WEBVTT 00:00:01.000 --> 00:00:04.000 Among the issues that some people asked that should be discussed in 00:00:04.000 --> 00:00:08.000 greater detail should be the structure of proteins. 00:00:08.000 --> 00:00:12.000 I'll touch on it very briefly this morning, different kinds of bonding, 00:00:12.000 --> 00:00:17.000 tertiary and quaternary structure, condensation or dehydration 00:00:17.000 --> 00:00:21.000 reactions. And, in fact, many of those issues should 00:00:21.000 --> 00:00:26.000 be addressed in the recitation sections. 00:00:26.000 --> 00:00:30.000 That's the ideal place to begin to clarify things which although they 00:00:30.000 --> 00:00:34.000 were mentioned here may not have been mentioned in the degree of 00:00:34.000 --> 00:00:38.000 detail that you really need to assimilate them properly. 00:00:38.000 --> 00:00:42.000 And I urge you to raise these issues with the recitation section 00:00:42.000 --> 00:00:46.000 instructors. That's exactly what they're there for. 00:00:46.000 --> 00:00:50.000 Having said that I just want to dip back briefly into protein structure, 00:00:50.000 --> 00:00:54.000 even though we turned our back on it at the end of last time, 00:00:54.000 --> 00:00:58.000 just to reinforce some things that I realized I should have mentioned 00:00:58.000 --> 00:01:01.000 perhaps in greater detail. Here for the example are different 00:01:01.000 --> 00:01:05.000 ways of depicting the three-dimensional structure of the 00:01:05.000 --> 00:01:08.000 protein. And, by the way, we see that these are 00:01:08.000 --> 00:01:11.000 beta pleated sheets in the light brown and these are alpha helices. 00:01:11.000 --> 00:01:15.000 There are two of them here in green, one going this way, 00:01:15.000 --> 00:01:18.000 the other going this way, a third one going this way. 00:01:18.000 --> 00:01:22.000 And the other blue areas are not structured, i. 00:01:22.000 --> 00:01:25.000 ., they're not structured in the sense that they are in any way 00:01:25.000 --> 00:01:29.000 obviously alpha helices or beta pleated sheets. 00:01:29.000 --> 00:01:32.000 Here's a space-filling model, a space-filling depiction of a 00:01:32.000 --> 00:01:36.000 protein. We talked about that last time. Here is a trace of the 00:01:36.000 --> 00:01:40.000 backbone, of the peptide backbone of the same protein where the side 00:01:40.000 --> 00:01:44.000 chains are left out, and obviously where one is only 00:01:44.000 --> 00:01:47.000 plotting the three-dimensional coordinates of each of the backbone 00:01:47.000 --> 00:01:51.000 atoms, CCN, CCN, CCN. Here is yet another way of 00:01:51.000 --> 00:01:55.000 plotting exactly the same protein in terms of indicating, 00:01:55.000 --> 00:01:59.000 as we just said, the structure of these alpha helices in 00:01:59.000 --> 00:02:03.000 the other regions. That is the secondary structure of 00:02:03.000 --> 00:02:07.000 this protein. And here's yet a fourth way of plotting, 00:02:07.000 --> 00:02:11.000 of depicting the same structure of the protein where roughly one is 00:02:11.000 --> 00:02:16.000 depicting the configuration of the amino acids in terms of a large 00:02:16.000 --> 00:02:20.000 sausage. Excuse me. If one were to use a space-filling 00:02:20.000 --> 00:02:24.000 model we'd go up to here. So these are just four ways of 00:02:24.000 --> 00:02:29.000 looking at the same protein with different degrees of simplification. 00:02:29.000 --> 00:02:33.000 Another point that I thought I would like to reinforce and make was the 00:02:33.000 --> 00:02:37.000 following. We've talked about transmembrane proteins in the past. 00:02:37.000 --> 00:02:41.000 That is, proteins which protrude through a membrane from one side to 00:02:41.000 --> 00:02:46.000 the other. And a point that I realized I'd like to make is that if 00:02:46.000 --> 00:02:50.000 we look at a transmembrane protein here's one that is starting out in 00:02:50.000 --> 00:02:54.000 the cytoplasm of a cell. And, by the way, the soluble part 00:02:54.000 --> 00:02:59.000 of the cytoplasm is sometimes called the cytosol. 00:02:59.000 --> 00:03:03.000 Here is the lipid bilayer that we talked about at length and here is 00:03:03.000 --> 00:03:07.000 the extracellular domain of this same protein. Now, 00:03:07.000 --> 00:03:11.000 how is all this organized? Well, the fact of the matter is we 00:03:11.000 --> 00:03:15.000 discussed the fact that this hydrophobic space in the lipid 00:03:15.000 --> 00:03:19.000 bilayer is so hydrophobic that it really doesn't like to be in the 00:03:19.000 --> 00:03:23.000 presence of hydrophilic molecules, including in this case amino acids. 00:03:23.000 --> 00:03:27.000 And what we see here is the fact that almost all of the amino acids 00:03:27.000 --> 00:03:31.000 in this region of the protein, which is called the transmembrane 00:03:31.000 --> 00:03:35.000 region of the protein because it reaches from one side to the other, 00:03:35.000 --> 00:03:39.000 are all hydrophobic or neutral amino acids which are reasonably 00:03:39.000 --> 00:03:43.000 comfortable in the hydrophobic space of the lipid bilayer. 00:03:43.000 --> 00:03:47.000 There happens to be two apparent violators of this, 00:03:47.000 --> 00:03:51.000 glutamine and histidine. You see these two here? I mean 00:03:51.000 --> 00:03:55.000 glutamic acid and histidine. Glutamic acid and histidine. 00:03:55.000 --> 00:03:59.000 One is negatively charged and therefore is highly hydrophilic. 00:03:59.000 --> 00:04:03.000 The other is positively charged and is therefore highly hydrophilic. 00:04:03.000 --> 00:04:07.000 And on the surface that would seem to violate the rule I just 00:04:07.000 --> 00:04:11.000 articulated. But the fact is that as it turns out in the particular 00:04:11.000 --> 00:04:15.000 protein these two charges, these two amino acids are so closely 00:04:15.000 --> 00:04:19.000 juxtaposed with one another that their positive and negative charges 00:04:19.000 --> 00:04:23.000 are used to neutralize one another. And as a consequence in effect 00:04:23.000 --> 00:04:27.000 there is no strong charging or polarity in this area 00:04:27.000 --> 00:04:32.000 or in this area. The take-home lesson is that somehow 00:04:32.000 --> 00:04:36.000 proteins manage to insert themselves and to remain stable in the lipid 00:04:36.000 --> 00:04:41.000 bilayer by virtue of either using only stretches of hydrophobic or 00:04:41.000 --> 00:04:45.000 nonpolar amino acids or they use tricks like this of neutralizing any 00:04:45.000 --> 00:04:50.000 charges that happen to be there. Note, by the way, that because 00:04:50.000 --> 00:04:55.000 there are hydrophilic amino acids down here and there turn out to be 00:04:55.000 --> 00:04:59.000 hydrophilic amino acid around here, arginine, and here there's a whole 00:04:59.000 --> 00:05:03.000 bunch of basic amino acids. Note that this keeps the 00:05:03.000 --> 00:05:07.000 transmembrane protein from getting pulled in one direction or the other 00:05:07.000 --> 00:05:10.000 because this arginine likes to associate with the negative 00:05:10.000 --> 00:05:14.000 phosphates on the outside of the phospholipids. 