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Molecular Biology Lecture IV Prof. Graham Walker
Biology > Introductory Biology/n * Email this page/nVideo Lectures - Lecture 12/nTopics covered: /nMolecular Biology IV/nInstructor: /nProf. Graham Walker/nTranscript - Lecture 12/nBy the time that Watson and Crick figured out the structure of DNA, you know, it was sort of obvious that since the two strands were complimentary you could see how it replicated. And they also could see that somehow the information must be encoded in the sequence of letters down the strands of the DNA. But it wasn't obvious what the code was and how it was arranged, how it worked. And in principle it was anything you could do with four-letters./nAnd so I pointed out the other day this was sort of a four-letter alphabet. And I think it's useful to think of it this way with A, G, C and T, and RNA as also being a four-letter alphabet. But proteins are actually a 20-letter alphabet because there are 20 different amino acids. And so somehow, since one of the key things that the DNA had to do, it somehow had to encode the information for making the proteins./nAnd there was a lot of work on protein biosynthesis at the time. And it looked pretty complicated. People had found that RNA seemed to be important. Cells that were making lots of protein had lots of RNA in them. And another thing they noticed was that if you looked in eukaryotic cells the DNA stayed in the nucleus. The proteins, most of them, were out in the cytoplasm./nAnd the evidence was that they were made out in the cytoplasm. So somehow the information had to get out of the nucleus where the DNA was and into the cytoplasm. And biochemists were breaking cells open and trying to make cellular extracts that would synthesize proteins. And I think it's fair to say at the time that it looked extremely complicated. And so thinking about how DNA encoded information and got translated into proteins was a very complex issue./nBut then actually there was a very interesting development that had a strong influence on Watson and Crick and led to them, Crick in particular, getting a key insight into the nature of this coding problem. There's a physicist, George Gamow, who some of you know. He proposed the "Big Bang Theory". A very strong theoretical physicist. And he wrote a letter to Watson and Crick. He thought he'd figured out the basis of the genetic code./nAnd his idea was you had these sequences of A, G, C and Ts. And so everywhere the two bases came together there was sort of like a little different shaped hole. So his idea was the amino acids would stick into these little holes. And he had a theory showing that you could encode the sequence of proteins by having the side chains in the amino acids stick into these little holes along the DNA./nNow, there turned out to be a number of problems with that. It didn't take into account the involvement of RNA, which there sort of was quite of bit of evidence for. And more importantly it didn't take into account the structure of the side chains of the amino acids, which you guys have been exposed to. But it had a very profound influence on Watson and Crick. They read this letter./nThey immediately realized the idea was wrong and went out and had a lunch at a pub, decided again how they actually thought there were 25 amino acids, but they realized some of them were just sort of special ones that were modified only in particular proteins and there were really 20 amino acids that were found universally in nature and amino acids. And what they, Crick in particular, realized was that maybe instead of having to think about protein synthesis through this very complex set of extracts and mixtures a biochemist would work on, that he could think about it at a purely theoretical level, which basically is up at this kind of level./nBut if you have a molecule that has four letters and it's going to be encoding proteins how does it do it? Can I work out sort of the basis or a possible theory for how that could happen without actually knowing all of the biochemical details? So Crick made a couple of simplifying assumptions. One was that the DNA only determined -- -- the linear sequence of amino acids and protein./nThat all this information about the 3-dimensional stuff came from the properties of the linear sequence once it was made. And I think you hopefully have enough understanding of hydrophobic and other sorts of interactions that would cause a linear sequence amino acid to take a particular confirmation. And the other assumption he made was that it must be universal. And it would be hard to see how life could have started if there wasn't some kind of code that was universal between organisms./nAnd if you start from those kinds of considerations then what you can see is you cannot just have a one-to-one correspondence between a letter in the nucleic acid alphabet and a letter down here. If A stood for valine that would be fine, but you could only have code for four amino acids that way. So if you had one-letter words in DNA there are four possibilities./nAnd so it could only make four. If you had two two-letter words then you'd have 16 possibilities, still not enough for all the amino acids. If you had a three-letter word -- -- then you could do 64, and in principle that would be all you'd need. It doesn't rule out there couldn't be five or six or seven-letter words. Or if you think about this as they were thinking about it at the time, even if it were let's say a three-letter word, is it a code where you have one word, then the next word, then the next word? Or could it be an overlapping word? And what about punctuation? And maybe another thing, you can see if it's AG, CT, etc./n, there's a frame of reference problem, because if I'm going to read them in groups of three, if I start here I'll get one word, but if I start one letter over the next group of three won't be the same. So somehow there would have to be a starting point. And so these are the sort of considerations that they had to take into account. And, in fact, Watson, excuse me. Francis Crick and another scientist Sydney Brenner and some other scientists worked out a very elegant genetic experiment that demonstrated that it was a three-letter code./nAnd I don't have the time to go into it in this course. If you take a genetics course it's a very beautiful experiment. The principle of the thing, which I could show you rather easily, is if you're writing a thing where you're reading in three-letter words, something like this. The cat ran out and, I don't know, ate the rat or something like that. And these were all just continuously run together, not separated out, but I've put them out here./nAs you can see they're three-letter words. If you lost one letter then it would change to sort of gibberish. You'd get stuff that looked like this. And if you put one in you'd have the same problem, but if you were to either take out three letters or put in three letters then, even though there'd be a little mess in here somewhere, say I took out two more of these, what we would now have from then is the rest of it would now make sense again./nAnd they did this sort of experiment genetically. They managed to figure out there were two kinds of mutations they could get in a particular way. Some were putting in a letter. Some were taking out a letter. And they didn't know at the time whether they were adding or deleting, but they could tell they were in the opposite directions. And then they found if they took three of one class, like three that would delete a letter and put them all together then things would more or less work./nOr if they put three that stuck in an extra letter then everything would more or less work. So there was a genetic proof of the three-letter part of the code before it was figured out exactly how the code itself worked. And so going from this sort of theoretical insight into the code to actually figuring out how proteins were made there was still quite a lot of stuff that had to happen. And one was the concept of messenger RNA./nAs I said, there'd been quite a lot of evidence that RNA was somehow involved in protein synthesis because cells that made a lot of protein made a lot of RNA. And it seemed to be in the right sort of place in the cell for the proteins to be made. So the idea merged that RNA was somehow a carrier of information from the DNA to the cytoplasm. So it could serve as a template for making proteins. So the idea that the cell copied the sequence of a portion -- -- of the DNA./nAnd we'd probably think of this as a gene right now. Into RNA. And the RNA would go into the cytoplasm. That's the part outside the nucleus. And then it would serve as a template -- -- for protein synthesis. Because of this thought that if you had a cell like this with a nucleus and the DNA in here, that if a piece of RNA were to go out into the cytoplasm and have those properties it would be functioning more or less as a messenger./nIt would be carrying the genetic information from inside the nucleus out into the cytoplasm. And so the term began to be used of a messenger RNA. And so over here I'll put an mRNA to indicate that. Now, one thing you can also see is we've talked about the structure of DNA and RNA. And it's essentially the same with one. This is the nucleotide, which is the fundamental building block of DNA./nAnd if you recall, in DNA there's a hydroxyl, excuse me, a hydrogen there, but in RNA there is this extra hydroxyl. This is 1 prime, 2 prime, 3 prime, 4 prime, excuse me. Let's just leave it like for the moment, 1, 2, 3, 4, 5. And so the DNA was deoxyribonucleic acid because it's missing this. But other than that the backbones are similar and the letters are almost the same./nThe A, the G and the C are exactly the same bases in DNA and RNA. The only difference is with the T and the uracil. So this is thymine which is found in DNA. And this is uracil -- -- which is found in -- -- RNA. So the base pairing is over on this part of the molecule. So whether or not you have a methyl group doesn't really change the base pairing. And so this process of copying information in DNA to information that's in RNA was seen as essentially the same kind of language, but it's just sort of like taking somebody's word processor file and writing out longhand./nYou'd be transcribing the information but it would be essentially the same kind of information in essentially the same form. So this is known as transcription. I'll take just one very brief thing. Some of you may wonder why did nature do it this way? Why didn't it just use uracil in DNA? So as a very brief aside, I think we understand pretty much why it does it./nAnd that is cytidine has this structure. So this is C which is found in DNA but it undergoes, all of your DNA is a chemical and it's able to undergo spontaneous kinds of damage. In fact, in every one of our human cells every day, 10,000 times in any given cell a base falls off totally just leaving the deoxyribose sitting there. And the cells have to fix it up. And we have DNA repair systems that do that./nBut another very common kind of thing that happens is that this NH2 group deaminates. And if you do that, if a C happens to deaminate in DNA it gives you a uracil. And if that ever happens, the cell is actually able to tell that something went wrong because uracil is not supposed to be in DNA and there are repair systems that constantly scan the DNA and take out any uracils that are in there. And the reason, if instead of using thymine it used uracil then the cell wouldn't know whether the uracil got there