000 --> 00:06:59.000 methionine, there's a cavity for this guy and there's a cavity 00:06:59.000 --> 00:07:05.000 for the next guy. And other tRNAs come into the cell 00:07:05.000 --> 00:07:11.000 carrying their next amino acid. Maybe it will be here a lysine that 00:07:11.000 --> 00:07:17.000 matches up with the codon and the anti-codon. And when the right tRNA 00:07:17.000 --> 00:07:23.000 fits in the next cavity over, the ribosome itself catalyzes a 00:07:23.000 --> 00:07:30.000 peptide bond between these amino acids. 00:07:30.000 --> 00:07:35.000 Then it chugs over by one, it translocates by one moving this 00:07:35.000 --> 00:07:40.000 bit of the complex to the left, and the peptide chain continues to 00:07:40.000 --> 00:07:45.000 grow out this end as each new codon is moved into position, 00:07:45.000 --> 00:07:50.000 a tRNA comes in bringing the right amino acid until finally a stop 00:07:50.000 --> 00:07:55.000 codon is hit. And what happens when you hit a stop codon? 00:07:55.000 --> 00:08:00.000 It stops. And is there a tRNA for a stop? 00:08:00.000 --> 00:08:02.000 It turns out there's not. There actually isn't. There's some 00:08:02.000 --> 00:08:05.000 other factor. There's a protein factor that helps recognize the 00:08:05.000 --> 00:08:08.000 stops. So, that just continues to chug on. Those of you who are 00:08:08.000 --> 00:08:11.000 computer scientists or mathematicians will recognize this 00:08:11.000 --> 00:08:14.000 is a two-tape Turing machine. It is the small two-tape Turing 00:08:14.000 --> 00:08:17.000 machine that I know to exist. If you don't know what that means, 00:08:17.000 --> 00:08:20.000 you can forget about that comment. In any case, but some of you know 00:08:20.000 --> 00:08:23.000 what that is. So, that's how it proceeds. 00:08:23.000 --> 00:08:26.000 That is your basic protein translation. 00:08:26.000 --> 00:08:28.000 And, I must say, what I really love about this was 00:08:28.000 --> 00:08:31.000 that Francis Crick kind of figured out what had to happen just on first 00:08:31.000 --> 00:08:34.000 principles and was able to think through it much more clearly and 00:08:34.000 --> 00:08:37.000 direct people to know what to look for in the laboratory. 00:08:37.000 --> 00:08:40.000 And if people had not had the clarity of thinking that Crick 00:08:40.000 --> 00:08:43.000 provided by saying, look, there's got to be this kind of 00:08:43.000 --> 00:08:46.000 adapter, I don't think they would have found it as quickly. 00:08:46.000 --> 00:08:49.000 But once he said this is what you've got to look for, 00:08:49.000 --> 00:08:52.000 golly, it was there. You can't do that very often, 00:08:52.000 --> 00:08:55.000 but Francis Crick seemed to have a very good track record of doing 00:08:55.000 --> 00:08:58.000 those things. OK. So, that was just finishing off 00:08:58.000 --> 00:09:02.000 translation. Now what I'd like to do is turn to 00:09:02.000 --> 00:09:08.000 variations on the theme as the major issue for today. 00:09:08.000 --> 00:09:14.000 How does this central dogma, DNA replicates, is transcribed into 00:09:14.000 --> 00:09:20.000 RNA and is translated into protein, vary amongst the different kinds of 00:09:20.000 --> 00:09:26.000 organisms that we might be interested in? 00:09:26.000 --> 00:09:32.000 The kinds of organisms we might be interested in, 00:09:32.000 --> 00:09:39.000 eukaryotes, prokaryotes, viruses. Sample eukaryote, 00:09:39.000 --> 00:09:46.000 MIT undergraduate. Prokaryote, E. coli. And virus, many possible 00:09:46.000 --> 00:09:53.000 viruses. The eukaryotes' big nucleated cells. 00:09:53.000 --> 00:10:00.000 So, in here we're going to have our nucleated cells. 00:10:00.000 --> 00:10:04.000 DNA living in there. In our prokaryotes we have no 00:10:04.000 --> 00:10:09.000 distinct nucleus. The DNA is not in a distinct 00:10:09.000 --> 00:10:14.000 nucleus, although it's not entirely freely floating around. 00:10:14.000 --> 00:10:19.000 It tends to be clustered together. In the virus the nucleic acid 00:10:19.000 --> 00:10:24.000 resides in some kind of a capsid, some kind of a, it could be a 00:10:24.000 --> 00:10:29.000 protein capsid. There are some of them that have 00:10:29.000 --> 00:10:34.000 lipid capsids with lipid particles around them, but some kind of a coat 00:10:34.000 --> 00:10:39.000 around nucleic acid there. Do they all do exactly the same 00:10:39.000 --> 00:10:44.000 things with regard to DNA replication, RNA transcription and 00:10:44.000 --> 00:10:49.000 protein translation? Well, not entirely. So, 00:10:49.000 --> 00:10:54.000 as a way, in a way to reinforce what we know about these, 00:10:54.000 --> 00:11:00.000 let's look at how they differ. DNA replication. Eukaryotes. 00:11:00.000 --> 00:11:06.000 What's the structure of one of your chromosomes? Is it a long line, 00:11:06.000 --> 00:11:12.000 a long linear molecule, or is it a circular molecule? 00:11:12.000 --> 00:11:18.000 How many of you have linear chromosomes? How many of you have 00:11:18.000 --> 00:11:24.000 circular chromosomes? I heard there were some people with 00:11:24.000 --> 00:11:30.000 circular. And how many of you are unsure about your chromosomes? 00:11:30.000 --> 00:11:38.000 OK. That's good. Well, then I'm pleased to inform 00:11:38.000 --> 00:11:47.000 you that you have long linear chromosomes. Every human chromosome 00:11:47.000 --> 00:11:55.000 is a long double-stranded molecule of DNA. Linear double-stranded DNA. 00:11:55.000 --> 00:12:02.000 They can be extremely long. You have 23 chromosomes, 00:12:02.000 --> 00:12:08.000 and together they make up three billion nucleotides of DNA. 00:12:08.000 --> 00:12:13.000 A typical chromosome could be 150 million bases long as an average 00:12:13.000 --> 00:12:19.000 size for a chromosome. And it's a single connected 00:12:19.000 --> 00:12:24.000 molecule. 150 million bases long in the human is a typical chromosome. 00:12:24.000 --> 00:12:30.000 One tricky little bit about replicating DNA. 00:12:30.000 --> 00:12:34.000 Let's just think back to our little model of replicating DNA. 00:12:34.000 --> 00:12:38.000 Let's come to the chromosome end here. It's five prime to three 00:12:38.000 --> 00:12:42.000 prime. Five prime to three prime. We're going to start replicating. 