00:05:14.000 --> 00:05:18.000 And the same thing is here. And all that means is that this 00:05:18.000 --> 00:05:21.000 transmembrane protein is firmly anchored in the lipid bilayer, 00:05:21.000 --> 00:05:25.000 a point we'll talk about later in greater detail when we talk about 00:05:25.000 --> 00:05:29.000 membrane structure. One other little point I'll mention 00:05:29.000 --> 00:05:33.000 here in passing, which we'll also get into in greater 00:05:33.000 --> 00:05:38.000 detail, is that once a protein has been polymerized that polymerization 00:05:38.000 --> 00:05:42.000 is not the last thing that happens to it once it's polymerized and 00:05:42.000 --> 00:05:46.000 folded into place because we know that proteins undergo what is called 00:05:46.000 --> 00:05:51.000 post-translational modifications. And, as we'll talk about in the 00:05:51.000 --> 00:05:55.000 coming weeks, the process of synthesizing a protein 00:05:55.000 --> 00:06:00.000 is called translation. And when we talk about 00:06:00.000 --> 00:06:04.000 post-translational modification what we're talking about is opening our 00:06:04.000 --> 00:06:08.000 eyes to the possibility that even after the primary amino acid 00:06:08.000 --> 00:06:13.000 sequence has been polymerized there are chemical alterations that can 00:06:13.000 --> 00:06:17.000 subsequently be imposed on the amino acid side chains to further modify 00:06:17.000 --> 00:06:22.000 the protein. One such modification, by example, is a proteolytic 00:06:22.000 --> 00:06:26.000 degradation. And when I talk about proteolytic degradation, 00:06:26.000 --> 00:06:31.000 I'm talking about the fact that one can break down a protein. 00:06:31.000 --> 00:06:35.000 Proteolysis is the breaking down of a protein. And when we talk about 00:06:35.000 --> 00:06:39.000 degradation we're talking about destroying what has been synthesized. 00:06:39.000 --> 00:06:44.000 In the case of many proteins, once they're synthesized there may 00:06:44.000 --> 00:06:48.000 be a stretch of amino acids at one end or the other that simply clipped 00:06:48.000 --> 00:06:53.000 off therefore creating a protein which is smaller than the initially 00:06:53.000 --> 00:06:57.000 synthesized product of protein synthesis, i.e. 00:06:57.000 --> 00:07:02.000 the initially synthesized product of translation. 00:07:02.000 --> 00:07:07.000 Here we see yet another kind of post-translational modification, 00:07:07.000 --> 00:07:12.000 because it turns out that in many proteins which protrude into the 00:07:12.000 --> 00:07:18.000 extracellular space there is yet another kind of covalent 00:07:18.000 --> 00:07:23.000 modification which is the process of glycosylation in which a series of 00:07:23.000 --> 00:07:28.000 sugar side chains, carbohydrate side chains is 00:07:28.000 --> 00:07:34.000 covalently attached to the polypeptide chain usually on serines 00:07:34.000 --> 00:07:39.000 or threonines using the hydroxyl of the side chain of serines or 00:07:39.000 --> 00:07:45.000 threonines to attach these oligosaccharide side chains. 00:07:45.000 --> 00:07:49.000 We know from our discussion the last time oligosaccharide means an 00:07:49.000 --> 00:07:53.000 assembly of a small number of monosaccharides. 00:07:53.000 --> 00:07:58.000 And each of these blue hexagons represents a monosaccharide which 00:07:58.000 --> 00:08:02.000 are covalently linked and also modify the extracellular domain of 00:08:02.000 --> 00:08:07.000 this protein as it protrudes into the extracellular space. 00:08:07.000 --> 00:08:11.000 So I'm just opening our eyes to the possibility that in the future we're 00:08:11.000 --> 00:08:15.000 going to talk about yet other ways in which proteins are modified to 00:08:15.000 --> 00:08:20.000 further tune-up their structure to make them more suitable, 00:08:20.000 --> 00:08:24.000 more competent to do the various jobs to which they've been assigned. 00:08:24.000 --> 00:08:29.000 Let's therefore return to what we talked about the last time, 00:08:29.000 --> 00:08:33.000 the fact that the structure of nucleic acids is based on 00:08:33.000 --> 00:08:38.000 this simple principle. Here, by the way, 00:08:38.000 --> 00:08:42.000 I'm returning to the notion of this numbering system. 00:08:42.000 --> 00:08:46.000 We're talking about a pentose nucleic acid. The fact that there 00:08:46.000 --> 00:08:50.000 are two hydroxyls here right away tells us that we're looking at a 00:08:50.000 --> 00:08:54.000 ribose rather than a deoxyribose which, as I said last time, 00:08:54.000 --> 00:08:58.000 lacks this sugar right there. Note, as we've said repeatedly, 00:08:58.000 --> 00:09:03.000 that the hydroxyl side chains of carbohydrates offer numerous 00:09:03.000 --> 00:09:09.000 opportunities for using dehydration reactions, or as they're sometimes 00:09:09.000 --> 00:09:14.000 called condensation reactions where you remove a water, 00:09:14.000 --> 00:09:19.000 where you take out a water, dehydration, or we can call them 00:09:19.000 --> 00:09:25.000 condensation reactions to attach yet other things. And, 00:09:25.000 --> 00:09:30.000 in fact, in principle there are actually four different hydroxyls 00:09:30.000 --> 00:09:35.000 that could be used here to do that. There's one here, 00:09:35.000 --> 00:09:39.000 there's one here, one here and one here. There are four different 00:09:39.000 --> 00:09:44.000 hydroxyls. The 1, the 2, the 3 and the 5 hydroxyl are, 00:09:44.000 --> 00:09:48.000 in principle, opportunities for further modification. 00:09:48.000 --> 00:09:53.000 In truth the 2-prime hydroxyl is rarely used, as we'll discuss 00:09:53.000 --> 00:09:57.000 shortly, but the main actors are therefore this hydroxyl here in 00:09:57.000 --> 00:10:02.000 which a condensation reaction has created a glycosidic bond. 00:10:02.000 --> 00:10:06.000 That is a bond between a sugar and a non-sugar entity. 00:10:06.000 --> 00:10:11.000 Glyco refers obviously to sugars like glycogen or glycosylation we've 00:10:11.000 --> 00:10:15.000 talked about before. Here a bond has been made between a 00:10:15.000 --> 00:10:20.000 base, and we'll talk about the different bases shortly, 00:10:20.000 --> 00:10:25.000 and the 1-prime hydroxyl of the ribose. Over here at the 5-prime 00:10:25.000 --> 00:10:30.000 hydroxyl yet another condensation reaction. 00:10:30.000 --> 00:10:34.000 Sometimes this is called an esterification reaction. 00:10:34.000 --> 00:10:39.000 And again esterification refers to these kinds of condensation 00:10:39.000 --> 00:10:44.000 reactions where an acid and a base react with one another, 00:10:44.000 --> 00:10:48.000 and once again through a condensation reaction, 00:10:48.000 --> 00:10:53.000 yield the removal of a water. And let's look at what's happening 00:10:53.000 --> 00:10:58.000 here, because not only is one phosphate group attached to the 00:10:58.000 --> 00:11:03.000 5-prime carbon, to the 5-prime hydroxyl. 00:11:03.000 --> 00:11:07.000 In fact, there are three. And they are located, and each of 00:11:07.000 --> 00:11:11.000 them has a name. The inboard one is called alpha, 00:11:11.000 --> 00:11:15.000 moving further out is beta, and furthest out is gamma. 00:11:15.000 --> 00:11:19.000 And it turns out that this chain of phosphates have very important 00:11:19.000 --> 00:11:23.000 implications for energy metabolism and for biosynthesis. 00:11:23.000 --> 00:11:27.000 Why? I'm glad I asked that question. Because these are all 00:11:27.000 --> 00:11:31.000 three highly negatively charged. This is negatively charged, 00:11:31.000 --> 00:11:35.000 this is and this is. And, as you know, negative charges repel one 00:11:35.000 --> 00:11:39.000 another. And as a consequence, to create a triphosphate linkage 00:11:39.000 --> 00:11:43.000 like this represents pushing together negative charged moieties, 00:11:43.000 --> 00:11:47.000 these three phosphates, even though they don't like to be next to one 00:11:47.000 --> 00:11:51.000 another. And that pushing together, that creation of the triphosphate 00:11:51.000 --> 00:11:55.000 chain represents an investment of energy. And once the three are 00:11:55.000 --> 00:11:59.000 pushed together that represents great potential energy much like a 00:11:59.000 --> 00:12:03.000 spring that has been compressed together and would just 00:12:03.000 --> 00:12:07.000 love to pop apart. These three phosphates would love to 00:12:07.000 --> 00:12:11.000 pop apart from one another by virtue of the fact that these negative 00:12:11.000 --> 00:12:16.000 charges are mutually repelling. But they cannot as long as they're 00:12:16.000 --> 00:12:20.000 in this triphosphate configuration. But once the triphosphate 00:12:20.000 --> 00:12:25.000 configuration is broken then the energy released by their leaving one 00:12:25.000 --> 00:12:30.000 another can then be exploited for yet other purposes. 