because it was supposed to be there as part of the sequence or whether it had arisen by deamination of a cytidine./nIt's a minor point but I think we do have an understanding as to why there's thymine in DNA and uracil in RNA. This isn't such a worry in RNA. OK. But anyway. So there's still a really big problem here, though, that Watson and Crick and others were grappling with. And it has to do, as I say, with this fact that the information up here is the first in DNA and RNA./nIt's written as a sequence of letters, if you will, chemical letters, but there are only four letters in the DNA alphabet and essentially the same four letters in the RNA alphabet. However, the protein language has got a totally different alphabet so it's somehow like sort of translating now from English to Japanese or something like that. Some really fundamental change had to happen because there was a real conversion from one kind of language to another./nAnd so this process is known as translation, as going from information that's written using a four-letter nucleic acid alphabet to information that's written using a 20-letter amino acid alphabet. And Crick on purely theoretical grounds figured, well, if you're going from one language to another what do you need? You need a translator? And what's a translator? A translator is someone who speaks both languages./nSo his idea was that if there was -- I'm going to just separate out, let's say this is the messenger RNA. And I, just for clarity here, have spaced out the three-letter words so we can see them. These would be three like G-A-C or something like that in the RNA. That there would be some kind of translator. And his idea was that it would be something that had a particular amino acid at one end and it had the complimentary nucleotides at the other end./nSo it could, if you will, read the genetic code that was written in the RNA using the nucleic acid alphabet, but it would also be speaking the amino acid language. Got the idea? So the idea was that this would be, they used the words adaptor or a translator. So that was on basically theoretical grounds. If you had to go from a four-letter language to a 20-letter language you needed some kind of translator or adapter./nNow, at that same time that these considerations were going on, biochemists began to find a class of small RNAs -- -- that had an amino acid -- -- attached. And so there were entities that had just the sort of properties that Crick had envisioned you'd need from theoretical considerations. These were given the name transfer RNAs or tRNAs as they're usually referred to now./nAnd I've told you that RNA has, since it's got nucleic acid bases, if you have a single strand of either an RNA or a DNA and you don't have a complimentary double-strand, then if there are complimentary sequences they can come together and pair just the same way that complimentary sequences can come together in DNA. And in the case of tRNAs, once the sequence of these was determined, oops./nThere we go. They folded up into a clover leaf shape. And the amino acid is attached up at the 3 prime end of the chain up here in what's known as the acceptor part of the molecule. And so that corresponds to this part up here. And here is what's known as the anticodon. Each of these three-letter words -- -- in nucleic acid language is called codon. And so something that had a complimentary sequence to a codon was called an anticodon./nSo if G-G-G is the codon then C-C-C would be the anticodon. Now, this is just a schematic, as you can see. It shows where the hydrogen bonds are that form this stuff. When the crystal structures were done, the first crystal structure of tRNA was actually done by Alex Rich. He's in the Biology Department at MIT. And he was in this picture I showed you talking to Matt Meselson./nAnd although we cannot see this terribly well, maybe you could hit the lights here, the crystal structure showed that the molecule didn't look like a clover leaf as in there. It had more this shape. And I'll show you this more clearly in this picture. I showed you this little part of the thing when I was showing you how an RNA could form. For example, if you copy the gene encoding a tRNA and, for example, the sequence here in green is complimentary to the sequence here, or the sequence here in sort of blue or purple was complimentary to the sequence here./nThat what can happen then, if you allow a single strand RNA like this to fold up, thermodynamically it will then go to the lower energy state which involves being able to make these hydrogen bonds. And I think you can sort of see the clover leaf. Here's one of the leaves. The other is down here and the others. It's a little bit distorted here. And the reason is, because I'm going to continue now to show you how this structure, once you get to the clover leaf, then it folds up to make other kinds of interactions and it takes that shape with the tRNA going on at this end and the anticodon being down here./nAnd what's happening now is they've morphed on the van der Waals surfaces so you can see what this would look like, 3-dimensional shape. The amino acid would be attached at that end and there is the anticodon that we'd be able to recognize, the codon in the RNA. I mean the physical reality is pretty close to this simple little depiction here. OK. So once this basic paradigm had been straightened out that gave rise to this idea then, putting it all together, that the information in DNA, that a portion of it would be copied into RNA and that would go out into the cytoplasm./nAnd then in the cytoplasm these translators, the tRNAs would be able to decode, read the nucleic acid information and use that to determine the linear order of amino acids in a protein. Crick, when he came up with this, gave this the term "the central dogma". And people still use this term to apply this idea of information flow going from DNA to RNA in protein. And it's still used to this day. There's actually sort of a little twist to that, because at the time that Crick proposed the term he actually thought that the word dogma meant "an idea for which there is not reasonable evidence"./nBut he was sort of amused years later to realize that a more reasonable definition of dogma is it is something that a true believer cannot doubt. So he kind of accidentally made an assertion that he was right, but fortunately he was right. Now -- -- the next big job, though, in working this out was to crack the code. And it's fine to know that it's a 3-letter code and it's fine to know it goes into RNA and then the tRNAs translate it, but if you cannot crack the code then you have no idea what any of the information means./nIt was sort of like before the Rosetta Stone they could look at the hieroglyphics in the Egyptian tombs and they could see that it was a lot of information and there were symbols and so on, but they didn't know what it meant until finally they got something that allowed them to relate it to a language they did know and they were able to work out the principles./nSo somehow scientists had then to crack the code. And there were two scientists who played a really big role. One was Marshall Nirenberg who was at NIH and is, in fact, still at NIH. And the other was a scientist who's on the same floor as me at MIT, Gobin Khorana. And they used two different approaches, but between these two approaches the genetic code was cracked. And what Nirenberg did was to take a protein synthesizing -- -- extract that he knew needed RNA in order to work./nSo that wasn't a surprise at this point because people were thinking the RNA would be the message. And at that point the ability to make synthesized nucleic acids was quite limited compared to what we do now. And so there were different ways of making them. Sometimes you could do it enzymaticly. But what Nirenberg, for example, was able to make was poly-U. So this was an RNA that was just UUUUUUU./nAnd then what he did was he set up 20 reactions, and in every reaction he put some of this extract, he put poly-U and he put 19 of the amino acids that were unlabeled. And then only one amino acid that had radiolabel in it. So he ran these 20 reactions and waited to see in any of these did he get protein made that would have been coded by the poly-U. And what he ended up with was polyphenylalanine./nWhich you may recall when we were talking about structures of amino acids, there's the basic backbone. And the polyphenylalanine is the one that has, if you will, a benzene ring hanging off the end. And so what that meant was that UUU must code for a Phe or phenylalanine. And if it's UUU in the RNA that must mean that the DNA that encodes this must have that sequence AAA and TTT. And you can see that one of the two strands of the DNA, since T base pairs the same as uridine, but one of the strands in the DNA is going to have the same sequence as one of the strands in the RNA./nNow, I'll just tell you one brief little anecdote. I heard Marshall Nirenberg at this meeting they had to celebrate the 50th anniversary of the discovery of DNA. And he posed something that I'd never thought about in my years of teaching this but might occur to you guys if we put it on a problem set. You all know something that benzene is nothing but sort of these, this as I call it, we even referred to it as a benzene ring, which is a very organic kind of solvent./nSo if we put a problem set, if you've made polyphenylalanine would you expect this to be soluble in water? Well, this is very, very hydrophobic, very, very water-hating. And your answer would be correct. If you said no, I wouldn't expect polyphenylalanine to be soluble in water. In fact, if it were in a protein you'd expect it to probably be in the core where all the hydrophobic interactions, the water-hating parts would go./nSo Marshall Nirenberg said in his talk, well, he had shown that he had radioactive phenylalanine, and he still had to prove chemically that he had polyphenylalanine. But he wasn't much of a biochemist so he walked down to the lab just below NIH and walked in the door and saw the first person he saw and said how do you solubilize polyphenylalanine? Just to make sure I got this right. And the guy said, oh, you just take 33% hydrobromic acid and glacial acetic acid and it works./nSo he went back upstairs and dissolved it. It turned out it dissolved in that. And he went on and characterized it. And he said it didn't occur to him or he didn't learn until about 15 or 20 years later that he just walked up to the only person in the world who knew how to solubilize polyphenylalanine. By total coincidence this guy who had talked to had been working away trying to figure out a way and had come up with this odd mix of hydro- bromic acid and glacial acetic acid./nAnd he just said of all the places in the world, he walked up to the one person who knew and got the answer. So the other part of the story then involves Gobin Khorana who I mentioned when I was telling you initially about the Nobel Laureates at MIT. And Gobin is a brilliant organic chemist. He synthesized DNA. You know, it was a point where a whole issue of a journal came out and there was nothing but his labs work and synthesizing DNA./nWell, he was good at nucleic acids. And one of the strategies that they could use chemically was they would make something like a dye nucleotide like CA. And then they were able to polymerize that to make a piece of RNA. So they could make an RNA that had the sequence CA, CA, CA, CA and so on. And what you can see from that is that there are two different codons in that./nOne is CAC and the other is