00:12:42.000 --> 00:12:47.000 We're getting to the end of chromosome number one. 00:12:47.000 --> 00:12:51.000 We've got a primer here, and the primer is going to be used 00:12:51.000 --> 00:12:55.000 to extend, extend, extend. We get right to the end. 00:12:55.000 --> 00:13:00.000 That's good. Tell me how we're going to replicate back. 00:13:00.000 --> 00:13:04.000 We need a little primer to start it, right? And where's that primer 00:13:04.000 --> 00:13:09.000 going to land? Maybe over here it will start 00:13:09.000 --> 00:13:14.000 replicating back. Oh, boy, we haven't done this 00:13:14.000 --> 00:13:19.000 figure. So, what do we have to do there? So, we need to primer a 00:13:19.000 --> 00:13:24.000 little further back. OK. But, you know what, 00:13:24.000 --> 00:13:29.000 the chance that we're going to get that right at the end, 00:13:29.000 --> 00:13:34.000 that we're going to get a primer exactly at the end is pretty low. 00:13:34.000 --> 00:13:37.000 And if we don't have a primer exactly at the end, 00:13:37.000 --> 00:13:41.000 what's going to be wrong with that copy of the chromosome? 00:13:41.000 --> 00:13:45.000 Too short. Now, big deal. So, it's short by maybe 20 bases. 00:13:45.000 --> 00:13:49.000 But that's just this cell division. What about next cell division? It 00:13:49.000 --> 00:13:53.000 will be short on average by a little bit, and then the next cell division 00:13:53.000 --> 00:13:57.000 and the next cell division. It's actually pretty tricky to 00:13:57.000 --> 00:14:01.000 replicate a linear chromosome on the lagging strand, 00:14:01.000 --> 00:14:05.000 unless you can land the primer in exactly the right place, 00:14:05.000 --> 00:14:09.000 which doesn't happen. So, a special little solution is 00:14:09.000 --> 00:14:15.000 used. The ends of chromosomes here are called telomeres, 00:14:15.000 --> 00:14:21.000 telo meaning end. These telomeres have very specific structures. 00:14:21.000 --> 00:14:27.000 In the human they repeat, T-T-A-G-G-G, again and 00:14:27.000 --> 00:14:32.000 again and again. At the end of the chromosome there's 00:14:32.000 --> 00:14:36.000 a special enzyme that will come along and add some extra telomere to 00:14:36.000 --> 00:14:41.000 the chromosome. That, sorry? Did I say leading 00:14:41.000 --> 00:14:46.000 strand? It's the, oh, yeah, sorry. It's the lagging, 00:14:46.000 --> 00:14:50.000 sorry. It's the leading strand. No, no, no, this is the lagging strand. 00:14:50.000 --> 00:14:55.000 This is the leading strand because it's running along happily not 00:14:55.000 --> 00:15:00.000 having to make a primer. The okazaki fragment should be here. 00:15:00.000 --> 00:15:04.000 I'll stick by that. We'll debate it later. 00:15:04.000 --> 00:15:08.000 Anyway, they, we get the point. But it's lagging because you've got 00:15:08.000 --> 00:15:12.000 the ogzocy fragments there. So, anyway, we have a problem of 00:15:12.000 --> 00:15:16.000 replication. And the way the cell solves it is the actual replication 00:15:16.000 --> 00:15:20.000 is shorter, but since it manages to stick some repeat at the end of the 00:15:20.000 --> 00:15:24.000 chromosome it adds back some more T-T-A-G-G-G, T-T-A-G-G-G, 00:15:24.000 --> 00:15:29.000 T-T-A-G-G-G, and it keeps dynamically adding more. 00:15:29.000 --> 00:15:33.000 What do you think would happen if you didn't, or what's the enzyme 00:15:33.000 --> 00:15:37.000 that adds telomeres? Telomerase. Telomerase adds that. 00:15:37.000 --> 00:15:41.000 What cells do you think need to have active telomerase? 00:15:41.000 --> 00:15:45.000 Rapidly dividing cells would need to have telomerase. 00:15:45.000 --> 00:15:49.000 Cells that are not rapidly dividing, cells that have stopped dividing can 00:15:49.000 --> 00:15:53.000 shut off their telomerase. But if a cell is going to go 00:15:53.000 --> 00:15:57.000 through lots and lots of cell divisions it's got to, 00:15:57.000 --> 00:16:01.000 it's got to tidy up its telomeres each time because they're 00:16:01.000 --> 00:16:06.000 getting too short. You've got to have an enzyme that's 00:16:06.000 --> 00:16:10.000 adding back ends of chromosomes. What cells do you think 00:16:10.000 --> 00:16:14.000 particularly care about having telomerase on them? 00:16:14.000 --> 00:16:19.000 Cancers. It turns out that this is not a trivial point. 00:16:19.000 --> 00:16:23.000 More than 90% of cancers turn on actively the telomerase gene, 00:16:23.000 --> 00:16:27.000 which would be a shut off in normal cells because the cell is 00:16:27.000 --> 00:16:32.000 not dividing anymore. Part of becoming a cancer is having 00:16:32.000 --> 00:16:36.000 to turn on this repair mechanism for the ends, this extension mechanism 00:16:36.000 --> 00:16:40.000 for the ends of your chromosomes. And so, various people are trying 00:16:40.000 --> 00:16:44.000 to make drugs to inhibit cancers by inhibiting this telomerase enzyme. 00:16:44.000 --> 00:16:49.000 So, understanding just your linear replication of chromosomes is a kind 00:16:49.000 --> 00:16:53.000 of useful thing even in dealing with things like cancer. 00:16:53.000 --> 00:16:57.000 Genome sizes. I mentioned, how big was the human genome? 00:16:57.000 --> 00:17:02.000 Three times ten to the ninth bases. The mouse genome? 00:17:02.000 --> 00:17:06.000 It's almost as big, about 2.7 times ten to the ninth 00:17:06.000 --> 00:17:11.000 bases, 2.7 million bases. The elephant genome? I actually 00:17:11.000 --> 00:17:15.000 just found this out last week because we just finished sequencing 00:17:15.000 --> 00:17:20.000 elephant DNA, and I can now tell you I think it's 3. 00:17:20.000 --> 00:17:25.000 . The dog is 2. times ten to the ninth. 00:17:25.000 --> 00:17:29.000 Anyway, it's about, for most mammals it's pretty close 00:17:29.000 --> 00:17:33.000 to three billion bases. And there is some fluctuation. 00:17:33.000 --> 00:17:37.000 Some are a little bigger. Some are a little smaller. 00:17:37.000 --> 00:17:41.000 It doesn't scale with sizing the animal, though, 00:17:41.000 --> 00:17:45.000 because the dog has a smaller genome, for example, than the mouse does, 00:17:45.000 --> 00:17:48.000 but the elephant is a big bigger than us. And check in later in the 00:17:48.000 --> 00:17:52.000 term, I'll tell you about the aardvark. We should know in a 00:17:52.000 --> 00:17:56.000 little while. But here are, for example, fruit flies. The fruit 00:17:56.000 --> 00:18:00.000 fly, it has a genome of two times ten to the eighth. 