00:12:30.000 --> 00:12:34.000 Keep in mind, just to reinforce what I said a second ago, 00:12:34.000 --> 00:12:38.000 the difference between a ribose and a deoxyribose is the presence or the 00:12:38.000 --> 00:12:42.000 absence of this oxygen. And now let's focus in a little 00:12:42.000 --> 00:12:46.000 more detail on the bases because the bases are indeed the subject of much 00:12:46.000 --> 00:12:50.000 of our discussion today. And we have two basic kinds of 00:12:50.000 --> 00:12:54.000 bases. They're called nitrogenous bases, these bases, 00:12:54.000 --> 00:12:59.000 because they have nitrogen in them. And if you look at the five bases 00:12:59.000 --> 00:13:03.000 that are depicted here you'll see that they are not aromatic rings 00:13:03.000 --> 00:13:08.000 with just carbons in them like a six carbon benzene. 00:13:08.000 --> 00:13:12.000 Rather all of them have a substantial fraction of nitrogens 00:13:12.000 --> 00:13:16.000 actually in the ring, two in the case of these pyrimidines. 00:13:16.000 --> 00:13:21.000 And here you see the number actually is four. 00:13:21.000 --> 00:13:25.000 In fact, one of these nitrogenous bases indicated here, 00:13:25.000 --> 00:13:30.000 guanine has actually a fifth one up here as a side chain. 00:13:30.000 --> 00:13:34.000 This is outside of the chain, it represents a side group. And if 00:13:34.000 --> 00:13:38.000 we begin now to make distinctions between the ring itself and the 00:13:38.000 --> 00:13:42.000 entities that protrude out of the ring, they really represent some of 00:13:42.000 --> 00:13:46.000 the important distinguishing characteristics. 00:13:46.000 --> 00:13:50.000 It's important that we understand that pyrimidines have one ring and 00:13:50.000 --> 00:13:54.000 these have two rings in them. The purines have a five and a six 00:13:54.000 --> 00:13:58.000 membered ring fused together, as you can see. The pyrimidines 00:13:58.000 --> 00:14:02.000 have only a six membered ring. And what's really important in 00:14:02.000 --> 00:14:06.000 determining their identity is not the basic pyrimidine or purine 00:14:06.000 --> 00:14:10.000 structure. It's once again the side chains that distinguish these one 00:14:10.000 --> 00:14:14.000 from the other. Here in the case of cytosine we see 00:14:14.000 --> 00:14:18.000 that there's a carbonyl here, an oxygen sticking out, and there's 00:14:18.000 --> 00:14:22.000 an amine over here. We see uracil which happens to be 00:14:22.000 --> 00:14:26.000 present in RNA but not DNA which has two carbonyls here and here. 00:14:26.000 --> 00:14:30.000 Obviously, therefore what distinguishes these two from one 00:14:30.000 --> 00:14:34.000 another is this oxygen versus this amine. 00:14:34.000 --> 00:14:37.000 And here we see the thymine which is present in DNA but not RNA. 00:14:37.000 --> 00:14:41.000 And this will become very familiar to you shortly. 00:14:41.000 --> 00:14:44.000 This looks just like uracil except for the fact that there's a methyl 00:14:44.000 --> 00:14:48.000 group sticking out here. Now, very important for our 00:14:48.000 --> 00:14:51.000 understanding of what's happening here is the fact that this methyl 00:14:51.000 --> 00:14:55.000 group, although it distinguishes thymine from uracil is itself 00:14:55.000 --> 00:14:59.000 biologically actually very important. 00:14:59.000 --> 00:15:03.000 It's there to be sure and it's a distinguishing mark of T versus U, 00:15:03.000 --> 00:15:07.000 but the business end of T versus U in terms of encoding information 00:15:07.000 --> 00:15:11.000 happens here with these two oxygens sticking out. They're the important 00:15:11.000 --> 00:15:15.000 oxygens, here and here. And therefore from the point of 00:15:15.000 --> 00:15:20.000 view of information content, as we'll soon see, T and U are 00:15:20.000 --> 00:15:24.000 essentially equivalent. It may be that one of them happens 00:15:24.000 --> 00:15:28.000 to be in RNA and the other in DNA, but from the point of view of 00:15:28.000 --> 00:15:32.000 understanding the coding information they carry it's these two carbonyls 00:15:32.000 --> 00:15:37.000 here and here which dictate essentially their identity. 00:15:37.000 --> 00:15:41.000 We have the same kind of dynamics that operate here in the case of A 00:15:41.000 --> 00:15:45.000 and G where once again this one has only an amine side chain and this 00:15:45.000 --> 00:15:49.000 one has a carbonyl and an amine side chain right here. 00:15:49.000 --> 00:15:53.000 Now, very important there is a confusing array of names that are 00:15:53.000 --> 00:15:57.000 associated with all this. I don't know if it you can, 00:15:57.000 --> 00:16:01.000 well, it reads reasonably well. Because once a base, 00:16:01.000 --> 00:16:05.000 and I just showed you bases which are unattached to the sugars, 00:16:05.000 --> 00:16:10.000 once bases are attached to the sugars they change their name 00:16:10.000 --> 00:16:14.000 slightly. So keep in mind that here, when we talk about these nitrogenous 00:16:14.000 --> 00:16:18.000 bases, the bases are just free molecules where in each case this 00:16:18.000 --> 00:16:23.000 lowest nitrogen is the one that participates in the formation of a 00:16:23.000 --> 00:16:27.000 covalent glycosidic bond with the ribose or the deoxyribose 00:16:27.000 --> 00:16:32.000 underneath it. And here we can see one indication 00:16:32.000 --> 00:16:37.000 of how that, you see this N, in all cases via a condensation 00:16:37.000 --> 00:16:42.000 reaction, forms a covalent bond with a five carbon sugar, 00:16:42.000 --> 00:16:47.000 once again deoxyribose or ribose. Once the base associates with the 00:16:47.000 --> 00:16:52.000 sugar, that is the base plus the sugar is called a nucleoside. 00:16:52.000 --> 00:16:57.000 So when we talk in polite company about a nucleoside we're not talking 00:16:57.000 --> 00:17:02.000 about free bases. We're talking about the covalent 00:17:02.000 --> 00:17:08.000 interaction of a pentose binding to a base. The pentose could be one or 00:17:08.000 --> 00:17:13.000 the other of these two. And that's what a nucleoside is. 00:17:13.000 --> 00:17:19.000 If on top of that we add additionally one or more phosphates 00:17:19.000 --> 00:17:24.000 then we even modify our language even further because a base attached 00:17:24.000 --> 00:17:30.000 to a sugar which in turn is attached to a phosphate is called 00:17:30.000 --> 00:17:34.000 a nucleotide. The nucleotide, 00:17:34.000 --> 00:17:38.000 the T is there to designate the fact that there's actually, 00:17:38.000 --> 00:17:42.000 in addition to the base and the sugar there's a phosphate which is 00:17:42.000 --> 00:17:46.000 attached and extends off the end. And there are slightly different 00:17:46.000 --> 00:17:50.000 names. For the purposes of this course we won't get into this very 00:17:50.000 --> 00:17:54.000 arcane nomenclature because it is, to be frank, and you know I always 00:17:54.000 --> 00:17:58.000 am frank with you, confusing. Here is U. 00:17:58.000 --> 00:18:02.000 And when uracil, the base becomes linked to a ribose 00:18:02.000 --> 00:18:07.000 it changes its name from uracil to uridine. Cytosine changes its name 00:18:07.000 --> 00:18:12.000 to cytidine when it becomes a nucleoside by a covalent linkage to 00:18:12.000 --> 00:18:17.000 either ribose or deoxyribose. Thymine becomes thymidine. And the 00:18:17.000 --> 00:18:21.000 same nomenclature exists, the shift in their names exists in 00:18:21.000 --> 00:18:26.000 the case of the purines as well, adenine becomes adenosine and so 00:18:26.000 --> 00:18:31.000 forth. We need to focus mostly on the 00:18:31.000 --> 00:18:35.000 notion of A, C, T, G and U. Those are the things we 00:18:35.000 --> 00:18:39.000 need to think about. And why is this nomenclature 00:18:39.000 --> 00:18:43.000 confusing? Well, here the nucleoside ends with osine, 00:18:43.000 --> 00:18:48.000 O-S-I-N-E. You see that here? You say that's easy to remember, 00:18:48.000 --> 00:18:52.000 but look up here. Here the base ends with O-S-I-N-E. 00:18:52.000 --> 00:18:56.000 And so this nomenclature which was cobbled together in the early 20th 00:18:56.000 --> 00:19:00.000 century will bedevil us and generations of biology students to 00:19:00.000 --> 00:19:05.000 come. Oh well, that's life. Now, one of the things we're 00:19:05.000 --> 00:19:09.000 interested in and which I talked about briefly last time was the 00:19:09.000 --> 00:19:14.000 whole notion of polymerization, i.e., how we actually polymerize a 00:19:14.000 --> 00:19:18.000 chain. Let's look at this illustration which I think is more 00:19:18.000 --> 00:19:23.000 useful. Recall the fact that I emphasized with great seriousness 00:19:23.000 --> 00:19:27.000 the fact that nucleic acid synthesis always occurs in a certain polarity. 