ACA. And the reason he made it was he was synthesizing it by polymerizing nucleotides. So in these same kinds of experiments I was describing before, what they found this synthesized was alternating histidine and threonine. And you cannot tell from that experiment alone. One of those must be histidine and one of them must be threonine, but you cannot tell from that experiment so more experiments were needed./nAnd what was learned from that experiment in that case was that CAC corresponded to histidine and ACA corresponded to threonine. So these kind of experiments were then put together to give what's known as the genetic code which is the three-letter words encoded in DNA that encode the sequence amino acids and proteins. And it's usually displayed as a table and you read it in this way. That this thing over here is the first base in the codon, across the top is the second base in the codon, and down over here is the third base./nSo if we go to C as the first, say the one for histidine we were just showing you. C is the first letter. A is the second letter, so this is the box that we're going to be looking at. And if C is the third letter we can see it encoded histidine or AC come back to A. Then the A is certainly threonine. But you can also see something else here. And that is because there were 64 possibilities with this three-letter word the code is what's known as degenerate./nThat is there are more words in the genetic code than are needed to specify the number of amino acids that have to be coded. So I just want to make a couple of points about this. So the genetic code -- It's degenerate. There are 61 codons that correspond to an amino acid. And that means that some, and I think threonine is a good example, there's more than one word in the genetic code that means threonine. There were three codons for which there was no corresponding amino acid./nAnd those mean stop. And that would make sense because if you're reading down a nucleic acid piece of RNA, at some point you'd have to end the protein. And so there are actually three that are used for that purpose. And although there's some small variation on this in nature there's usually one amino acid that's used for starting a protein, and that's methionine. And it's AUG right there. Now, some of this stuff probably sounds like it's been around forever, and that's certainly true of some of the stuff you hear in your chemistry, math and physics courses./nI just want to drive this home. When I was an undergrad Watson's first book called Molecular Biology of the Gene had come out, so when I was your age, and I realize that I look ancient but, you know, at least I'm still here. When I was an undergrad I had Watson's book. This was the genetic code that was in the book, the genetic code as of May 1965. And you'll notice there are gaps in here. And all the things that are underlined were things for which there was a tentative assignment./nSo although you may take this and think that it's been knowledge that's been around forever, it wasn't even complete in the textbook when I was an undergrad. OK. So one of the things then that's important to think about the nucleic acid stuff, this is the basis of how proteins are encoded in the DNA. But everything else has to be there, too./nAnd the genetic code, that's what we've been talking about, is universal. But there are other languages -- -- written in the DNA that are not universal. And one of them was that little example I gave you with an origin of replication. E. coli only starts DNA replication at one very particular point in its chromosome, so it is a particular sequence of DNA. It's actually about 250 nucleotides long./nSo you could think of that as a language. It's like starting a chromosome replication language. It's only got one word in it, and the word is 250 nucleotides long. Another place that's very important, and that is if you're going to make an RNA copy, if you're going to do transcription of a piece of DNA -- And I'll call this the coding sequence. This would be the sequence of three-letter words that we'd specify the amino acid of the protein./nIf you were going to make an RNA copy of that, you would have to somewhere have something here that's a sequence up here that means start transcription. And one at the end, some other sequence of letters in the nucleic acid that would mean stop transcription. This is given the technical term that's referred to as a promoter. The stop one is referred to as a terminator./nAnd these, we'll say more about this. Because the beauty of having this system of making an RNA copy is it provides a beautiful point of regulation. Because the cell can determine whether or not it's going to make a particular protein by whether or not it chooses to make the protein or not. And so having this RNA intermediate and being able to control transcription is a really important part of the whole regulation that makes life possible./nThe transcription is carried out by an enzyme that's known as RNA polymerase. And let me make one more point. These promoters and terminators are not universal. So when we talk about recombinant DNA a little bit in the course, if I take a mouse gene and I put it in E. coli. Even though the genetic code is the same, we might have all the same sequence of amino acids specified, you won't get the RNA made because the sequences that say start transcription and stop transcription are different between a mouse and a bacterium even though the genetic code is the same./nSo you can kind of see from first principles. If you're doing recombinant DNA and you wanted to express the mouse protein in E. coli, you would have to fiddle around with the sequences up here and the sequences down there, the parts that are not universal. You guys with me? OK. So what does an RNA polymerase do? It recognizes this sequence, and then it teases the strands apart to make a little bubble like this./nSo let's say ATAGCTA. So the other strand then would be TATCGTA. And then RNA polymerase, unlike a DNA polymerase, can begin a chain de novo. Remember an important thing about DNA polymerases was they had to have a primer terminus to get started. That was they had to use the Okazaki fragments. So this is DNA. This would be 5 prime, 3 prime, 3 prime and 5 prime. And what an RNA polymerase can do, it uses DATP, DGTP, DCTP and DUTP./nIt uses triphosphates, excuse me. Get rid of these. Excuse me. My mistake. No deoxies here. Of course this is RNA. It uses ATP, GTP, CTP and UTP as the substrates. So it uses triphosphates just the same way DNA polymerases do. And then it's able to start a chain de novo. And it synthesizes the RNA in a 5 prime to 3 prime direction, the same direction that a strand of DNA is made by DNA polymerase./nSo it would copy here. And so it would put in an A opposite a T. And then because it's RNA it will put in a U opposite an A, and then an AGCAU and so on. So this right here is the beginning of the RNA that's being synthesized by the RNA polymerase. This strand is known as the transcribed strand. And by default then that one is the non-transcribed strand. And what you can see by doing this, it's making an RNA the same sequences up here, except that everywhere there's a T there's now a U in the RNA./nSo the final thing then is how this information gets all put together to make proteins. And protein synthesis is done by an machine known as the ribosome. It's made up of some special large RNAs -- -- called rRNAs, some proteins as well. These make up the ribosome. And then it needs a mRNA and then it needs the various tRNAs, each of which carries an amino acid that's appropriate to its anticodon./nAnd in a very briefly sort of way this is -- And you can see this in your textbook, what the ribosome does is it takes, let's consider this is the mRNA. I'm just going to take three codons here. And this mRNA treads into the ribosome. And I'll sort of show it's able to recognize the first codon and the second codon. Remember, of course, there's no spacing like this in the RNA./nAnd then in the context of this large factory it's able to find the tRNA that has amino acid one and the anticodon that would correspond to this. The tRNA that has the next amino acid attached and its anticodon. So you can see what's happened. It's been able to order the first amino acid encoded by that codon and put it physically right next to the next amino acid that's coded here./nAnd then it catalyzes -- -- the formation of a peptide bond. And what happens when that does is the way this amino acid is joined to the tRNA there's energy stored in that bond. And so thermodynamically that allows this bond formation to go. And now you end up essentially with this. And what happens now is everything clicks over one. So you could think of it as this whole RNA shifts over one so the one that used to be here is now sticking outside./nHere's part of the ribosome. Here's the next codon. What we have here is the tRNA that's got amino acid two joined to amino acid one. The next codon specifies the next amino acid which is three. And the process is then able to go on like that. Now, the structure of the ribosome, the crystal structure of the ribosome was just finished. And I guess we've got as many lights out as we can do right now./nIt's absolutely remarkable. It's mostly RNA. The gray stuff and the blue stuff are two huge RNAs that are all folded up in 3-dimensional space. And these things that are sort of stuck on the outside, these purple things here or the dark blue things here that sort of look like cherries stuck on the outside of a cake, those are proteins. So most of this is RNA, big balls of RNA with proteins kind of decorating the outside./nThe mRNA is a green thing that snakes through. There's the mRNA. See it snaking through? And maybe you can recognize in the middle this tRNA. There's an orange one and a yellow one. Those correspond to the two tRNAs I depicted here. And I'm just going to see if I can stop this. There's a viewpoint I'd like you to see when it comes around again here in just a second./nI'll see if I can catch it there. Right there. Here's one of the tRNAs in yellow. And its end is right there. And there's the other tRNA. And its end is right there. So this corresponds to the point at which there's going to be an amino acid formed. And something is going to catalyze the formation of that bond. Well, the next picture sort of shows what happens if you pull that apart./nAnd what you'll see is that here's the end of one end of the tRNA, there's the other end, and there's nothing near it except for RNA. So RNA is actually catalyzing the formation of the peptide bond. Another way to say that would be that the ribosome, which is the protein synthesizing factory, is a ribozyme. Remember I said most of the chemical reactions that need catalysts are carried out by proteins but there are a few that are carried out by RNA where RNA is the catalyst? And remarkably the formation of the bond, which is at the heart of proteins which are so important for all life, is catalyzed by RNA./nIf you look at what makes proteins, what do you see? You see huge balls of RNA, a mRNA threading through two tRNAs, and the enzyme activity or the catalytic activity is encoded by the RNA as well. As I said, people think possibly there was an RNA world that preceded our present-day world with DNA, RNA and protein. And who knows? But this sort of look at a ribosome could at least make you see that that's a plausible explanation that RNA might have been running the show for a while before anything else got involved./nAnyway, we'll see you on Friday then.