00:18:00.000 --> 00:18:04.000 I'm giving, I'm being quite approximate. In fact, 00:18:04.000 --> 00:18:08.000 I'll make it, I'll give you 1. times ten to the eighth. 150 00:18:08.000 --> 00:18:12.000 million bases. Yeast, by contrast, 00:18:12.000 --> 00:18:17.000 has a genome of 1.2 times ten to the seventh. So, that's 12 million, 00:18:17.000 --> 00:18:21.000 150 million give or take, and about three billion, 00:18:21.000 --> 00:18:25.000 so 3,000 million. So, genome sizes can vary quite 00:18:25.000 --> 00:18:30.000 dramatically amongst different eukaryotes. 00:18:30.000 --> 00:18:36.000 Now, what about prokaryotes? How do the prokaryotes differ? 00:18:36.000 --> 00:18:43.000 Prokaryotes differ because their genomes are typically not linear 00:18:43.000 --> 00:18:49.000 chromosomes. The typical prokaryotic chromosome is a 00:18:49.000 --> 00:18:56.000 double-stranded circle. It's a double-stranded circular DNA. 00:18:56.000 --> 00:19:02.000 Now, the double-stranded circular 00:19:02.000 --> 00:19:07.000 DNA doesn't have this problem of telomeres. You just keep 00:19:07.000 --> 00:19:12.000 replicating around and you get to the end. So, there you have a much 00:19:12.000 --> 00:19:17.000 simpler replication system than having to worry about your ends of 00:19:17.000 --> 00:19:22.000 chromosomes. You also have much smaller genomes. 00:19:22.000 --> 00:19:27.000 The typical prokaryotic genome size, it's on the order of a few million 00:19:27.000 --> 00:19:31.000 bases. E. coli, 4.6 million bases. There are, for example, 00:19:31.000 --> 00:19:35.000 mycobacteria, such as the mycobacteria that caused 00:19:35.000 --> 00:19:39.000 tuberculosis or leprosy, have on the order of, well, 00:19:39.000 --> 00:19:43.000 actually, not quite them, but other mycobacteria have on the order of 00:19:43.000 --> 00:19:46.000 about a million bases or so. Mycobacteria, M. genitalia has 00:19:46.000 --> 00:19:50.000 actually slightly less than a million basis. 00:19:50.000 --> 00:19:54.000 So, these are basically several million bases. 00:19:54.000 --> 00:19:58.000 So, there's a huge variation in genome size. 00:19:58.000 --> 00:20:02.000 Your genome is about a thousand times bigger than E. 00:20:02.000 --> 00:20:07.000 coli's genome. Now, you do actually have one circular chromosome. 00:20:07.000 --> 00:20:11.000 Do you know what it is? I speak about the 23 pairs of human 00:20:11.000 --> 00:20:16.000 chromosomes. There's actually one more human chromosome. 00:20:16.000 --> 00:20:20.000 The mitochondria have their own chromosome. It's a circle. 00:20:20.000 --> 00:20:25.000 That's very odd that you would have one chromosome that's a circle that 00:20:25.000 --> 00:20:30.000 looks like a bacterial chromosome. Do you know why that is? 00:20:30.000 --> 00:20:34.000 The mitochondria arose as a symbiotic bacterium that became a 00:20:34.000 --> 00:20:38.000 symbiont of eukaryotic cells about 1. billion years ago. 00:20:38.000 --> 00:20:42.000 It was a bacterium taken up into another cell, and that's how 00:20:42.000 --> 00:20:46.000 eukaryotes evolved. And we can even see that little 00:20:46.000 --> 00:20:50.000 signature of it having been a prokaryote from the fact that it's 00:20:50.000 --> 00:20:54.000 got one of these circular prokaryotic looking chromosomes. 00:20:54.000 --> 00:20:58.000 Now, it, because it's living in your cells, has thrown out all sorts 00:20:58.000 --> 00:21:02.000 of genes that it doesn't need anymore because the main, 00:21:02.000 --> 00:21:06.000 the nucleus supplies most of the proteins. 00:21:06.000 --> 00:21:10.000 So, your mitochondrial genome is a circle that's a mere 16, 00:21:10.000 --> 00:21:14.000 00 bases long. It's a very small circle encoding a very limited 00:21:14.000 --> 00:21:18.000 number of genes, but it's, in fact, 00:21:18.000 --> 00:21:22.000 the residue of the bacterial symbiont that lead to the formation 00:21:22.000 --> 00:21:26.000 of euks. Now, viruses, what do viruses have? 00:21:26.000 --> 00:21:30.000 Do they have double-strained linear chromosomes? Which is it? 00:21:30.000 --> 00:21:38.000 Is it double-stranded linear DNA or is it double-stranded circular DNA? 00:21:38.000 --> 00:21:46.000 Circular DNA. So, who votes for linear? Who votes for circular? 00:21:46.000 --> 00:21:54.000 Who's undecided? Ah, the undecided are very larger here. So, 00:21:54.000 --> 00:22:01.000 the answer is both. Some viruses have double-stranded 00:22:01.000 --> 00:22:07.000 linear DNA. Some viruses have double-stranded circular DNA. 00:22:07.000 --> 00:22:14.000 It's worse than that, though. Some viruses have single-stranded 00:22:14.000 --> 00:22:20.000 linear, circular DNA. Ha? How does that work? 00:22:20.000 --> 00:22:26.000 Some viruses actually infect the cell injecting DNA, 00:22:26.000 --> 00:22:32.000 and it's just single-stranded. As soon as it gets into the cell, 00:22:32.000 --> 00:22:36.000 however, it's replicated to make a double-stranded DNA which can then 00:22:36.000 --> 00:22:41.000 be transcribed, et cetera, et cetera, 00:22:41.000 --> 00:22:46.000 et cetera. But it travels around as a single-stranded piece of DNA. 00:22:46.000 --> 00:22:50.000 And it's actually weirder than that. Some viruses, 00:22:50.000 --> 00:22:55.000 viruses being very small can experiment with all 00:22:55.000 --> 00:23:02.000 sorts of things. Some viruses actually consist not of 00:23:02.000 --> 00:23:10.000 DNA at all but of RNA, single-stranded RNA. How does it do 00:23:10.000 --> 00:23:18.000 that? So, in other words, in the capsid there's a single 00:23:18.000 --> 00:23:26.000 strand of RNA. When it gets into the cell, 00:23:26.000 --> 00:23:32.000 what does it do? Sorry? It creates DNA. 00:23:32.000 --> 00:23:36.000 How does it create DNA? From the RNA. How's it going to do 00:23:36.000 --> 00:23:41.000 that? Well, how is it going to turn itself into DNA? 00:23:41.000 --> 00:23:46.000 It needs an enzyme to do that? Reverse transcriptase. You would 00:23:46.000 --> 00:23:50.000 like to reverse the transcription process, and you would like to name 00:23:50.000 --> 00:23:55.000 that reverse transcriptase. And where are you going to get this 00:23:55.000 --> 00:24:00.000 reverse transcriptase from? Laying around. Laying around where? 