00:19:27.000 --> 00:19:32.000 It goes in a certain direction. You cannot add nucleotides on one 00:19:32.000 --> 00:19:36.000 end or the other end willy-nilly. You can only add them onto the 00:19:36.000 --> 00:19:41.000 3-prime end. And keep in mind that the reason why this is defined as 00:19:41.000 --> 00:19:45.000 the 5-prime end is that this is, the last hydroxyl sticking out at 00:19:45.000 --> 00:19:49.000 this end comes out of the 5-prime carbon right here, 00:19:49.000 --> 00:19:54.000 the 5-prime hydroxyl. And conversely at this end we're 00:19:54.000 --> 00:19:58.000 adding another base at the 3-prime hydroxyl, at this end, 00:19:58.000 --> 00:20:03.000 which creates the 3-prime end of the DNA or the RNA. 00:20:03.000 --> 00:20:08.000 In fact, the polymerization always occurs between the 5-prime end of a 00:20:08.000 --> 00:20:13.000 deoxyribonucleotide indicated here where the bases remain anonymous and 00:20:13.000 --> 00:20:18.000 the 3-prime hydroxyl. That's the way it always happens. 00:20:18.000 --> 00:20:24.000 And here we begin to appreciate the role of the high energy 00:20:24.000 --> 00:20:29.000 phosphate linkage. Because this high energy 00:20:29.000 --> 00:20:34.000 triphosphate linkage, which is synthesized elsewhere in 00:20:34.000 --> 00:20:39.000 the cell like a coiled spring and which contains a lot of potential 00:20:39.000 --> 00:20:43.000 energy by virtue of this mutual negative repulsion of the phosphate 00:20:43.000 --> 00:20:48.000 groups, this energy is used to form the bond here between the phosphate 00:20:48.000 --> 00:20:53.000 in this condensation reaction and the 3-prime hydroxyl. 00:20:53.000 --> 00:20:58.000 So that requires an investment of energy. And the resulting linkage 00:20:58.000 --> 00:21:03.000 which is formed is sometimes called a phosphodiester linkage. 00:21:03.000 --> 00:21:06.000 Why phosphodiester? Well, obviously it's phospho. 00:21:06.000 --> 00:21:10.000 And there actually are two esterifications that are occurring 00:21:10.000 --> 00:21:14.000 here. If we look at one of these phosphodiester bonds we see that an 00:21:14.000 --> 00:21:17.000 ester linkage has been made with this hydroxyl and an ester linkage 00:21:17.000 --> 00:21:21.000 has been made with this hydroxyl. And for that reason it's called a 00:21:21.000 --> 00:21:25.000 phosphodiester linkage. Therefore we come to realize that 00:21:25.000 --> 00:21:29.000 polymerization of nucleic acids doesn't take place spontaneously. 00:21:29.000 --> 00:21:33.000 It requires the investment of a high-energy molecule, 00:21:33.000 --> 00:21:38.000 the investment of the energy that it carries. And when this linkage is 00:21:38.000 --> 00:21:42.000 formed the diphosphate here, the beta and the gamma phosphates 00:21:42.000 --> 00:21:47.000 float off into interstellar space. It's only the alpha phosphate that 00:21:47.000 --> 00:21:52.000 is retained to form the resulting diphosphate, a phosphodiester 00:21:52.000 --> 00:21:56.000 linkage. And this process can be repeated literally thousands and 00:21:56.000 --> 00:22:01.000 millions of times. An average human's chromosomes 00:22:01.000 --> 00:22:06.000 contains on the order of tens, fifty, a hundred mega-bases of DNA. 00:22:06.000 --> 00:22:10.000 A mega-base is a million bases or a million nucleotides. 00:22:10.000 --> 00:22:14.000 So there you can understand that there's no limit to the extent of 00:22:14.000 --> 00:22:18.000 elongation of these various kinds of molecules. Now, 00:22:18.000 --> 00:22:22.000 note by the way yet another feature of this which is that the 00:22:22.000 --> 00:22:26.000 distinguishing feature between DNA and RNA, the most important 00:22:26.000 --> 00:22:30.000 distinguishing feature is this 2-prime hydroxyl. 00:22:30.000 --> 00:22:34.000 And here we're talking about DNA, but we could almost in the same 00:22:34.000 --> 00:22:38.000 breath be talking about the way that RNA gets polymerized. 00:22:38.000 --> 00:22:42.000 Why? Because this 2-prime hydroxyl or this 2-prime hydrogen in this 00:22:42.000 --> 00:22:46.000 case is out of the line of fire. The business action is happening 00:22:46.000 --> 00:22:50.000 right along here. Look where the business action is 00:22:50.000 --> 00:22:54.000 in terms of the backbone. The 2-prime hydroxyl is off to the 00:22:54.000 --> 00:22:58.000 side. And whether it's oxygen or just whether it's OH, 00:22:58.000 --> 00:23:02.000 that is in ribose, a hydroxyl group or just a hydrogen, 00:23:02.000 --> 00:23:06.000 as is indicated here in the case of deoxyribose, is irrelevant 00:23:06.000 --> 00:23:10.000 to the polymerization. And therefore we can guess or intuit, 00:23:10.000 --> 00:23:14.000 and just because we guessed doesn't mean it's wrong, 00:23:14.000 --> 00:23:18.000 often it's right, it doesn't really make much 00:23:18.000 --> 00:23:22.000 difference whether we look at DNA or RNA. Here's a polymerization scheme 00:23:22.000 --> 00:23:26.000 of RNA and it's absolutely identical to that of DNA. 00:23:26.000 --> 00:23:30.000 In this case it's ribonucleotide triphosphates that are used for the 00:23:30.000 --> 00:23:35.000 polymerization reaction. Now here I just uttered the phrase 00:23:35.000 --> 00:23:41.000 ribonucleoside triphosphates. Why did I say that? Well, 00:23:41.000 --> 00:23:47.000 ultimately only the good Lord knows why I said that. 00:23:47.000 --> 00:23:53.000 But let's look at this phrase. I said ribonucleoside triphosphate 00:23:53.000 --> 00:23:59.000 rather than ribonucleotide triphosphate because the fact that I 00:23:59.000 --> 00:24:05.000 added this on the end makes the T there unnecessary. 00:24:05.000 --> 00:24:09.000 The T is there to indicate the phosphate being attached to the 00:24:09.000 --> 00:24:13.000 ribose or the deoxyribose. But if I'm adding this phrase over 00:24:13.000 --> 00:24:17.000 here, triphosphate, that obviates, that makes 00:24:17.000 --> 00:24:21.000 unnecessary my saying ribonucleotide triphosphate. If I'm looking at UTP 00:24:21.000 --> 00:24:25.000 or ATP, I would say I'm a ribonucleotide if I don't mention 00:24:25.000 --> 00:24:29.000 the triphosphate. But the moment this comes from my 00:24:29.000 --> 00:24:33.000 lips then we'll say ribonucleoside indicating that a ribonucleoside, 00:24:33.000 --> 00:24:37.000 that is a base and a sugar are then attached to one or more 00:24:37.000 --> 00:24:41.000 phosphate linkages. Now, the ultimate basis of the 00:24:41.000 --> 00:24:45.000 biological revolution comes from the realization that these different 00:24:45.000 --> 00:24:50.000 bases have complementarity to one another. That is they like to be 00:24:50.000 --> 00:24:54.000 together with one another. And if we look at this and we think 00:24:54.000 --> 00:24:58.000 about the DNA double helix we come to realize that these bases have 00:24:58.000 --> 00:25:03.000 affinities for one another. And the general affinity is one 00:25:03.000 --> 00:25:07.000 purine likes to be facing opposite one pyrimidine. 