Tags // Molecular Biology Lecture Graham Walker
Added: April 2, 2009, 1:55 am
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Molecular Biology Lecture III Prof. Graham Walker
Biology > Introductory Biology/n * Email this page/nVideo Lectures - Lecture 11/nTopics covered: /nMolecular Biology III/nInstructor: /nProf. Graham Walker/nTranscript - Lecture 11/nSo just trying to remind you that the replication fork looks something like this where 5 prime to 3 prime and 5 prime to 3 prime. This is what's known as the leading strand because DNA, the synthesis of the new strand can go -- Which is going 5 prime to 3 prime is going in the same direction as the movement of the replication fork. The other strand, which is known as the lagging strand, the DNA synthesis is actually going backwards to the movement of the replication fork, which means it has to go and then start up here and go again./nAnd it's continually jumping. And I told you that the little RNA primer is used to start each strand. And then the DNA polymerase is able to elongate that. And then at the end these little nicks in here, the RNA has to be removed, fill in the gap and then it's sealed up by the enzyme DNA ligase, which we'll talk about when we talk about recombinant DNA./nSomeone asked, I had mentioned why this strategy of using RNA was beneficial, and that has to do with the fact that the fidelity, which is going to be the next thing I'm going to focus on of DNA replication is not, you can get a much higher accuracy if you have the end of a primer already there and then carry out the chemistry in there. No enzyme has ever achieved the accuracy that you see in DNA replication if it's starting a strand./nSo RNA polymerase, which constantly starts strands to make RNA copies, as we'll talk about, is not as accurate as DNA replication. And by putting a little bit of RNA, because the cell has to start a new strand. Before it gets here there's no strand at all on this lagging strand so it needs to make this little RNA primer. It needs to make a little primer. And by making it out of RNA then it can tell what doesn't belong there. It doesn't matter if it's not quite as accurate as the rest of DNA replications because it's going to take it out anyway and fill it in using the DNA polymerase./nAnd if you think about that maybe you can see one of the reasons that the cell has chosen or nature has chosen through evolution to use little RNAs to begin the strands. OK. Well, in any case, the fidelity of DNA replication is really pretty amazing. Incidentally, just speaking of DNA, many of you wrote some very thoughtful things about Vernon Ingram's visit. I didn't give him a whole lot of warning and he had to go and change his schedule and move meetings around in order to come to talk to you./nAnd it was very nice of you. Many of you wrote some very thoughtful things, which I'm going to pass onto him. I want him to know that many of you appreciated his visit. I also saw a lot of you react to his advice about crowded labs. That has been my experience, too. And one thing about the scientific process is it's not just one person. You're in with a group of people, just as Vernon described, and that group of people becomes the creative engine that drives all the science within that lab./nAnd so you're not only picking your project, you're looking for a group of people to work with. And, as Vernon said, if the lab is really doing hot stuff they tend to attract a lot of people. So a crowded lab can sometimes be a really good indicator. No absolutes, and there's an exception to everything, but that was a good piece of advice he gave you if you're looking for UROPs sometimes./nOK. So, anyway, DNA fidelity. Remember I said we've gone from, our bodies have somewhere from like 10 to 20 billion miles of DNA in them if we could take all the human DNA and stretch it out? But that fidelity is done at an error rate of about one mistake to every ten to the minus tenth nucleotides replicated. Which I said if you were typing all the time it would be like sort of making one mistake every 38 years./nSo it's an astonishing degree of fidelity. Something that's beyond anything within our experience. And there are three principles that go. One is polymerase is really good at the base pair recognition telling that an A is paired with a T or a G is paired with a C. And discriminating against everything else. There's a phenomenon known as proofreading, and I'll tell you how that works./nAnd then there's a third system called mismatch repair. And all three of these contribute to this very, very low-frequency of errors, one mistake for approximately every ten to the tenth nucleotides replicated. So the first thing is I've pointed out to you several times that if you draw the hydrogen bonds between an A and a T base pair, the two hydrogen bonds or the three hydrogen bonds between a G and C base pair, that the shapes of this pair and that pair are virtually identical./nYou can pick them up and lay it right down on top. Now, if you actually look at it you'll see you could draw some base pairs between, for example, a G and a T. In fact, you can draw two hydrogen bonds, which is the same as between an A and a T. But the one thing I hope you can see, just from the shapes even without being able to see the individual atoms, is that a GT base pair doesn't have the same shape as the correct base pairs./nSo when I showed you that little movie the other day where this is the template nucleotide, this is the incoming nucleotide and there's this alpha helix that's swinging up. What's happening in there is that the enzyme is checking the way that the incoming nucleotide is the correct shape to go with the base pair. And you can sort of see it's flipping it right into a very narrow little slot in the enzyme./nSo it's not only asking for sort of hydrogen bonds, it's asking for the exact shape. If you just did it by thermodynamic grounds you'd make about one mistake in a hundred because that's about the discrimination between the correct base pairs and some of these other ones. This works so well. You get more like one mistake in ten to the fourth or ten to the fifth./nWe're still quite a distance away from the ten to the tenth, but this is one of the things. It's looking for the correct shape of the base pair. Now, the second thing that helps with fidelity is a phenomenon known as proofreading -- -- exonuclease. Things called a nuclease. That means it can degrade DNA. And the exo works at an end. And, furthermore, the directionality of this proofreading was something that puzzled people initially because it's going 3 prime to 5 prime./nAnd when people started to purify DNA polymerases or complexes of DNA polymerases involved in replication there seemed to be a puzzle because the polymerase, as I've told you, goes 5 prime to 3 prime, but the same enzyme complex had an exonuclease that went in the opposite direction. So this seemed very peculiar at first in the sense if you were trying to polymerize DNA in this way why in that same enzyme would you have something that wanted to degrade DNA in the other way? And the answer turned out that this was known as a proofreading exonuclease, as I've put up here./nAnd here's the principle of how it works. Suppose you were replicating the DNA and there was a G. And if you put a C in there it very quickly goes on and continues the replication. If it puts in a T, let's say, this is not a very good base pair. It wouldn't have the right shape. So when the enzyme came up looking for that 3 prime hydroxyl, which would be right at the end of that T, things are not in the right place./nAnd so the polymerase activity slows down. And as that primer terminus, if it sits there for a little bit, it's able to just peel off the DNA, flip up, and there's this function that does just what you'd do if you were typing and you made a mistake. You'd just hit the delete key and take off the last nucleotide that you did. And I have a little movie showing you that./nThis is a crystal structure. This is the DNA template. And the polymerase catalytic activity site is right here. And in this little movie it's just added an incorrect base pair and the polymerase is sort of stalled. And the actual nuclease function is physically separate on the protein structure. But what you'll see in the movie is that if the polymerase cannot go very well eventually this thing will come up and it will chop off one nucleotide, come back and try it again./nLet's see. I think if we do this, oopsy-daisey. Let me see if I can get this to work here. Nope, it's not working. OK. Well, anyway, I'm going to skip it for right now. I don't want to waste time. But, in any case, the end would go up here and it would take off one nucleotide. So there at least are two of the ways that the polymerase is able to work with such fidelity./nIt selects for the correct base pair shape. And then after it's done an addition it sort of looks back, just as if you were a very slow typist, and every time you typed a letter you looked back and said did I make a mistake? And if you made a mistake then you'd delete and then just try again. And that gets the cell another maybe two orders of magnitude of accuracy. So we're up to about one mistake in ten to the seventh base pairs replicated./nThe third system, which is called mismatch repair, turns out to be very important for a whole variety of reasons. And before I tell you about it, I want to first introduce the idea of DNA repair in general. One of the things that's wonderful about DNA -- -- as you've learned, is it's got the information in two copies. It's in a complimentary form but it's like having the photograph and the negative./nAnd if your kid sister pokes a hole with a pair of scissors through the picture of your boyfriend or your girlfriend, you're not really in trouble as long as you've got the negative because you can get the information back again. And that same principle applies in DNA repair. So if you have some kind of lesion in DNA, and this might have come from going outside in the sunlight, your DNA absorbs in the UV and it undergoes photoreactions, they tend, for the most part, to just effect one of the two strands of DNA./nOr if you smoke, which I hope none of you do, there are many chemicals in smoke that will react with DNA, and they'll modify one strand. And so what the cell has is a system that has many kinds of repair systems, but it has a special type of repair system known as nucleotide excision repair. And you could think of this as a protein machine that constantly scans the DNA looking for little distortions./nAnd if it finds it then what it needs to do is it needs to make cuts, remove the DNA and make a little gap. And now you can see what it can do now. Once it's got a little gap the information over here is a complimentary form. So if a DNA polymerase were to come along it could fill in that gap and seal it up and then you'd be back to ordinary DNA, the lesion would be gone. And I made a silly little PowerPoint thing here to show it./nSo if you were to, say, damage the guanine with something, say one of the carcinogens you find in cigarette smoke, you could think of this protein machine as being a sort of pair of scissors that have a conditionality in them. As this protein machine scans along the DNA the scissors aren't activated until it recognizes there's a distortion here, at which point then it senses that there's some bump in the DNA./nAnd it's very cleaver the way it does it because the nuclease