00:24:00.000 --> 00:24:04.000 I mean the cell is just sitting there with reverse transcriptase 00:24:04.000 --> 00:24:08.000 waiting to obligingly reverse transcribe this virus? 00:24:08.000 --> 00:24:12.000 Your RNA. So make it how? With ribosomes. So, in other words, 00:24:12.000 --> 00:24:17.000 if I'm an RNA, why don't I encode the sequence for 00:24:17.000 --> 00:24:21.000 reverse transcriptase and actually translate myself. 00:24:21.000 --> 00:24:25.000 So, if you were really cleaver, you might decide to put in the 00:24:25.000 --> 00:24:30.000 genetic code for reverse transcriptase. 00:24:30.000 --> 00:24:36.000 And when that message gets into the cell, it will first act as an mRNA, 00:24:36.000 --> 00:24:42.000 a messenger RNA, translate, make, here's the reverse transcriptase 00:24:42.000 --> 00:24:48.000 enzyme, which is then going to go, and it's going to reverse transcribe 00:24:48.000 --> 00:24:54.000 this thing into, say, DNA. So, wow. 00:24:54.000 --> 00:25:00.000 Now, that's a good one. This is a plus strand virus. 00:25:00.000 --> 00:25:05.000 It encodes its own reverse transcriptase in its instructions. 00:25:05.000 --> 00:25:10.000 There actually are minus-strand viruses that don't, 00:25:10.000 --> 00:25:15.000 but what they do is instead in their own code, in their own package bring 00:25:15.000 --> 00:25:20.000 a longer reverse transcriptase. So, either you can encode your own 00:25:20.000 --> 00:25:25.000 reverse transcriptase or in the package you can include your own 00:25:25.000 --> 00:25:30.000 reverse transcriptase. Do you know any viruses? 00:25:30.000 --> 00:25:36.000 And then the reverse transcriptase is then used to transcribe the DNA, 00:25:36.000 --> 00:25:43.000 the RNA into DNA, and eventually into a double-stranded DNA which, 00:25:43.000 --> 00:25:49.000 in some of the viruses, can then be slammed into and inserted into your 00:25:49.000 --> 00:25:56.000 own chromosomes. So, a DNA copy of the virus can be 00:25:56.000 --> 00:26:03.000 installed into your own chromosomes, which is somewhat insidious. 00:26:03.000 --> 00:26:08.000 What viruses do you know that do this? HIV. More generally 00:26:08.000 --> 00:26:13.000 retroviruses are the class of these viruses that can, 00:26:13.000 --> 00:26:19.000 in fact, run this replication process from RNA to DNA and install 00:26:19.000 --> 00:26:24.000 DNA copies of them in your genome. And how do you then get the DNA 00:26:24.000 --> 00:26:30.000 copy out of your genome? You don't. 00:26:30.000 --> 00:26:33.000 It doesn't come out. Retroviral insertions don't come 00:26:33.000 --> 00:26:37.000 out. That's one of the issues in dealing with AIDS is once this DNA 00:26:37.000 --> 00:26:40.000 copy is in a cell it's not coming out. We have no way to remove it. 00:26:40.000 --> 00:26:44.000 We have to make sure that the virus is shut down by other mechanisms 00:26:44.000 --> 00:26:47.000 that might inhibit its products, et cetera, but once its stuck a DNA 00:26:47.000 --> 00:26:51.000 copy into your chromosomes, you know, there's no way of getting 00:26:51.000 --> 00:26:55.000 it out. So, if we had to try to inhibit the 00:26:55.000 --> 00:27:00.000 action of the AIDS virus, we might wish to make inhibitors of 00:27:00.000 --> 00:27:05.000 this aspect of replication, inhibitors or reverse transcription. 00:27:05.000 --> 00:27:10.000 And, of course, as probably many of you know, some of the important AIDS 00:27:10.000 --> 00:27:15.000 drugs are reverse transcriptase inhibitors, very important to 00:27:15.000 --> 00:27:20.000 limiting the replication of the AIDS virus. And there are many other 00:27:20.000 --> 00:27:25.000 kinds of weirdnesses. Viruses pretty much explore, 00:27:25.000 --> 00:27:30.000 everything you possibly can do, viruses come up with ways to do. 00:27:30.000 --> 00:27:35.000 Let's take now the process of transcription. 00:27:35.000 --> 00:27:40.000 We have replication up there. Let's look at transcription. And 00:27:40.000 --> 00:27:45.000 this time let's start with prokaryotes. For the simple aspect 00:27:45.000 --> 00:27:50.000 of transcribing genes, the prokaryotic genome looks just 00:27:50.000 --> 00:27:55.000 like the simple model I gave you. There is some kind of a promoter 00:27:55.000 --> 00:28:00.000 that tells RNA polymerase to come sit down here. 00:28:00.000 --> 00:28:07.000 RNA polymerase hops on, RNA polymerase begins to copy in RNA, 00:28:07.000 --> 00:28:15.000 and eventually it hits the signal that says to terminate transcription. 00:28:15.000 --> 00:28:22.000 OK. This is not a stop codon which is about translation. 00:28:22.000 --> 00:28:30.000 This is a termination of transcription. 00:28:30.000 --> 00:28:35.000 And this RNA then goes off. A perfectly happy thing, a 00:28:35.000 --> 00:28:40.000 messenger RNA, mRNA. So, there's nothing weird 00:28:40.000 --> 00:28:46.000 about proks compared to the simple description that we gave before. 00:28:46.000 --> 00:28:51.000 But eukaryotes are different. There are some funny things that 00:28:51.000 --> 00:28:57.000 happen in the eukaryote. Well, first off it starts the same. 00:28:57.000 --> 00:29:03.000 There's a promoter. RNA polymerase sits down there, 00:29:03.000 --> 00:29:09.000 it starts transcribing, it makes an mRNA, it hits the transcriptional 00:29:09.000 --> 00:29:16.000 termination signal, it stops, and then this RNA gets 00:29:16.000 --> 00:29:22.000 processed in interesting ways. The first thing that happens is 00:29:22.000 --> 00:29:29.000 three modifications happen. The first one is at the five prime 00:29:29.000 --> 00:29:35.000 end, remember five prime to three prime, a funny modification is put 00:29:35.000 --> 00:29:41.000 on. It's a, if the message, say, were A-U-C-U-G-G-C et cetera, 00:29:41.000 --> 00:29:47.000 a G triphosphate is put on backwards. It's actually a methyl G 00:29:47.000 --> 00:29:53.000 triphosphate is put on backwards, so going in the other direction. 