00:25:07.000 --> 00:25:12.000 One pyrimidine opposite one purine. And if we have two pyrimidines 00:25:12.000 --> 00:25:16.000 facing one another they're not close enough to one another to kiss. 00:25:16.000 --> 00:25:21.000 And if we have two purines they're too close to one another, 00:25:21.000 --> 00:25:25.000 they're bumping into one another, they take up too much space. And 00:25:25.000 --> 00:25:30.000 therefore the optimal configuration is one purine and one pyrimidine. 00:25:30.000 --> 00:25:34.000 And you can see these two pairings here in the case of what happens 00:25:34.000 --> 00:25:39.000 with DNA. In fact, the realization of this diagram 00:25:39.000 --> 00:25:44.000 right here is what triggered the discovery of DNA in 1953. 00:25:44.000 --> 00:25:49.000 This diagram right here is what triggered the biological revolution. 00:25:49.000 --> 00:25:53.000 And though it's been depicted in many, many ways it's worthwhile 00:25:53.000 --> 00:25:58.000 dwelling on it because this is perhaps the most important diagram 00:25:58.000 --> 00:26:02.000 that we'll address all semester. Although this doesn't mean we have 00:26:02.000 --> 00:26:06.000 to spend all semester assimilating it. It's not so complicated. 00:26:06.000 --> 00:26:10.000 It's relatively straightforward. And let's look at its features. 00:26:10.000 --> 00:26:14.000 Let's dwell on them momentarily because this is a microscopic 00:26:14.000 --> 00:26:17.000 snapshot of what DNA is composed of. You all know it's a double helix 00:26:17.000 --> 00:26:21.000 and therefore there are two strands of DNA in a double helix. 00:26:21.000 --> 00:26:25.000 And one of the interesting things about the double helix, 00:26:25.000 --> 00:26:29.000 although we're not showing it yet, we're just showing a little section 00:26:29.000 --> 00:26:33.000 of a double helix, is the polarity of the two chains 00:26:33.000 --> 00:26:36.000 that constitute the double helix. Let's look at that polarity. 00:26:36.000 --> 00:26:40.000 This one is running in one direction and this one, 00:26:40.000 --> 00:26:44.000 the opposite one, the complementary one is running in the other 00:26:44.000 --> 00:26:47.000 direction. And therefore we talk about the double helix as being 00:26:47.000 --> 00:26:51.000 anti-parallel. Well, I guess I should have a 00:26:51.000 --> 00:26:55.000 bandage on the other finger to convince you but you get the idea. 00:26:55.000 --> 00:26:59.000 They're running in opposite directions. 00:26:59.000 --> 00:27:03.000 They're not both pointed the same. And the other thing to indicate is, 00:27:03.000 --> 00:27:08.000 to repeat what I said just seconds ago, that there's a complementarity 00:27:08.000 --> 00:27:13.000 between the purines and the pyrimidines. So we use the word 00:27:13.000 --> 00:27:18.000 complementary with great frequency, with great promiscuity in biology. 00:27:18.000 --> 00:27:22.000 Complementarity refers to the fact that A and T here or A and U because 00:27:22.000 --> 00:27:27.000 I said U and T are functionally equivalent, they like to 00:27:27.000 --> 00:27:32.000 be opposite one another. There's a purine and a pyrimidine. 00:27:32.000 --> 00:27:36.000 And the converse is the case with C and G, they like to be opposite one 00:27:36.000 --> 00:27:40.000 another. Now, there is specificity here. 00:27:40.000 --> 00:27:44.000 You might say any purine can pair up with any pyrimidine, 00:27:44.000 --> 00:27:48.000 but it's not the case. For instance, A doesn't like to be opposite C and 00:27:48.000 --> 00:27:52.000 T doesn't like to be opposite G. So one of the things we have to 00:27:52.000 --> 00:27:56.000 memorize this semester, and it's not many and it's not hard, 00:27:56.000 --> 00:28:00.000 is that A and T are opposite one another, or A and U, 00:28:00.000 --> 00:28:04.000 and G and C are opposite one another. That's one of the essential concepts 00:28:04.000 --> 00:28:08.000 in molecular biology. There are now a thousand things you 00:28:08.000 --> 00:28:11.000 need to learn, but if you don't understand that 00:28:11.000 --> 00:28:14.000 then ultimately sooner or later you'll find yourself in a swamp, 00:28:14.000 --> 00:28:18.000 literally or figuratively. Now, let's look at the different between 00:28:18.000 --> 00:28:21.000 these two. One of the interesting things is, to state the obvious, 00:28:21.000 --> 00:28:24.000 the way they're associating with one another, hand in glove, 00:28:24.000 --> 00:28:28.000 is via hydrogen bonds. That's not any covalent interaction, 00:28:28.000 --> 00:28:31.000 which means they're reversible. We talked about that. 00:28:31.000 --> 00:28:35.000 Which means that if we were to take a solution of double stranded DNA 00:28:35.000 --> 00:28:39.000 and boil it we would break those hydrogen bonds. 00:28:39.000 --> 00:28:43.000 Remember they only have 8 kilocalories per mole and boiling 00:28:43.000 --> 00:28:47.000 water has far higher energetic content. And consequently if we 00:28:47.000 --> 00:28:51.000 heat up a DNA double helix and we break those double bonds of DNA that 00:28:51.000 --> 00:28:55.000 hold the two strands together, the two strands come apart, the DNA 00:28:55.000 --> 00:28:59.000 ends up being denatured, that is the two strands are 00:28:59.000 --> 00:29:03.000 separated one from the other. In fact, if there ever were a 00:29:03.000 --> 00:29:07.000 covalent cross-link between the two strands that's really bad news for a 00:29:07.000 --> 00:29:11.000 cell carrying such a DNA double helix. A covalently cross-link from 00:29:11.000 --> 00:29:15.000 one strand to the other DNA double helix represents often a sign that a 00:29:15.000 --> 00:29:19.000 cell should go off and die because it has a very hard time dealing with 00:29:19.000 --> 00:29:23.000 that by virtue of the fact, as we will soon learn or as you 00:29:23.000 --> 00:29:27.000 already know, the cell has, with some frequency, to pull apart 00:29:27.000 --> 00:29:31.000 these two strands. And therefore this association must 00:29:31.000 --> 00:29:35.000 be tight enough so that it's stable at body temperature but not so tight 00:29:35.000 --> 00:29:39.000 that it cannot be pulled apart when certain biological conditions call 00:29:39.000 --> 00:29:43.000 for it. You see that in fact here there are three hydrogen bonds and 00:29:43.000 --> 00:29:47.000 here there are only two hydrogen bonds. That also has its 00:29:47.000 --> 00:29:51.000 implications. It turns out to be the case that the disposition of 00:29:51.000 --> 00:29:55.000 this hydrogen and this oxygen here, they're far enough apart that for 00:29:55.000 --> 00:29:59.000 all practical purposes they don't really make very good 00:29:59.000 --> 00:30:03.000 hydrogen bonds. And therefore we think of this as 00:30:03.000 --> 00:30:07.000 having two and this having three. And if you were to try to put C 00:30:07.000 --> 00:30:12.000 opposite A or G opposite T you'd see that they cannot form hydrogen bonds 00:30:12.000 --> 00:30:16.000 well with one another. Instead they kind of bump into one 00:30:16.000 --> 00:30:20.000 another, and therefore are not complementary to one another at all. 00:30:20.000 --> 00:30:25.000 There's another corollary that we can deduce from this diagram, 00:30:25.000 --> 00:30:29.000 and that is the following. If it's always true that A equal 00:30:29.000 --> 00:30:36.000 C and G equal T -- 00:30:36.000 --> 00:30:40.000 A equals T and G equals C. By the way, this is an interesting 00:30:40.000 --> 00:30:45.000 story. This is the Chargaff Rule. Because about a year or so before 00:30:45.000 --> 00:30:49.000 Watson and Crick figured out the structure of the double helix there 00:30:49.000 --> 00:30:54.000 was a guy named Erwin Chargaff in New York at Columbia University who 00:30:54.000 --> 00:30:58.000 one day figured out that if you looked at a whole bunch of nucleic 00:30:58.000 --> 00:31:03.000 acids, different DNAs from different cell types -- 00:31:03.000 --> 00:31:09.000 And in certain cell types what he found was that G was equal to, 00:31:09.000 --> 00:31:16.000 for example G equals 20% of the bases. Therefore, 00:31:16.000 --> 00:31:23.000 obviously we know C must equal also 20% because there always has to be a 00:31:23.000 --> 00:31:29.000 C opposite a G in the double helix, right? G and C always have to be 00:31:29.000 --> 00:31:36.000 equal. And Chargaff discovered that, in fact, A in such DNA always was 00:31:36.000 --> 00:31:43.000 30% and T was also 30%. Well, these together make up 100% 00:31:43.000 --> 00:31:49.000 which is, we're not in higher math yet, but A and T were always the 00:31:49.000 --> 00:31:55.000 same. If you looked at another type of DNA he might find that G equals 00:31:55.000 --> 00:32:00.000 23% and C also equals 23%. And in this same DNA then A would 00:32:00.000 --> 00:32:05.000 equal 27%, I guess, and T also equals 27%. 00:32:05.000 --> 00:32:10.000 And I hope that adds up to 100%. So he looked at a whole bunch of 00:32:10.000 --> 00:32:15.000 DNAs and they always tracked one another, A always tracked T, 00:32:15.000 --> 00:32:21.000 G always tracked C. And then in 1953 up comes these two guys from 00:32:21.000 --> 00:32:26.000 Cambridge, England, Watson and Crick whom Chargaff 00:32:26.000 --> 00:32:31.000 regarded as upstarts, as smart-asses who thought they knew 00:32:31.000 --> 00:32:35.000 all the answers. And Watson and Crick said, 00:32:35.000 --> 00:32:39.000 gee, this Chargaff rule really is very interesting because it suggests 00:32:39.000 --> 00:32:42.000 something about the structure of DNA. These cannot just be coincidences. 00:32:42.000 --> 00:32:46.000 There's something profoundly important they said, 00:32:46.000 --> 00:32:50.000 correctly, in the fact that there was always an equivalence between A 00:32:50.000 --> 00:32:53.000 and T and between G and C. And that represented one of the 00:32:53.000 --> 00:32:57.000 conceptual cornerstones of their elucidating the structure 00:32:57.000 --> 00:33:01.000 of the double helix. And so Chargaff who died last year 00:33:01.000 --> 00:33:05.000 or the year before last, at an advanced age, was for the next 00:33:05.000 --> 00:33:09.000 fifty years a very bitter man, because he was this far away from 00:33:09.000 --> 00:33:13.000 figuring out this far. Not this far, but this far away 00:33:13.000 --> 00:33:17.000 from figuring out, making the most important discovery 00:33:17.000 --> 00:33:22.000 in biology in the 20th century. He had the information right there. 00:33:22.000 --> 00:33:26.000 And if he thought a little bit about information theory and thought 00:33:26.000 --> 00:33:30.000 a little bit about the way information content is encoded he 00:33:30.000 --> 00:33:34.000 could have already predicted, not the detailed structure of the 00:33:34.000 --> 00:33:39.000 double helix, but at least the way in which it encodes information. 00:33:39.000 --> 00:33:42.000 Because, to state the obvious, and as many of you know already, 00:33:42.000 --> 00:33:46.000 if one looks at the structure of a double helix one can, 00:33:46.000 --> 00:33:50.000 in principle, depict it in a two or a three-dimensional cartoon. 00:33:50.000 --> 00:33:54.000 Here's the way one can think of it. This is the way we've been talking 00:33:54.000 --> 00:33:58.000 about it over the last couple of minutes. It's a two-dimensional 00:33:58.000 --> 00:34:02.000 double helix. And from the point of view of 00:34:02.000 --> 00:34:06.000 information encoding, it doesn't really matter whether we 00:34:06.000 --> 00:34:10.000 draw it this way or that way. It happens that the double helix is 00:34:10.000 --> 00:34:14.000 turned around like that, it's twisted around. It's very 00:34:14.000 --> 00:34:18.000 difficult for biological molecules to be totally flat for an extended 00:34:18.000 --> 00:34:22.000 period. And the helix is, in fact, something that is 00:34:22.000 --> 00:34:26.000 frequently resorted to. Witness the alpha helix in the 00:34:26.000 --> 00:34:30.000 protein. So these are turned around. It turns out that each of these 00:34:30.000 --> 00:34:34.000 constitutes a base pair, and each of these base pairs is, 00:34:34.000 --> 00:34:39.000 in fact, 3.4 angstroms apart. 3.4 angstroms thick. 00:34:39.000 --> 00:34:44.000 So you have ten of them, the DNA helix advances 3.4 angstroms 00:34:44.000 --> 00:34:49.000 every ten turns. And ten turns is roughly, 00:34:49.000 --> 00:34:54.000 oh, I'm sorry. Ten base pairs is roughly one turn of the alpha helix. 00:34:54.000 --> 00:34:59.000 So if you go here and you count up ten, we should start again at the 00:34:59.000 --> 00:35:04.000 same orientation. Another ten is another turn. 00:35:04.000 --> 00:35:09.000 Another ten is another turn. In fact, I'm just recalling that I 00:35:09.000 --> 00:35:15.000 was once a TA in 7. 1 in 1965. And there was a physics 00:35:15.000 --> 00:35:20.000 professor who became a biologist who always talked about these double 00:35:20.000 --> 00:35:25.000 helices. And he always talked about the measurements of different DNA 00:35:25.000 --> 00:35:30.000 molecules. Now, you may know that the term angstrom 00:35:30.000 --> 00:35:36.000 is named after a Danish person named Angstrom. 00:35:36.000 --> 00:35:40.000 That's why it got its name. So whenever this professor, 00:35:40.000 --> 00:35:45.000 whom I never corrected, God forbid, ever talked about something that was 00:35:45.000 --> 00:35:50.000 ten angstroms long, he called these ten angstra. 00:35:50.000 --> 00:35:54.000 Now, as you know, when you go in a Latin verb from singular to plural 00:35:54.000 --> 00:35:59.000 it's “-um” to “-a”, right? So he pretended this was a 00:35:59.000 --> 00:36:04.000 Latin word. What's a good word? 00:36:04.000 --> 00:36:08.000 Sorry? What's a common Latin word we use? Sorry? 00:36:08.000 --> 00:36:12.000 Millennium. Yeah, millennium, millennia. 00:36:12.000 --> 00:36:16.000 So he went from angstrom to anstra. And it went on for a whole year. I 00:36:16.000 --> 00:36:20.000 never said anything but I knew better. OK, anyhow. 00:36:20.000 --> 00:36:24.000 Here you see the genius of Watson and Crick. And, 00:36:24.000 --> 00:36:28.000 by the way, Angstrom was a Dane, as I said, and not a Roman soldier. 00:36:28.000 --> 00:36:32.000 So here we see. OK. So here is the genius of their 00:36:32.000 --> 00:36:36.000 discovery. And the elegance of it is not how complicated it is. 00:36:36.000 --> 00:36:41.000 The elegance of it is how simple it is, because information we see is 00:36:41.000 --> 00:36:46.000 encoded in two strands. The information is redundant 00:36:46.000 --> 00:36:50.000 because if we know the sequence of one strand we can obviously predict 00:36:50.000 --> 00:36:55.000 the sequence in the other strand because it's a complementary 00:36:55.000 --> 00:37:00.000 sequence. If we always realize that A is 00:37:00.000 --> 00:37:05.000 opposite T and G is opposite C we can know directly that a sequence in 00:37:05.000 --> 00:37:10.000 one strand, which may be A, C, T, G, G, C and the other strand 00:37:10.000 --> 00:37:16.000 moving in the other anti-parallel direction the sequence is like this. 00:37:16.000 --> 00:37:21.000 I don't need to know the sequence of the other strand. 00:37:21.000 --> 00:37:26.000 I can predict it by using these rules of complementary 00:37:26.000 --> 00:37:31.000 sequence structure. And that, in turn, 00:37:31.000 --> 00:37:35.000 obviously has important implications. If we look at the three-dimensional 00:37:35.000 --> 00:37:39.000 structure, this is more of what's called a space-filing model. 