activities, the things that are going to cut the DNA are actually some distance away, a few nucleotides away from the lesion. So even if this is distorting the DNA, the scissors are able to work out here and out here. It makes two cuts. That was a huge surprise. Nobody expected that when they started to do the biochemistry./nAnd then in principle once you cut it now you can remove this little nucleotide and then a DNA polymerase can just come in, and following those A pairs with T, G pairs with C, copy it along and then would seal it up to get to the end. And I've actually shown you a picture of what happens if a human is missing that system. When I was showing you how profound an effect you could get from just losing one single gene or a mutation affecting one single gene, this disease called xeroderma pigmentosum./nThey're a variety of different groups. And the one on the left is an example. That's someone who is missing one of the genes that encodes one of the proteins involved in nucleotide excision repair. And this is really, really important for fixing up the damage we get all the time in sunlight. So if you miss that repair system and you got out in the sun then you get all kinds of lesions and people are very susceptible to skin cancer./nAnd I told you fortunately now you don't find people with this disease looking like that because at least in developed countries we recognize it. They're kept out of the sun. And these were the kids who I said are called "children of the moon" because they, for example, go to summer camps where they do everything at night so they won't get exposed to sunlight./nBut that's what happens to us if we miss that excision repair. And, again, what makes that possible is that the information is there twice in a double-stranded DNA. I also showed you a little movie early on when I was showing you, I'm going to actually run this in QuickTime because it works a little more smoothly, I think. So I showed you this when we were talking about DNA because I wanted you to sort of get that sense of what it was like to kind of fly down the groove of a DNA./nBut what I didn't emphasize was this protein that was bound to the DNA. That's a protein that's a DNA repair protein. And it's one of these things that looks for lesions in the DNA. And as we fly along the major groove this little green thing is actually the lesion that that protein is looking for. And it sort of puts fingers down into the groove and it's able to sense that./nAnd you can sort of see how this protein is bound to DNA. This is a lesion that we get all the time from oxidative damage. And remember I said oxygen is bad for DNA? So our bodies have to have systems that are able to do that. So DNA repair is very important for life. We'll just finish flying down the major groove one more time here. OK. I'm going to go back to PowerPoint. OK./nSo mismatch repair is a form of repair that's got that same idea. Let's think about it if we had a replication fork here, and let's say there was a G here and the T got misincorporated, but in this case it wasn't removed by the proofreading which happens about one in ten to the seventh times. Now if that strand is fixed up, excuse me, is continued then you'd end up with a GT base pair. And the next time you copied it this strand would give rise to a GC but this one would give rise to an AT./nAnd then you'd have a mutation that now would have changed. And if it affected an important gene that could be bad for you. So the cell has what's known as a mismatch repair -- -- that works in exactly the same logic as here. That it basically comes along. It scans the DNA. It finds the bump because this is not a proper base pair./nAnd then it fills it in and you're back to ordinary DNA with a GC base pair. There's one little wrinkle. For this system to work it has to do one other thing that's different from that kind of DNA repair. Can anybody see what it is? Why don't you talk to the person next to you and see if you can figure it out. This system must be doing something else in order for this to work./nOK, you can ask somebody. What do you think? What if I removed the gene? Would that work? What would happen if I took the gene instead? Say I made the little gap over on this strand instead, cut it here? Yeah. So which one is the one that's right, the old strand or the new strand? The old strand, yeah. See, this is the old and this is the new. And the term that's usually used, it's known as the daughter strand, the new strand./nSo the other thing this system has to do is it not only has to be able to detect that there's an incorrect little base pair in there, but it also has to know which is the parental strand, the template strand, and which is the daughter strand, the newly synthesized strand. And this system makes the assumption that the strand that's old is the one that's correct and the mistake is on the new one./nYou guys see that? OK. So that gets another two or three orders of magnitude in accuracy and that's what brings it up. Now, the people who made this, who formulated this model for mismatch repair, complete with the feature that it needed to recognize the old and new strand, that's a bit of a trick, if you think about it because it's DNA on both sides. And there are several different ways used in nature, so I'm not going to go into it, but there's at least a couple of different ways of doing that trick./nYou could sort of see if you were the replication fork and you talked to that you could certainly, just from the geometry of that, if you wanted, you could probably keep track of who's old and new. E. coli has a very cute trick, but it's not universal so I won't go into it, but the people who did the seminal stuff, I had to just quickly show you a couple of pictures./nWhen I showed you that picture of the DNA 50th, the guy sitting in the front row was Miroslav Radman who was one of the two people. He's a European scientist originally from Croatia. And he collaborated with someone you've heard about before, Matt Meselson, who was up at Harvard. And it was with the Meselson-Stahl experiment that showed the semi-conservative mechanism of DNA repair./nThis was a little reception. And Matt was talking to Alex Rich who's in the MIT Biology Department. And I was amused because remember how Vernon told you how Francis Crick would run up and down the stairs in the Cambridge lab and he was talking all the time? And I've heard Vernon say you could never really tell whether an idea came from Watson or Crick because they'd just talk, talk, talk all the time./nSo this was at sort of nice reception at the DNA 50th. And within a couple of minutes, I looked over and there were Miroslav Radman and Matt Meselson talk, talk, talk. They were in the corner drawing pictures on a board. I also showed you actually a picture of one of the genes that's involved in recognizing this mismatch, because there's a protein that recognizes that mismatch and it's given the name of MutS./nAnd when I was showing you some proteins it had one that had a lot of alpha helices. This is actually a picture of MutS. It's a dimer. That's why some of it's green and some of it's blue. And this is DNA viewed end on and it's recognizing a GT mismatch in DNA in that picture. Now, this may sound very esoteric, you know, and obviously important for life and an important part of sort of understanding how life works if you're interesting in studying molecular biology./nIt may not seem to have very much connection to your real life. But, in fact, in this case mismatch repair does because it affects the frequency with which, if you lose it, then when you replicate your DNA you're going to make more mistakes. And I need to just give you a very quick introduction to cancer so you can see why this is important. Cancer comes from the fact that remember a human cell or a multi-cell like us that has many kinds of different cells starts out from one cell./nAnd I talked about first you get the embryonic stem cells that can become anything. And the cells become successively more and more and more specialized as they go along. So ultimately a cell that's in your retina or in, say, the lining of your colon needs to know that's where it belongs. And it also needs to know that it cannot just keep replicating. So if this is actually showing a little picture of the lining of your intestine./nAnd there's a single layer of cells right along the inside edge of your intestines. This is the cells through which all the nutrient exchange happens and everything else when your body extracts nutrients as food stuff passes through your intestine. And so what happens with cancer is a cell that's normally a part of your body has to obey a whole set of rules./nAnd what you can think of when someone starts to develop cancer is that what started out as an ordinary cell undergoes some kind of successive changes in its DNA that gradually causes it to forget the rules that make it be part of an organized body system. So if we take a look here at all these different cells. But let's imagine just one of the gets a change that makes it forget to stop, or it should know to stop replicating when it touches its neighbors, but if a cell were to lose that control what would happen? Well, it would then begin to proliferate./nAnd then what happens in cancer is the cell will, now there are more of them, and one cell with acquire an additional mutation that will lead to a further loss of growth control. You can see now the cells are starting to become sort of funny shapes. And then one of the cells in here will undergo yet another change. And right at this point, up until now, the cancer has, even though the cells are dividing and have lost some of their growth control they're still staying in the same place./nSo that would be sort of, you know, like a wart or something like that, or what you would hear as a benign tumor. You can go in surgically and take it away. But then the other thing that can happen is cells can forget where they're supposed to be in the body. And when that happens they say the cells metastasize and become metastatic or a malignant tumor. And what that means is the cell is beginning to, it's acquired yet another change that's made it forget which part of the body it's supposed to be in./nAnd they've signified it here as being a change in this cell that then leads to, you can see here right now it's starting to invade into the whole intestine. Or if one of those cells comes off loose in your bloodstream it can land somewhere else in your body and then start to grow there. And that's what happens when somebody has metastatic cancer. You cannot really cure it because now there are cancer cells all over the body./nAnd that usually is a very difficult situation to get any kind of cure on. So to put this in perspective, you needed to have a number of changes to go from an ordinary cell to a metastatic cancer cell. So each one of these changes there was some kind of change in the DNA. Either there was a mutation or maybe a chromosome was lost or something like this so that you need a series of successive genetic alterations./nSo there was a very key insight that a number of people had after we understood the mechanism of mismatch repair. Because some people realized that if a human cell had lost mismatch repair then the frequency of each one of these changes would go up. It wouldn't affect what the change was. It wouldn't actually have anything to do, if you lost mismatch repair it wouldn't affect directly the ability of this cell