00:29:53.000 --> 00:30:00.000 You have the triphosphate bond there, a methyl G. 00:30:00.000 --> 00:30:04.000 And the only thing that you share care about that, 00:30:04.000 --> 00:30:09.000 I don't care if you know the structure, is that there's a funny 00:30:09.000 --> 00:30:13.000 cap. This thing is called a cap that is put on this message. 00:30:13.000 --> 00:30:18.000 And that cap is very important to signaling to the cell this is a 00:30:18.000 --> 00:30:23.000 messenger RNA to be dealt with in a certain way, to get the ribosome to 00:30:23.000 --> 00:30:27.000 hop on, to get this thing processed properly, et cetera. 00:30:27.000 --> 00:30:32.000 At the other end of the message a long string of As is added 00:30:32.000 --> 00:30:37.000 to messenger RNAs. This long string of As is called, 00:30:37.000 --> 00:30:41.000 very sensibly, a poly A tail. The poly A tail is added to the 00:30:41.000 --> 00:30:46.000 messenger RNA, and very often, 00:30:46.000 --> 00:30:51.000 I mean it's, if you wanted to purify messenger RNAs from your own human 00:30:51.000 --> 00:30:55.000 cells, you can actually use poly T as a reagent because it turns out, 00:30:55.000 --> 00:31:00.000 because messenger RNAs have a poly A tail, they'll bind to 00:31:00.000 --> 00:31:04.000 and stick to poly T. So, people actually purify messenger 00:31:04.000 --> 00:31:08.000 RNAs by binding them to poly T and they get the poly A tail. 00:31:08.000 --> 00:31:11.000 But it is broadly believed that the reason for this poly A tail is not 00:31:11.000 --> 00:31:15.000 to make things convenient for molecular biologists to purify 00:31:15.000 --> 00:31:18.000 messages. To the contrary, it is an important function for the 00:31:18.000 --> 00:31:22.000 cell. And it turns out that this is important in regulating the 00:31:22.000 --> 00:31:25.000 stability of messages. If, in fact, you don't have a poly 00:31:25.000 --> 00:31:29.000 A tail, if you contrive to make the same message without the poly A tail, 00:31:29.000 --> 00:31:33.000 the message will be degraded rather rapidly. 00:31:33.000 --> 00:31:36.000 And the lengths of the poly A tails control aspects of the degradation, 00:31:36.000 --> 00:31:39.000 et cetera. So, in a complex eukaryotic cell, 00:31:39.000 --> 00:31:43.000 already it's how to attach a little signal at the front, 00:31:43.000 --> 00:31:46.000 some signals at the back that says process me in a certain way, 00:31:46.000 --> 00:31:49.000 et cetera, don't degrade me yet. You could even imagine that this 00:31:49.000 --> 00:31:53.000 poly A tail could serve as a little bit of a clock for how long that 00:31:53.000 --> 00:31:56.000 message sticks around. It's not quite that simple but 00:31:56.000 --> 00:31:59.000 there are ways to do it. But all of these pale in comparison 00:31:59.000 --> 00:32:03.000 to the third way in which eukaryotic messages differ from prokaryotic 00:32:03.000 --> 00:32:10.000 messages. These small modifications are, 00:32:10.000 --> 00:32:21.000 as I say, small. The most striking way in which they differ is that 00:32:21.000 --> 00:32:33.000 only a small portion often of the gene, here's my gene, 00:32:33.000 --> 00:32:44.000 matters for the protein that is made. So, my mRNA gets made. 00:32:44.000 --> 00:32:54.000 It includes the whole long sequence. And then the cell comes along and 00:32:54.000 --> 00:33:05.000 splices this message together. So, this is the immature RNA. 00:33:05.000 --> 00:33:13.000 It is processed by clipping out this, clipping out this, 00:33:13.000 --> 00:33:22.000 clipping out this. And what you get is a splice where the mature message 00:33:22.000 --> 00:33:30.000 throws this stuff out, splices between here and here, 00:33:30.000 --> 00:33:39.000 splices here, splices here, splices here, and you get a 00:33:39.000 --> 00:33:47.000 much shorter mRNA. And this is a mature mRNA. 00:33:47.000 --> 00:33:53.000 This splicing is a remarkable phenomenon. In fact, 00:33:53.000 --> 00:34:00.000 it was discovered by Phil Sharp here, for which he won a Nobel prize. 00:34:00.000 --> 00:34:04.000 This splicing is a very complex operation. First off, 00:34:04.000 --> 00:34:08.000 how does, well, actually, what accomplishes splicing? 00:34:08.000 --> 00:34:12.000 It should be splicase, right? But it turns out it's not a single 00:34:12.000 --> 00:34:17.000 enzyme. It's a big body of stuff. So, instead it's the splicosome, OK. 00:34:17.000 --> 00:34:21.000 Everything is either ase or some or something like that. 00:34:21.000 --> 00:34:25.000 So, it turns out it's the splicosome that does that. 00:34:25.000 --> 00:34:30.000 It's just wonderful how all those names work out. The splicosome. 00:34:30.000 --> 00:34:36.000 The splicosome comes along and splices it. How does the splicosome 00:34:36.000 --> 00:34:42.000 know how to do this? Well, there are kind of codes. 00:34:42.000 --> 00:34:48.000 It turns out that there are some information encoded along in these 00:34:48.000 --> 00:34:54.000 messages. It turns out that there is, you know, slight biases. 00:34:54.000 --> 00:35:00.000 Typically the sequence just after where the slice starts here is a GU 00:35:00.000 --> 00:35:06.000 and the sequence here is an AG, but that's obviously not enough 00:35:06.000 --> 00:35:10.000 information, right? It's not enough bases of information 00:35:10.000 --> 00:35:14.000 to get this right. And so there's a little more 00:35:14.000 --> 00:35:18.000 preferences for what bases use, but the truth is we don't fully know. 00:35:18.000 --> 00:35:21.000 Our best picture right now involves some cellular factors help 00:35:21.000 --> 00:35:25.000 recognizing the parts that are supposed to stay in some sequences 00:35:25.000 --> 00:35:29.000 here. But the truth is we don't have the simple codes. 