00:37:39.000 --> 00:37:44.000 This is the way the x-ray crystallographer would actually 00:37:44.000 --> 00:37:48.000 depict it. We talked about space-filling models before. 00:37:48.000 --> 00:37:53.000 One of the things we appreciate is the fact that the phosphates are on 00:37:53.000 --> 00:37:57.000 the outside and these bases are in the inside. And because these bases 00:37:57.000 --> 00:38:01.000 are able also to stack with one another via hydrophobic interactions 00:38:01.000 --> 00:38:05.000 importantly the bases are protected. The face where they interact is 00:38:05.000 --> 00:38:09.000 protected from the outside world. What do I mean by that? Well, 00:38:09.000 --> 00:38:13.000 let's go back to this figure right here. You see the interaction faces 00:38:13.000 --> 00:38:16.000 between A and T or C and G they're not on the outside of the helix. 00:38:16.000 --> 00:38:20.000 They're hidden in the middle. And that's important because it means 00:38:20.000 --> 00:38:23.000 that these interactions between A and C and G and T, 00:38:23.000 --> 00:38:27.000 you can see it up here as well, are biochemically protected from any 00:38:27.000 --> 00:38:31.000 accidents that might happen on the outside. 00:38:31.000 --> 00:38:35.000 They're sheltered from that. And that's important because the 00:38:35.000 --> 00:38:39.000 information content in DNA must be held very stable, 00:38:39.000 --> 00:38:43.000 very constant. If it isn't then we have real trouble like cancer. 00:38:43.000 --> 00:38:47.000 And therefore whenever a cell divides and copies its DNA, 00:38:47.000 --> 00:38:51.000 its three billion base pairs of DNA, whenever that happens the number of 00:38:51.000 --> 00:38:55.000 mistakes that are made is only three or four or five out three billion. 00:38:55.000 --> 00:39:00.000 A stunningly low rate. And this DNA can sit around. 00:39:00.000 --> 00:39:04.000 I told you about Neanderthal DNA that can sit around for 30, 00:39:04.000 --> 00:39:08.000 00 years and it's chemically relatively stable. 00:39:08.000 --> 00:39:12.000 In part, a testimonial to the fact that this base pairing, 00:39:12.000 --> 00:39:16.000 the face where the two bases interact across one another, 00:39:16.000 --> 00:39:20.000 this is shielded from the outside world because it's tucked into the 00:39:20.000 --> 00:39:24.000 middle, these interaction faces here. This is the inside of the helix. 00:39:24.000 --> 00:39:29.000 Here the sugar phosphate groups are on the outside. 00:39:29.000 --> 00:39:33.000 In fact, when Watson and Crick were struggling with the structure of the 00:39:33.000 --> 00:39:37.000 double helix they were in a horse race with a man named Linus Pauling 00:39:37.000 --> 00:39:41.000 who was really the inventor, the discoverer of the hydrogen bond 00:39:41.000 --> 00:39:45.000 pretty much who actually got two Nobel Prizes in his lifetime who 00:39:45.000 --> 00:39:49.000 ended his life believing that if you took enough vitamin C grams of it 00:39:49.000 --> 00:39:53.000 every day you would never get sick. I don't know what he died of, but 00:39:53.000 --> 00:39:57.000 probably like Dr. Atkins he probably died of an 00:39:57.000 --> 00:40:02.000 illness he was trying to ward off. Or he might have died of kidney 00:40:02.000 --> 00:40:06.000 failure from all the vitamin C he was putting into his body. 00:40:06.000 --> 00:40:10.000 Who knows? Anyhow, I digress. The fact is that Pauling thought 00:40:10.000 --> 00:40:14.000 that, in fact, DNA was constituted of a triple 00:40:14.000 --> 00:40:18.000 helix, with three strands, and that the bases were facing 00:40:18.000 --> 00:40:22.000 outward. Well, of course, now we can snicker, 00:40:22.000 --> 00:40:26.000 now we can laugh, but at the time nobody had any idea. 00:40:26.000 --> 00:40:30.000 Now we realize it's only a double helix and the bases 00:40:30.000 --> 00:40:33.000 are facing inward. And, of course, 00:40:33.000 --> 00:40:37.000 because Pauling worked with that preconception, 00:40:37.000 --> 00:40:41.000 he was never able to figure what was actually going on, 00:40:41.000 --> 00:40:45.000 even though Watson and Crick thought that he had the answer and was about 00:40:45.000 --> 00:40:49.000 to scoop them. Implicit in what I've just said is 00:40:49.000 --> 00:40:53.000 the notion that the structure of DNA, which we'll talk about later, 00:40:53.000 --> 00:40:57.000 allows it to be copied, i.e., now we're referring in passing, 00:40:57.000 --> 00:41:01.000 and we'll get into this in greater detail later, to the whole 00:41:01.000 --> 00:41:05.000 process of replication. Because if we have genetic material 00:41:05.000 --> 00:41:09.000 and we've created in a certain sequence we must be able to make 00:41:09.000 --> 00:41:13.000 more copies of it. Keep in mind that each one of us, 00:41:13.000 --> 00:41:18.000 as I mentioned to you some lectures ago, we start out with a fertilized 00:41:18.000 --> 00:41:22.000 egg with one human genome, and through our lifetimes we produce 00:41:22.000 --> 00:41:26.000 how many cells? Anybody remember? 00:41:26.000 --> 00:41:31.000 I did mention it, right? Is there one soul who remembers it? 00:41:31.000 --> 00:41:36.000 Remember the whole story of Sodom and Gomorrah where the Lord says if 00:41:36.000 --> 00:41:41.000 there's one soul, one righteous soul in the city I 00:41:41.000 --> 00:41:46.000 will spare the city. And of course there wasn't so he 00:41:46.000 --> 00:41:52.000 wiped them all out. 30 trillion? Well, 00:41:52.000 --> 00:41:57.000 sorry. What do we do for him? Something nice. [APPLAUSE] 00:41:57.000 --> 00:42:02.000 Excellent. OK. You'll remain anonymous, 00:42:02.000 --> 00:42:06.000 though. You won't be on that video. OK. Ten to the sixteenth cell 00:42:06.000 --> 00:42:10.000 divisions in a human lifetime. And on every one of those occasions 00:42:10.000 --> 00:42:14.000 the double helix is copied. I'm telling you that only to give 00:42:14.000 --> 00:42:18.000 you the most dramatic demonstration of the fact that if you have one set 00:42:18.000 --> 00:42:22.000 of DNA molecules you need to be able to copy it, you need to be able to 00:42:22.000 --> 00:42:26.000 replicate it. And that replicative ability is inherent in the double 00:42:26.000 --> 00:42:30.000 helix as Watson and Crick immediately said and as they noted 00:42:30.000 --> 00:42:33.000 at the end of their paper when -- I think the last sentence says it 00:42:33.000 --> 00:42:37.000 has not escaped our attention that this structure, 00:42:37.000 --> 00:42:41.000 i.e., the structure of the double helix, allows for copying, 00:42:41.000 --> 00:42:44.000 allows for replication. Because if you pull the two strands apart, 00:42:44.000 --> 00:42:48.000 recall we said earlier that in certain biological situations you 00:42:48.000 --> 00:42:51.000 need to do that, if the two strands are pulled apart 00:42:51.000 --> 00:42:55.000 not by putting them in boiling water but by enzymes whose dedicated 00:42:55.000 --> 00:42:59.000 function it is to separate the two strands. 