to stop dividing when it touches its neighbors./nBut it would increase the chances that a mutation somewhere would have that effect. And if every one of these steps goes now a hundred or a thousand times faster, you can see that if somebody loses mismatch repair in a cell then the chances of that cell becoming into a cancer are very high. So there was a kind of human cancer, it's a susceptibility to colon cancer called hereditary nonpolyposis colon cancer./nYou don't need to remember the name. It's often abbreviated HNPCC for people who cannot remember the name. But it was a kind of susceptibility to cancer that ran in families. So it was thought to be genetically determined in some way. And one of the interesting things was a number of the people who had this disease would show a kind of instability of the genome if they looked in the tumors./nThey just looked at the DNA. It seemed to be undergoing changes at a much faster rate. And the insight that came out was that the people who had this disease had, for example, a mutation affecting what we can think of as a human homolog of MutS. And we'll talk about genetics of humans in a small number of weeks, but I think most of you know that for most genes, except for the genes associated with the sex chromosomes, you get one copy of a gene from mom and another copy of a gene from dad./nSo under most circumstances we would have two good copies of this gene encoding a human homolog of MutS. What does that human homolog of MutS do? The same thing as the bacteria. It recognizes a mismatch in DNA and fixes it up. So it turned out that what the people with this disease have is they have one of the genes. The gene they got from mom or the gene they got from dad is broken. So they're still OK. They have one copy of mismatch repair in every cell./nBut if a cell ever had lost that copy of the good version now that cell and all of its descendents would mutate at something like a hundred or a thousand times the normal probability. And so they would progress down this pathway. And so the polyposis means that if they look in the colons of people who have this disease they find lots and lots of little growths or polyps that are on their way to progressing down this disease./nEven in these people it takes quite a while. And so once they knew that they were able to go in and through colonoscopies find these cancers and remove them. And most of you will not have that disease, but this is now a kind of cancer that's pretty much preventable as long as it gets detected. It can take in a normal person as long as 20 years or something for an initial cell that underwent this initial change to go all the way down to becoming metastatic./nSo when you get older, and this certainly applies to most of your parents or in this age group, you should ask them if they've had a colonoscopy. It's not the world's most fun procedure because, you know, they stick a probe and look inside your intestine, but it isn't that bad. And what they do is if they see one of these little polyps they can catch it before it's progressed far enough to be metastatic./nAnd then there's no problem. I had my first one done about, I don't know, three or four years ago and they found one. And they took it out and I'm fine. But if it had been left there and allowed to progress then some years down the line I would have gotten colon cancer. And I'm going to have to go back and get checked again in another year or two./nBut it is something that you should check with your parents because everybody should have a colonoscopy. My hope is by the time you guys reach an age when this comes they'll probably have some kind of little blood test or something where you won't have to go through this indignity. But right at the moment it's something everyone should do, I think. I just wanted to make one other comment about basic research because there's another thing here./nActually, my lab was the first lab to clone the MutS gene. We cloned it, we sequenced it, and we looked in the databases. And at that time in the late eighties there was nothing else that looked like it. I thought it would be like, there were some sort of similar mutants, and here's what it looked like. This is a culture of E. coli. And there are about ten to the ninth cells per ml./nAnd we plated about ten to the ninth or ten to the eighth on a plate with a drug on it. And you can see they almost all died, but there were maybe three or four that survived. And then their descendents were able to grow up and form a colony. This is how we recognized something was defective and what we now know as mismatch repair. If you took this mutant of E./ncoli and plated it out, you'd see you got a lot more drug-resistant colonies. That's the difference that I was describing, the importance of mismatch repair. If you don't have mismatch repair you can see, you get a lot more mistakes that show up as mutants. So I was studying that. And we cloned the MutS and MutL genes which is another gene that's involved in this. Didn't see anything in the database, but there were very similar mutants in streptococcus pneumonia that people had isolated./nRemember streptococcus pneumonia in the transformation experiments? So I thought, well, maybe these are the same genes on an evolutionary basis. So I phoned some labs, and I found one that was sequencing what turned out to be a homolog of MutS. We tried to publish our papers in a medium fancy journal because I thought this was a pretty cool result that two bacteria that were evolutionarily diverged had this conserved mechanism for mismatch repair, but the reviewer said, you know, this is a pretty specialized topic, it's not of general interest, it should go in, the phrase they use is "a more specialized journal"./nSo it was published in the Journal of Bacteriology which is a really wonderful journal, but it basically deals with bacteria. And about a week after that paper came out my phone rang and it was a guy from Emory. And he said, "I work on mouse. We were sequencing a gene," it doesn't matter what, "and we sequenced in the wrong direction. And we seem to have something called MutS./nDo you know anything about MutS?" And a couple of days after that I got a phone call from somebody at NIH. And they said the same thing, "We were trying to sequence this gene in humans. We kind of sequenced in the wrong direction and found MutS." So within a week of the paper coming out I knew there were mouse and human homologs. And that led from these sorts of studies, which my first graduate student worked on, to the identification of the human homologs./nAnd then not me but others made the connection between mismatch repair and cancer. But this is the way a lot of things happen with basic research. This doesn't look like anything that's very important. And it sure doesn't look like it's going to lead to an insight into cancer, but this is very much the way it goes. I've had this happen twice with another set of genes in my life that turned out to be important for cancer as well./nAnd, as I said, what happens, if you lose mismatch repair, then all these alterations happen much more quickly and the cells can become cancerous. I've included a couple of outtakes because I actually made this slide with my son's pillowcase on our dining room counter. And our cats, who you saw at some point earlier in the year, thought this was the weirdest thing they had ever seen, when I brought these plates home./nSo, OK, anyway. All right. So one other thing to tell you about DNA replication before I move on, and that is -- -- the initiation of DNA replication. In E. coli there's one great big piece of DNA. And it's all one giant circular chromosome. And if you realize what I've told you about DNA replication, I've talked to you only about once you have a replication fork established how you keep it going. But, as you might guess, a really important point of biological control is the initiation of DNA replication./nAnd so the way cells do that is they have a special sequence in their DNA. It's written just with Gs and Cs and As and Ts, but it's a word sort of written in a different language than the kind of genetic code we're going to be talking about in the next couple of lectures. And what it means is "start replication here". And so in E. coli these are called an origin of DNA replication./nAnd, for example, in E. coli it's a stretch of DNA that's about 250 base pairs long. And it's got a sequence that lets proteins bind and they kind of are able to make a little bubble like this. And it's at the edges of this little bubble where it's able to start a replication fork. And one of the secrets to control of cell division is that cells are able then to control whether the protein that sees the origin is there or not./nAnd it won't start a new round of replication unless everything is right. Then it can make the things that initiate a new round. And after that it finishes. Our eukaryotic cells with a lot more DNA use the same thing. The same idea, but there tend to be multiple origins. And you get a little bubble and another little one down here./nAnd once you get the replication forks established then these kind of merge. And then eventually we end up with the two strands of DNA. But I just mention that in passing because it's an example of how even though the DNA is nothing but Gs and Cs and As and Ts, you can kind of write words in there that mean different things. Some of them on the genetic code tell you what the order of amino acids in the cell are, but everything else has to be encoded in the DNA, too./nAnd here's a really nice example of how that works. Now, we're going to switch at this point from worrying about how DNA is replicated to how information is stored and interpreted. And there's a figure that most of you have probably seen, DNA goes to RNA goes to protein. This is the usual direction of information flow. The information for making proteins is encoded in the DNA, as we'll talk about in more detail, and an RNA copy of some piece of that, one gene's worth usually, gets made in RNA./nAnd then that information in the RNA is used to direct the sequences of amino acids that appear in a protein. And this is a four letter alphabet, if you want, A, G, T and C. This is a four letter alphabet, A, G, U and C, where the uracil and the thymine have the same base pairing capacity. And this is a 20 letter alphabet. All those 20 amino acids that you were looking at, at the chart over at the back of the exam. So from the point of view of information storage and information flow there are some interesting things that had to come up in order for the information to flow in that way./nBut before I do that I want to just get you to think about DNA as an information storage device. This is MIT. I'm almost sure in this room there are some people that are experts in high density information storage. And even if you're not most of us have now a lot of experience with it. Your computer can do gigabytes of information. Your iPod probably has a 40 megabyte hard drive in it or something like that./nSo you have some experience with high density information storage. So here's the question. How much DNA would it take to encode everybody who's alive on earth today, 6 billion and a bit people? And let's argue that all we need is a single cell's worth of DNA because everybody started out a single fertilized egg and went on. Yeah? OK./nEnough DNA to fill one human being. Anybody else got any sense? All right. This is, I think, the most amazing demo. I did this when I was teaching for the first time. The amount of DNA it would take to encode everybody who's alive on earth, one cell of everybody who's alive on earth today is this little thing in here, which you probably cannot see even, but I took a picture of it. There are about six times ten to the minus twelfth grams of DNA in a human cell./nAnd if you multiple that out by 6 billion people it comes out to 36 milligrams of DNA. And I weighed out 40 something milligrams of DNA. So there's actually more DNA there than you need to encode everybody who's alive on earth today. And I don't know how this hits you, but I've been working on DNA my entire life. And every time I do this, you know, I think I understand this molecule, but I don't really think I do at some more fundamental level./nIt's absolutely amazing how much information is stored in that molecule. So the one point I will, actually, I think it's close enough. Why don't we just call it a day, and I'll pick this stuff --