00:35:29.000 --> 00:35:33.000 Because if we had the simple codes, we'd be able to take a long stretch 00:35:33.000 --> 00:35:37.000 of DNA and figure out exactly where the splices go based on just 00:35:37.000 --> 00:35:42.000 computer analysis. And we can't do that so well. 00:35:42.000 --> 00:35:46.000 These bits that stay in are called exons. The bits that go out are 00:35:46.000 --> 00:35:51.000 called introns. This is the source of extraordinary 00:35:51.000 --> 00:35:55.000 confusion for students because you might think that the bits that are 00:35:55.000 --> 00:36:00.000 excised are the exons, but they're not. 00:36:00.000 --> 00:36:04.000 The bits that stay in are the exons. Why are they called exons if they 00:36:04.000 --> 00:36:08.000 stay in and ex is a prefix meaning out? Well, because the introns are 00:36:08.000 --> 00:36:12.000 named because they're intervening sequences. Once the introns, 00:36:12.000 --> 00:36:17.000 the intervening sequences were named as intervening sequences or introns, 00:36:17.000 --> 00:36:21.000 you were stuck then having to name the things that stay in as exons. 00:36:21.000 --> 00:36:25.000 This was all done by a Harvard professor, don't blame me. 00:36:25.000 --> 00:36:30.000 In any case, a good friend Harvard professor. 00:36:30.000 --> 00:36:37.000 But, nonetheless, I'm not sure that this was the best 00:36:37.000 --> 00:36:44.000 way to name them. But you're stuck with it. 00:36:44.000 --> 00:36:52.000 So, for a typical human gene, typical human gene, the length of 00:36:52.000 --> 00:36:59.000 the gene itself might be 30, 00 bases. But the mature RNA, 00:36:59.000 --> 00:37:07.000 the mature mRNA might be one and a half, 1,500 bases. 00:37:07.000 --> 00:37:11.000 That's remarkable. Out of 30,000 letters in the 00:37:11.000 --> 00:37:15.000 initial transcript that is made, the genes start, the promoter, and 00:37:15.000 --> 00:37:19.000 the transcription will stop 30, 00 bases away. The cell goes 00:37:19.000 --> 00:37:24.000 through the trouble of making an RNA of 30,000 bases long, 00:37:24.000 --> 00:37:28.000 and then it trims it down by throwing out 28, 00:37:28.000 --> 00:37:33.000 00 of the bases, keeping only 1, 00 bases at the end. 00:37:33.000 --> 00:37:37.000 Now, this may seem profligate but it ain't nothing compared to some 00:37:37.000 --> 00:37:42.000 extreme cases. The clotting factor gene, 00:37:42.000 --> 00:37:47.000 the factor 8 gene, the gene that has mutated in individuals with 00:37:47.000 --> 00:37:52.000 hemophilia, that gene is 200, 00 bases long, and it gets spliced 00:37:52.000 --> 00:37:57.000 down to a mere 10, 00 bases. 190,000 bases are thrown 00:37:57.000 --> 00:38:02.000 away. But that's nothing compared to 00:38:02.000 --> 00:38:08.000 Duchene muscular dystrophy. The Duchene muscular dystrophy is 00:38:08.000 --> 00:38:13.000 the all time winner. That gene makes an immature initial 00:38:13.000 --> 00:38:19.000 RNA of 2 million bases. RNA polymerase hops on at the 00:38:19.000 --> 00:38:24.000 promoter and it gets off at the end of the Boston Marathon on here 2 00:38:24.000 --> 00:38:30.000 million bases later having made an RNA of 2 million bases long. 00:38:30.000 --> 00:38:36.000 Calculate the speed of RNA polymerase and you'll find out that 00:38:36.000 --> 00:38:42.000 it's at it for hours. It hops on and it stays on for 00:38:42.000 --> 00:38:48.000 hours until it gets to the other end. And then for all its troubles this 00:38:48.000 --> 00:38:54.000 gene is spliced down to 16, 00 bases in the mature message. 00:38:54.000 --> 00:39:00.000 Yup? How would it increase the chance of mutations? Yup. 00:39:00.000 --> 00:39:05.000 So, splicing mutations could be a problem. Some diseases could arise 00:39:05.000 --> 00:39:10.000 from errors in splicing. Do you think that happens? 00:39:10.000 --> 00:39:15.000 Sure does. There could be mutations that create, 00:39:15.000 --> 00:39:20.000 that change a splicing, or mutations that create a new 00:39:20.000 --> 00:39:25.000 splicing, and all of that could screw up the gene. 00:39:25.000 --> 00:39:30.000 Why do this? What in the world is going on? 00:39:30.000 --> 00:39:35.000 Just think about the energetic cost. I mean count up the ATPs involved 00:39:35.000 --> 00:39:40.000 in synthesizing a nucleotide, and then the ATPs involved in adding 00:39:40.000 --> 00:39:45.000 nucleotides up. You know, think about this totally 00:39:45.000 --> 00:39:50.000 wasted energy. What is the point? 00:39:50.000 --> 00:39:55.000 I might be able to encode multiple proteins with the same gene. 00:39:55.000 --> 00:40:00.000 How would I do that? Ooh, wouldn't that be cleaver? 00:40:00.000 --> 00:40:05.000 I might be able to take a single gene and make a mix and match 00:40:05.000 --> 00:40:10.000 product. It might be, do you mean like one type of cell 00:40:10.000 --> 00:40:15.000 might splice up that message one way to produce a certain protein, 00:40:15.000 --> 00:40:20.000 but a different cell type might splice the same gene another way to 00:40:20.000 --> 00:40:25.000 produce a different protein? Ooh. So, you're proposing, if I 00:40:25.000 --> 00:40:30.000 understand you correctly, alternative splicing. 00:40:30.000 --> 00:40:34.000 Alternative splicing could create multiple proteins, 00:40:34.000 --> 00:40:38.000 multiple distinct proteins. It might be, for example, that you 00:40:38.000 --> 00:40:42.000 might make one protein that has a cytoplasmic tail and another protein 00:40:42.000 --> 00:40:46.000 that doesn't have cytosplasmic tail or a different tail or, 00:40:46.000 --> 00:40:50.000 or, this is true. This actually happens. It's very cleaver. 00:40:50.000 --> 00:40:54.000 Anything that can happen does happen somewhere, 00:40:54.000 --> 00:40:58.000 and it's fairly regularly used. A typical gene in the human being 00:40:58.000 --> 00:41:02.000 has at least two alternative splice forms, on average. 00:41:02.000 --> 00:41:05.000 Most, many don't, but there are some that have large 00:41:05.000 --> 00:41:08.000 numbers. The most extreme is there's a gene known, 00:41:08.000 --> 00:41:11.000 drosophila, that has more than a thousand alternative splice forms. 00:41:11.000 --> 00:41:15.000 How does it know, how does the cell know whether to splice it one way in 00:41:15.000 --> 00:41:18.000 the liver and one way in a heart or something? We don't fully know but 00:41:18.000 --> 00:41:21.000 there's machinery and signals people are trying to work out for that. 00:41:21.000 --> 00:41:25.000 Now, I don't want to confuse you too much about it. 00:41:25.000 --> 00:41:28.000 You know, mostly, when we give you a gene, you should think about it 00:41:28.000 --> 00:41:31.000 spliced out introns, exons. But the truth is it is more 00:41:31.000 --> 00:41:35.000 complicated than that. There can be alternative splicing 00:41:35.000 --> 00:41:39.000 that allows genes to be used in multiple ways. 00:41:39.000 --> 00:41:43.000 Sometimes they don't make multiple proteins. They may splice into 00:41:43.000 --> 00:41:46.000 portions of the mRNA that are not translated, but, 00:41:46.000 --> 00:41:50.000 there is that, but, boy, it's a huge amount of overhead 00:41:50.000 --> 00:41:54.000 here just to do that. Is it justified? Yes? 00:41:54.000 --> 00:41:58.000 That is by computer if I just gave you the sequence? Not quite. 