00:42:59.000 --> 00:43:04.000 Then when that happens one can begin to create two new daughter double 00:43:04.000 --> 00:43:10.000 helices by simply adding on new bases and thereby replicating the 00:43:10.000 --> 00:43:16.000 DNA. And how that happens is, of course, as you know, IO 00:43:16.000 --> 00:43:22.000 "Intuitively Obvious". OK. Uh-oh, we're in a dyslexic 00:43:22.000 --> 00:43:28.000 moment. Now, the fact is I emphasized with great vigor 00:43:28.000 --> 00:43:33.000 and conviction -- And remember, class, 00:43:33.000 --> 00:43:37.000 when somebody is convinced of something more often than not 00:43:37.000 --> 00:43:41.000 they're just wrong in a loud voice. But I nevertheless emphasized with 00:43:41.000 --> 00:43:45.000 great conviction that T and U are, from an information standpoint, 00:43:45.000 --> 00:43:49.000 functionally equivalent. They're replaceable, 00:43:49.000 --> 00:43:53.000 interchangeable. And therefore if we want we can 00:43:53.000 --> 00:43:57.000 make an RNA copy of a DNA molecule by realizing that if this were DNA 00:43:57.000 --> 00:44:01.000 we could make an RNA that was complementary to a DNA strand 00:44:01.000 --> 00:44:05.000 realizing that when the RNA molecule was being polymerized, 00:44:05.000 --> 00:44:09.000 instead of using T one would use U. All the other three bases are 00:44:09.000 --> 00:44:13.000 functionally equivalent. And so we could, in principle, 00:44:13.000 --> 00:44:17.000 and indeed it happens transiently, we could make a DNA-RNA hybrid helix 00:44:17.000 --> 00:44:21.000 where a DNA molecule is wrapped around an RNA molecule because the 00:44:21.000 --> 00:44:25.000 two molecules are functionally equivalent. The only difference 00:44:25.000 --> 00:44:29.000 between the two strands would be, well, there are two differences. 00:44:29.000 --> 00:44:33.000 One, in the RNA strand we'd have a U instead of a T. 00:44:33.000 --> 00:44:37.000 And, two, in the RNA strand all the sugars would be ribose rather than 00:44:37.000 --> 00:44:41.000 deoxyribose. Right on. OK. Good. So this structure, 00:44:41.000 --> 00:44:45.000 the simplicity of the structure gives one enormous power in encoding 00:44:45.000 --> 00:44:50.000 all kinds of information and replicating it. 00:44:50.000 --> 00:44:54.000 What it means, as we'll discuss also in great detail later, 00:44:54.000 --> 00:44:58.000 is that if we have a certain sequence of bases in the double 00:44:58.000 --> 00:45:02.000 helix of DNA an RNA molecule could be made to copy one of the two 00:45:02.000 --> 00:45:07.000 strands to make a complementary copy. 00:45:07.000 --> 00:45:11.000 And that RNA molecule could then leave the DNA double helix having 00:45:11.000 --> 00:45:15.000 lifted one of the sequences from it and then move to another part of the 00:45:15.000 --> 00:45:19.000 cell where it might do something interesting. And therefore to 00:45:19.000 --> 00:45:23.000 extract information out of the double helix doesn't necessarily 00:45:23.000 --> 00:45:27.000 mean to destroy it. If one can copy one of the two 00:45:27.000 --> 00:45:31.000 double strands in a complementary form as an RNA molecule that may 00:45:31.000 --> 00:45:35.000 enable the information that is encoded in the DNA to be copied 00:45:35.000 --> 00:45:39.000 without destroying the double helix itself. 00:45:39.000 --> 00:45:43.000 Again, that process, which we'll also talk about later, 00:45:43.000 --> 00:45:48.000 is called the process of transcription. 00:45:48.000 --> 00:45:52.000 And so in the course of this morning I have uttered the three 00:45:52.000 --> 00:45:57.000 words which represent the cannon, the basic fundaments of molecular 00:45:57.000 --> 00:46:02.000 biology. What are the three words? Replication, transcription and 00:46:02.000 --> 00:46:06.000 translation. Transcription means when you make an RNA copy of a 00:46:06.000 --> 00:46:11.000 strand of the DNA double helix. Let's just add a couple more 00:46:11.000 --> 00:46:15.000 footnotes to what I've been saying just so we are on firm ground for 00:46:15.000 --> 00:46:20.000 subsequent discussions. It turns out that often in RNA 00:46:20.000 --> 00:46:25.000 molecules they can form intramolecular double helices. 00:46:25.000 --> 00:46:29.000 There's no reason why you cannot make a double helix out of RNA as 00:46:29.000 --> 00:46:34.000 you can make out of DNA. And therefore you see often in many 00:46:34.000 --> 00:46:38.000 kinds of RNA molecules they will hydrogen bond to themselves using 00:46:38.000 --> 00:46:42.000 these complementary sequences. And this is called a hairpin, by 00:46:42.000 --> 00:46:46.000 the way for obvious reasons. And so many RNA molecules, most of 00:46:46.000 --> 00:46:51.000 them in fact have these intramolecular hydrogen bonded 00:46:51.000 --> 00:46:55.000 double helices with confers on them very specific structure. 00:46:55.000 --> 00:46:59.000 One other aspect of the two versus three hydrogen bonds 00:46:59.000 --> 00:47:04.000 is the following. If a double helix has many Gs and Cs 00:47:04.000 --> 00:47:09.000 then it's going to have more hydrogen bonds holding it together 00:47:09.000 --> 00:47:13.000 than if it has few Gs and Cs. So let's look at the Chargaff 00:47:13.000 --> 00:47:18.000 example. Chargaff who lived for fifty years stewing in his own bile 00:47:18.000 --> 00:47:23.000 in bitterness because he couldn't figure this out, 00:47:23.000 --> 00:47:28.000 which is exactly what happened by the way. 00:47:28.000 --> 00:47:32.000 And so here this has a higher G plus C content, the one on the right than 00:47:32.000 --> 00:47:36.000 this one. This is 23% or 46% G plus C. This is 40% G plus C. 00:47:36.000 --> 00:47:40.000 If it's 46% G plus C that means there are more hydrogen bonds 00:47:40.000 --> 00:47:44.000 holding the two strands together. And it turns out that if you want 00:47:44.000 --> 00:47:48.000 to denature a double helix that has high G plus C content you need to 00:47:48.000 --> 00:47:52.000 put in more energy, you need to heat the double helix up 00:47:52.000 --> 00:47:56.000 to a higher temperature. It's more difficult to pull the 00:47:56.000 --> 00:48:00.000 strands apart. One other side comment on what I 00:48:00.000 --> 00:48:04.000 wanted to say is the following. The presence or the absence of this 00:48:04.000 --> 00:48:08.000 hydroxyl here in RNA has an important consequence for the 00:48:08.000 --> 00:48:12.000 stability of RNA and DNA. Let's look at what happens to an 00:48:12.000 --> 00:48:16.000 RNA chain when a hydroxyl ion, which happens to be floating around 00:48:16.000 --> 00:48:20.000 at a low concentration, happens to attack this 00:48:20.000 --> 00:48:24.000 phosphodiester bond. What happens is that this 00:48:24.000 --> 00:48:28.000 phosphodiester bond will tend to cyclize. It's forming this 00:48:28.000 --> 00:48:32.000 five membered ring. And ultimately that will resolve and 00:48:32.000 --> 00:48:37.000 break causing a cleavage of the RNA chain. This phosphodiester bond now 00:48:37.000 --> 00:48:42.000 forming a cyclic structure here as an intermediate representing the 00:48:42.000 --> 00:48:46.000 precursor to the ultimately cleaved chain. That means that if you take 00:48:46.000 --> 00:48:51.000 RNA molecules and you put them in alkali they will fall apart very 00:48:51.000 --> 00:48:56.000 quickly for this very reason. What happens to DNA molecules when 00:48:56.000 --> 00:49:01.000 you put them in alkali? Nothing. They're alkali resistant 00:49:01.000 --> 00:49:05.000 because there isn't a hydroxyl there to form this five membered ring. 00:49:05.000 --> 00:49:09.000 And therefore alkali cannot cleave apart the DNA or the DNA 00:49:09.000 --> 00:49:13.000 phosphodiester bond. If we imagine that OH groups, 00:49:13.000 --> 00:49:18.000 that hydroxyls, are present at a certain, albeit a certain 00:49:18.000 --> 00:49:22.000 concentration, albeit a low concentration in 00:49:22.000 --> 00:49:26.000 neutral water we can see that even at neutral pH with a certain 00:49:26.000 --> 00:49:31.000 frequency RNA molecules will slowly hydrolyze. 00:49:31.000 --> 00:49:35.000 They'll certainly be slowly broken down by the hydroxyl ions. 00:49:35.000 --> 00:49:40.000 DNA molecules, however, will not. And that represents yet another 00:49:40.000 --> 00:49:45.000 important biochemical reason why DNA is chemically stable and why it can 00:49:45.000 --> 00:49:50.000 carry information over years, decades or tens of thousands of 00:49:50.000 --> 00:49:55.000 years, because the phosphodiester linkage in DNA rather than RNA is 00:49:55.000 --> 00:50:00.000 very stable chemically and can hold these adjacent nucleotides together, 00:50:00.000 --> 00:50:05.000 one to the other. See you on Friday morning.