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Added: April 2, 2009, 1:45 am
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Molecular Biology I Lecture Prof. Graham Walker
Biology > Introductory Biology/n * Email this page/nVideo Lectures - Lecture 9/nTopics covered: /nMolecular Biology I/nInstructor: /nProf. Graham Walker/nTranscript - Lecture 9/nSo today we're going to continue our focus on DNA which I'm personally enthusiastic about at least in terms of being such a fascinating molecule. And I told you the story last time of how we actually came to understand that DNA was the genetic material. And I still see comments that, oh, God, all this stuff is not relevant to the exam. We're trying to construct the exams in ways that test whether you got the concepts and not just whether you memorized every term that you ran into in the textbook./nSo I'm hoping that you will see some greater purpose in why I'm trying to talk about some of this. And also I'm sure some of you will forget the details of transformation, of DNA replication we're going to go into as we sort of burrow into it over the next lecture or so, but what I am hoping you may remember ten years from now, even those who don't go in biology, is how experiments are done, how real people do them./nAnd that was partly what I was trying to tell you. And you guys are pretty good at figuring out the basic principle that someone had to somehow show that a DNA molecule in one organism could change some organism to have a new characteristic. And as I sort of told you with the work from Frederick Griffith. And then his initial stuff wasn't devoted to that at all./nIt was trying to solve a very pressing problem which is dealing with pneumonia in a pre-antibiotic era. And then the finding that he got, that this odd result that something in a heat-killed extract could be transferred to a live bacterium sort of set things up for Avery and his colleagues after a number of years of work to make a very powerful argument that DNA was the genetic material./nBut, as I said at the end of last lecture, that paper was published in the 1940s. And people didn't immediately say oh, wow, DNA is the genetic material. Often, and we'll see it again with genetics, there's sometimes sort of the body of science the average person thinks about. Science needs to reach to a certain state before an idea can take hold, even if there's evidence supporting it./nPart of the problem was that chemists had isolated DNA. And the way they used to isolate DNA was really rough on it. Crack the cells open. And what happened, it would all get broken down into little pieces of DNA. And people had worked out the basic chemical structure that it was the deoxyribose and how the things were joined together, but nobody had ever seen anything more than just these little pieces of DNA./nAnd there was a widely held conception that it was just an anonymous tetranucleotide of G, A, T and C. It wasn't clear why the cells made it, but it didn't look like anything that could encode information. Whereas, as I said, something like proteins, those seem to be very different. And so the world wasn't quite ready for it. Another thing, and this came from one of the comments here, was someone said they didn't know bacteria could take up DNA from the environment./nAnd, in fact, most bacteria can. It happens that streptacoccucci and some other bacteria at certain phases in their lifetime develop this capacity to take up DNA from the outside. Given what I've told you about a membrane and how hard it is to get things across it, you could imagine it's not trivial to get a DNA molecule which is huge from one side to the other. So it doesn't normally happen./nAnd what happens if you go into a lab and you're cloning something or other, and we'll talk about how to take a couple pieces of DNA and join them together in a test tube and then put them back into a bacterium. If we put it into E. coli that doesn't normally take up DNA you'll find that it's sort of basically black magic. You cook them up with some divalent cations at very high concentrations, you do temperature shifts and various things, or you give them a big jolt of electricity, and the next thing you know you get some DNA inside./nAnd it's not a very efficient process, but all you need is one molecule to get in one bacterium and then you're in business. So that was another reason that this wasn't accepted right away. Because this was not a phenomenon that could easily be repeated with other bacteria. So it looked like it was something perhaps special to streptococcus. And what did really change people's understanding, or at least bring people to the understanding that DNA could possibly be the genetic material came about from the discovery of the actual structure of -- How the structure of DNA as a long molecule with complimentary strands and the double helix, the little pictures I showed you with the base pairs, which you know about, and how the two strands which now I'm going to start emphasizing run in opposite directions./nWe'll come back to that in a little bit, but the 5 prime to 3 prime direction is this way on one end and 5 to 3 on the other. And just remember back here that there's the 5 prime carbon and that's the 3 prime carbon. So this is the 5 to 3 prime direction of the strand. And then it twists up in 3-dimensional space to form this double helix. And you've seen that movie several times./nSo once that structure was discovered then people began to see how these could possibly encode information. It was clearly not just a tetranucleotide of G, A, T, Cs. But we didn't move immediately to that understanding. And today, again sort of trying to show you how biological experiments are done and how they're done by real people, I want to just go on and tell you the key things that happened next. So someone who was very struck by the results of Avery when they came out was Erwin Chargaff who was at Columbia./nAnd, in fact, my colleague Boris Magasanik whose office is next to mine was a post-doc in Chargaff's lab. So I've got a neighbor of mine who worked with Chargaff. And Chargaff was very struck by this result from Avery and his colleagues that you could take DNA and put it in another organism. And here are a couple of quotes from his writing. One that I liked./nI've sort of had a sense of this in my own research career, this kind of thing. "I saw before me in dark contours the beginning of a grammar of biology." He didn't really know quite how it worked but he sort of sensed that somehow here where you could get down to the language that biology was written. So he started some experiments. And I started with the conviction that if different DNA species exhibited different biological activities there should exist chemically demonstrable differences between deoxyribonucleic acids./nSo he was able to start just doing some simple chemical experiments to try and look at DNAs from a whole variety of sources and see what he could learn. And this was not at the structural level. This was just at the chemical level. But one thing he learned was that the base content of DNA, that's the A, G, C, T part of it varied widely between organisms. So this was what Chargaff found in his lab, key findings. And that was important because if DNA was just a molecule of GATC, just a tetranucleotide that every organism made then you'd expect to find the same base composition in all organisms./nHe didn't, so that finding essentially buried the monotonous tetranucleotide hypothesis. Another thing he found was that DNA was the same in different tissues -- -- from the same organism -- -- but the proteins varied. And that's a characteristic you'd expect of something that was the genetic information from the cell that all cells have to have sort of the major blueprint. And if you had, even though proteins look like an attractive possibility for that because they had so much variation, this kind of finding wasn't consistent with it and it supported the idea that DNA was the genetic material./nWell, the other thing he could do was he could measure the A, T, G and C content of all these different DNAs. And he noticed some similarities then. And he extracted out of that a couple of generalizations. One was that if you looked at the ratio of the purines, those are the ones with the two rings, adenosine and guanidine over the pyrimidines, those are the ones with the single ring which were C and T, it was about one./nAnother thing he noticed was that the ratio of A to T was about one and the ratio of G to C was about one. Now, that was an important clue but it didn't lead to any immediate breakthrough, even though maybe now that you know the structure you can see, gee, if I had been there maybe I would have been smart enough to jump on that number. So instead the work that led to the structure of DNA now introduces a couple of other characters who you've heard of a lot, Jim Watson and Francis Crick./nAt the time that Avery made his discovery reporting DNA was transformation, and Jim Watson described himself later as a precocious college boy in Chicago who was consumed by ornithology. So he was into bird watching. That's what he was excited about at the time Avery did his experiment. And Francis Crick at that point was a physicist, and he was in the British Navy designing Naval mines./nSo that's where those two players were at the time of Avery's results. So then both Francis Crick and Jim Watson ended up in Cambridge, England about 1950. I think Crick got there around 1949 and Jim Watson got there in 1951. Francis Crick was a grad student, 35 years old at the time. I'll show you pictures in a minute. 