00:41:58.000 --> 00:42:01.000 Almost. Maybe. Sort of. It turns out that the 00:42:01.000 --> 00:42:04.000 computer programs for automatically recognizing the matter of the human 00:42:04.000 --> 00:42:07.000 genome are sort of, they're mediocre, not very good. 00:42:07.000 --> 00:42:09.000 We have some idea of the signals, and various people have trying to 00:42:09.000 --> 00:42:12.000 write better and better algorithms for doing that, 00:42:12.000 --> 00:42:15.000 but the cell knows what it's doing and we don't fully know, 00:42:15.000 --> 00:42:18.000 as evidenced by the fact that we can't write a clean computer program 00:42:18.000 --> 00:42:21.000 to do it yet. We need to get information from the cell or from 00:42:21.000 --> 00:42:24.000 evolution or various other things like that, and that's the ultimate 00:42:24.000 --> 00:42:27.000 test. If we knew what we were talking about we'd just be able to 00:42:27.000 --> 00:42:30.000 write a computer program and splice it out. 00:42:30.000 --> 00:42:34.000 And we don't. There's another reason why people think these big 00:42:34.000 --> 00:42:38.000 introns and exons, these big introns are helpful, 00:42:38.000 --> 00:42:42.000 and that is an evolutionary reason. The evolutionary reason is a little 00:42:42.000 --> 00:42:47.000 bit harder to follow, but let me try it on you. 00:42:47.000 --> 00:42:51.000 Suppose a random event happens and a chromosome breaks, 00:42:51.000 --> 00:42:55.000 that happens, and suppose a random breakage sticks one part of a 00:42:55.000 --> 00:43:00.000 chromosome to some other part of the chromosome. 00:43:00.000 --> 00:43:04.000 If it lands smack dab in the middle of the coding sequence of a gene 00:43:04.000 --> 00:43:09.000 that's bad new. But it turns out that if it lands 00:43:09.000 --> 00:43:13.000 in the introns of two different genes and sticks them together it 00:43:13.000 --> 00:43:18.000 could make a new gene that would still work. By having a random 00:43:18.000 --> 00:43:23.000 break between two genes in their introns and slamming them together, 00:43:23.000 --> 00:43:27.000 you could make a gene that had a bunch of exons from one gene and a 00:43:27.000 --> 00:43:32.000 bunch of exons from another gene. And this intervening sequence in the 00:43:32.000 --> 00:43:38.000 middle and it would get spliced up. Evolution might like that because 00:43:38.000 --> 00:43:44.000 it would be a very easy way for evolution to build new genes that 00:43:44.000 --> 00:43:49.000 had a portion of one protein and a portion of another protein. 00:43:49.000 --> 00:43:55.000 This kind of mix and match domain swapping could be very useful. 00:43:55.000 --> 00:44:01.000 And when we look across genomes, we see lots and lots of examples of 00:44:01.000 --> 00:44:07.000 genes that have a similar first half but different second halves. 00:44:07.000 --> 00:44:10.000 Or have some portion in the middle, a domain that we recognize, that we 00:44:10.000 --> 00:44:13.000 see in multiple proteins. And so, in fact, an argument for 00:44:13.000 --> 00:44:17.000 why we have all of this intronic DNA, one that's impossible to prove but 00:44:17.000 --> 00:44:20.000 is an argument is that from an evolution point of view, 00:44:20.000 --> 00:44:24.000 this allows a great deal of evolutionary innovation. 00:44:24.000 --> 00:44:27.000 You have to be careful that you say those organisms that have this extra 00:44:27.000 --> 00:44:31.000 space are able to mix and match and create more new kinds of combination 00:44:31.000 --> 00:44:34.000 proteins, et cetera, et cetera, and therefore survived 00:44:34.000 --> 00:44:38.000 better, et cetera, et cetera, et cetera. 00:44:38.000 --> 00:44:41.000 Why don't bacteria have this? Sorry? They're not as complicated. 00:44:41.000 --> 00:44:45.000 That's one though is we can take a sort of condescending attitude to 00:44:45.000 --> 00:44:48.000 these bacteria. They're not very, 00:44:48.000 --> 00:44:52.000 they're just not so complicated. There's another point of view which 00:44:52.000 --> 00:44:56.000 is bacteria are far more sophisticated than we are because 00:44:56.000 --> 00:45:00.000 they're under incredibly rigorous evolutionary selection. 00:45:00.000 --> 00:45:04.000 You might argue that if I'm a bacteria, can I really afford all 00:45:04.000 --> 00:45:08.000 this extra DNA? Now, the metabolic cost of all that 00:45:08.000 --> 00:45:12.000 extra DNA is huge to a bacteria which competes on replication. 00:45:12.000 --> 00:45:16.000 It's got to divide every 20 minutes, and trying to put in all these extra 00:45:16.000 --> 00:45:20.000 bases would be very news. So, you might imagine, just to be, 00:45:20.000 --> 00:45:24.000 you know, stand things on its head, that early life all had introns and 00:45:24.000 --> 00:45:28.000 bacteria, in the process of competing to be more and more 00:45:28.000 --> 00:45:31.000 efficient go rid of their introns. There's actually a large camp of 00:45:31.000 --> 00:45:34.000 people who think it went that way, that early life evolved with introns, 00:45:34.000 --> 00:45:37.000 and then bacteria, in the pressure to compete, 00:45:37.000 --> 00:45:41.000 got rid of them. And there's some evidence to support that. 00:45:41.000 --> 00:45:44.000 Bacteria don't have introns. Small eukaryotes like yeast that 00:45:44.000 --> 00:45:47.000 sort of do compete on replication have some introns, 00:45:47.000 --> 00:45:50.000 but a small number. There are only about 250 introns in 00:45:50.000 --> 00:45:53.000 yeast. Only about 5% of the genes have an intron and they're small. 00:45:53.000 --> 00:45:57.000 Bigger eukaryotes have bigger introns. And the bigger you get, 00:45:57.000 --> 00:46:00.000 often on average the bigger the genome sizes are the more 00:46:00.000 --> 00:46:03.000 you can tolerate it. And so I actually think, 00:46:03.000 --> 00:46:07.000 I actually probably favor this notion that introns were the 00:46:07.000 --> 00:46:10.000 original state and they've been gotten rid of. 00:46:10.000 --> 00:46:14.000 And the more pressure you're under to replicate rapidly the less you 00:46:14.000 --> 00:46:17.000 can tolerate this interesting and complicated innovation. 00:46:17.000 --> 00:46:21.000 Anyway, that's another way that things differ. 00:46:21.000 --> 00:46:24.000 And then, finally, viruses can do it either way. 00:46:24.000 --> 00:46:28.000 Viruses, depending on whether they are prokaryotic viruses or 00:46:28.000 --> 00:46:31.000 eukaryotic viruses, are able to replicate, 00:46:31.000 --> 00:46:35.000 are able to either do or don't have splicing. 00:46:35.000 --> 00:46:41.000 Last topic. Translation. Here eukaryotes are relatively 00:46:41.000 --> 00:46:48.000 simple. You get a message, you get a gene, you get an mRNA. 00:46:48.000 --> 00:46:54.000 The mRNA goes to the ribosome. Here's a ribosome. 00:46:54.000 --> 00:47:01.000 The ribosome goes to the mRNA, actually, and it starts turning out 00:47:01.000 --> 00:47:09.000 one protein as it chugs along. Prokaryotes differ in an interesting 00:47:09.000 --> 00:47:18.000 way. I get a promoter here that is transcribed into my mRNA, 00:47:18.000 --> 00:47:27.000 but it turns out that the mRNA can encode multiple independent proteins, 00:47:27.000 --> 00:47:36.000 protein one, protein two, protein three on the same mRNA. 00:47:36.000 --> 00:47:40.000 And a ribosome will hop on here and synthesize this one. 00:47:40.000 --> 00:47:44.000 A ribosome will hop on here and synthesize this one, 00:47:44.000 --> 00:47:48.000 and a ribosome will hop on here and synthesize that one. 00:47:48.000 --> 00:47:52.000 And you have what is called a polycistronic message. 00:47:52.000 --> 00:47:56.000 Poly, many. Cystronic, cystrons were an old name for coding 00:47:56.000 --> 00:48:00.000 regions of genes here. Polycystronic messages. 00:48:00.000 --> 00:48:03.000 Why would you want to do that, have a single mRNA that encodes 00:48:03.000 --> 00:48:06.000 multiple distinct proteins, each starting with its own ribosome 00:48:06.000 --> 00:48:09.000 start site there? Efficiency. Maybe, 00:48:09.000 --> 00:48:12.000 in fact, these would be, how about, oh, this would be cleaver, 00:48:12.000 --> 00:48:15.000 make them multiple steps in a biochemical pathway? 00:48:15.000 --> 00:48:18.000 Have them coded on a single messenger so then you'd only have to 00:48:18.000 --> 00:48:21.000 worry about regulating that once. If you have the regulatory 00:48:21.000 --> 00:48:24.000 machinery to turn on, you'll make all the enzymes for the 00:48:24.000 --> 00:48:27.000 pathway. And that's exactly what bacteria do. They tend to put all 00:48:27.000 --> 00:48:30.000 the enzymes for a pathway on a single message so when they want to 00:48:30.000 --> 00:48:33.000 call up, let's digest hexose this morning, they have a whole thing 00:48:33.000 --> 00:48:37.000 that will let them be able to do that, poly-cystronic. 00:48:37.000 --> 00:48:41.000 That's because they're small genomes. They're pressed for space. 00:48:41.000 --> 00:48:46.000 And, because of that, they have to slam a lot into a single unit. 00:48:46.000 --> 00:48:50.000 And this single unit that has multiple genes encoded in a single 00:48:50.000 --> 00:48:55.000 message is called an operon, and we'll talk more about that. 00:48:55.000 --> 00:49:00.000 Last of all viruses. Viruses. Viruses have very little room. 00:49:00.000 --> 00:49:05.000 Their genomes can be tiny. A typical virus might have a genome 00:49:05.000 --> 00:49:10.000 of 5,000 bases to 10, 00 bases to, in some cases, 00:49:10.000 --> 00:49:15.000 200,000 bases, but it hasn't got a lot of room. It wants to pack a lot 00:49:15.000 --> 00:49:21.000 of protein coating information in. And some viruses have come up with 00:49:21.000 --> 00:49:26.000 the most extraordinary way of doing that. Some viruses have gone to the 00:49:26.000 --> 00:49:32.000 extreme of having RNAs that get made from them that have a sequence -- 00:49:32.000 --> 00:49:39.000 I'm just going to pick up in the middle of the sequence here. 00:49:39.000 --> 00:49:46.000 A-C-U-A-C-U-A-C-U-A-C-U. You might decide to read the sequence like 00:49:46.000 --> 00:49:53.000 this, that those are the codons, and you'd get a certain protein. 00:49:53.000 --> 00:50:00.000 But I might also decide to read that sequence C-U-A-C-U-A-C-U-A. 00:50:00.000 --> 00:50:04.000 And, of course, I'm giving this in a repeating form 00:50:04.000 --> 00:50:08.000 because it's easy to note. I could give you any sequence and I 00:50:08.000 --> 00:50:12.000 could read it in this reading frame, I could read it in this reading 00:50:12.000 --> 00:50:17.000 frame, or I could read it as U-A-C-U-A-C-U-A-C. 00:50:17.000 --> 00:50:21.000 In other words, there are three reading frames that, 00:50:21.000 --> 00:50:25.000 in principle, you could translate a protein from. In a typical 00:50:25.000 --> 00:50:30.000 prokaryotic gene or eukaryotic gene only one of those is used. 00:50:30.000 --> 00:50:34.000 You start at the first AUG and that sets the reading frame. 00:50:34.000 --> 00:50:39.000 But some viruses are so pressed for space and are so cleaver and are so 00:50:39.000 --> 00:50:43.000 efficient that they make messages that have tricks that they actually 00:50:43.000 --> 00:50:48.000 use two or, in some cases, all three reading frames, which is 00:50:48.000 --> 00:50:53.000 an extraordinary packing of information density into a simple 00:50:53.000 --> 00:50:57.000 message. So, the basic point. We have a simple model. DNA is 00:50:57.000 --> 00:51:02.000 replicated. Transcribed into RNA. 00:51:02.000 --> 00:51:06.000 Translated into protein. But there are a lot of important 00:51:06.000 --> 00:51:11.000 variations between eukaryotes, prokaryotes and viruses. And 00:51:11.000 --> 00:51:15.000 understanding them can be useful for treating cancer, 00:51:15.000 --> 00:51:20.000 for treating AIDS, and for treating viral and bacterial 00:51:20.000 --> 00:51:25.000 infections. Next time.