35 years old at the time and still working on his PhD./nSo he was a pretty elderly grad student, if you want to think of it that way. And Jim Watson was a young hot-shot. He had done his PhD working with Salvador Luria who was at Indiana University at the time. Salvador Luria was one of the Nobel Laureates at MIT. He founded the Cancer Center, which is still here right across from the main biology building. And Jim was a very, very bright and brash young guy, and he had done his PhD with Salva and then he went to Cambridge as well./nAnd the reason they both went to Cambridge was they were attracted by the power of x-ray crystallography. Now, I said a little work about that earlier, that if you take x-rays and you bounce them off a crystal and then measure the diffraction pattern you can work backwards by Fourier transforms and whatnot to figure out what the underlying crystal structure is. For the purposes of this course the mechanics of how that's done, we don't have to worry about that right now./nYou just need to know that you can work backwards from the diffraction pattern to figure out what the underlying structure was. And I told you, when I introduced to proteins, that the first clues that there were these regions of secondary structure, alpha helices and beta sheets came because people saw characteristic reflections in these diffraction patterns of certain proteins./nAnd I also told you the story of how Linus Pauling had gone to Oxford, had gotten sick and tired of reading detective novels, started to try and explain the refractions in a certain class of proteins and came up with a model for the alpha helix. And so that was the sort of thing that inspired Watson and Crick. They were both interested in how one could get the structure of DNA. Now, Cambridge also had a very good x-ray crystallogram group./nAnd just in passing it's interesting as to why they didn't come up with the structure of the alpha helix. There were two things. One was just lack of basic knowledge. I told you that the peptide bond, if you remember I emphasized that you cannot rotate it because the electrons are distributed. Pauling was an outstanding chemist. He knew that fact./nAnd the folks at Cambridge who were doing that didn't learn this until later, so their models were far less constrained because they could have rotation around that bond. And the other one was just an experimental thing that the size of the photographic plates they used in the Cambridge lab were too small in the sense that they missed a key reflection that Pauling knew about and they didn't know about./nSo this combination led to them being scooped by the other group. But nevertheless the group at Cambridge was absolutely outstanding and at one of the top places in the world to do. And I showed you a couple of pictures when I was showing you the transition state. Sort of what you get out of working backwards from these diffraction patterns is they can measure regions of electron density, and then you fit atoms or fit molecules to the patterns that you see./nAnd if it's all working you can explain why there are bumps here. There's an oxygen here and so on. There's another one. This is an ATP that's bound actually in a pocket in a protein. But you can sort of see how beautifully the patterns of electron density deduced from the x-ray crystallography will match the chemical structures that we put on the board. So that was the idea, they were going to work out the structure of DNA./nNow, the thing about Watson and Crick, who at this point looked like this, they didn't look inordinately distinguished. In fact, Jim probably looked like, you've probably seen people who look approximately like that around MIT. He would have fit in right here and no one would have noticed. They were not actually x-ray crystallographers. They were just trying to model other people's data. And the best DNA crystallography data was from a young woman Rosalind Franklin, who was working in London./nA very somewhat uneasy alliance with Maurice Wilkins. And in trying to read the history it's a bit complicated because, at least some of what I've read, I think that when Rosalind Franklin arrived at the lab she was told this DNA structure problem was hers. And Maurice Wilkins in whose lab she was working was told that he was sort of working for her. So there was a bunch of confusion in this./nBut, in any case, Rosalind Franklin was collecting crystallographic data. And Watson and Crick located some distance away in Cambridge were trying to come up with models that could explain the structure of DNA. And they learned about Rosalind's data. And it was her data that they used to work out the basis, her crystallographic data that they used when they put together their structure./nSo if it hadn't been for her they wouldn't have been able to make their discovery. So part of the reason I'm dwelling on this is I think their discovery of the structure of DNA was arguably one of the great intellectual advances of our time. It just opened doors. The whole field of molecular biology became possible once people suddenly saw that DNA was complimentary strands./nYou could almost immediately see how you could copy genetic information. It laid the groundwork for what later turned out to be, you know, recombinant DNA and everything else. So much of this pivots around this one discovery. And I think I wouldn't be doing justice to this finding, which you all have heard about for years and years, if I had let you walk away from here thinking this was too young geniuses who sat down in a room with some crystallographic data and emerged with a structure that sort of changed the course of the study of biology./nAnd, as you can see, changes our society and everything else. There are a couple of accounts of this, there are numerous accounts. One that I found pretty interesting is called "The Eighth Day of Creation," if you ever want to read an interesting book on science. This was Horace Judson's effort to try and put together a history of this happening./nAnd with all history he's ultimately -- You know, there are some judgment calls by the historian, but this one certainly he tried to be pretty fair-handed and even-handed and he tried to get at the heart of what was going on. Watson wrote a book called "The Double Helix". Jim Watson's a very colorful character, quite brash particularly when he was younger, and that's reflected in this book./nIt's an interesting read. Probably more balanced point of view for sure in "The Eighth Day of Creation". And there are now a lot of other books. But what I did, just to try and do this in about a minute or two, was I took a couple of the key things that happened during their adventure of trying to work out the structure of DNA and just kind of ran some of their missteps together, because even though this was a marvelous discovery it just didn't happen./nSo they started out, they were inspired by Linus Pauling's discovery of the alpha helix. And I don't know if you can remember the story, but what Pauling decided to do when he was lying in bed and with a strip of paper trying to work out the structure that was giving these reflections in the crystal structure, he said I'm going to start by ignoring the side chains. So that was a brilliant move in the case of the alpha helix because he was then able to figure out that that hydrogen bond between the carbonyl and the amino group, you could see how if you got helix going it would repeat at exactly the way that would give the reflections that were observed in the crystallography data./nSo that was how Watson and Crick sort of did it. Linus Pauling had shown the way. So they decided they would ignore the side chains of DNA. So they started out by saying we won't consider the As,Ts, the Gs and the Cs. Well, given what you know about the structure of DNA that was not a helpful move in trying to work out the structure of DNA. Another thing, for example, that happened was that Jim Watson has no lack of self-confidence./nAnd so it turned out when he went to hear scientific talks he didn't take notes. And so he went to hear a talk on x-ray crystallography given by Rosalind Franklin, but he didn't quite remember the numbers right. He got the facts a little jumbled, and he and Francis spent a while trying to design models to data that wasn't the right data. It was just not quite remembered right, so there was kind of an inefficiency there./nAnd then Jim had a bias almost to the end that the phosphate backbones they knew would somehow be on the inside and the bases would be on the outside of the structure. So if that's your sort of starting place then it's sort of hard. So Watson, excuse me, Francis Crick was beginning to suspect that maybe the bases were important. So he hired a young mathematician. And he said, "Can you see if you could work out whether there would be any chemical attraction between any pairs of bases?" And the young mathematician came back and said that he thought G might go with C and A with T./nAnd given what happened here you might have thought that a light bulb would have gone off, but it didn't. And, in fact, Chargaff visited them and the light bulb went off for nobody. And, in fact, Chargaff wasn't a terribly big fan of what Watson and Crick were trying to do. So the pieces are piling up but still not there. Then a big experimental advance came from Rosalind Franklin./nAnd that was she discovered that the DNA that they had been diffracting was actually a mixture of two forms. So there were actually two structures in the mix that were contributing to the diffractions. She was able to separate out the two kinds of DNA, DNA-A and DNA-B she called it. And so now this gave a much clearer diffraction pattern, and that's the diffraction pattern that she saw./nAnd Watson and Crick managed to get a look at this data. And it's a little complicated how that happened, but Crick realized almost right away that there were two strands running in opposite directions. So he know knew it was 5 to 3 in one direction and 5 to 3 in the other direction like that. So you might have thought they were home-free, but no. Jim Watson immediately built a model that paired like with like, A with A, T with T, G with G./nThey wrote it up and they were ready to submit the paper. And they gave a presentation to their colleagues at the lab in Cambridge. And they were shot down. And one of the key things was they learned the chemical fact that most of the textbooks were wrong at that time in the way that they depicted the structure of guanine. If you look in your textbook, excuse me, here. So if you were to look in a textbook today you'd see guanine like this, but there is another way you could draw this./nSo this you may remember when we were talking about phosphoenolpyruvate that this is an enol form and this is a keto form. And this is the way most of the textbooks were showing guanine at the time. So they were looking at the structure of guanine in textbooks. And if you were trying to work out schemes for putting bases together you can see what's going on up here would be very different./nAnd if we have a hydrogen here versus if we have an oxygen, if you're trying to say make hydrogen bonds at that particular position, I think all of you understand hydrogen bonds well enough to see how that would throw you off. So once that insight came, once they learned that then the rest of the structure came pretty fast. And there's a movie about this. One of the nice things in it was sort of trying to recreate the experience where I think it was Watson who was shuffling these base pairs around./nAnd he suddenly realized that you could set up base pairs with A and T and with G and C, and when you looked at them you could see they were geometrically exactly the same shape. You could just take the shape of the G and C pair and lay it right down on the A and T pair. And then you could see how you could build either a G-C or an A-T pair into the repeating structure of this DNA and it would be compatible./nSo they built a model and they thought, we can just hit the lights for a second here maybe. I just want you to see what that first model looked like. It looks like something you could hack together in a chemistry lab. They had the bases cut out of metal. And you can see just, you know, here the retort sort of stands using chemistry and various clamps that you would use for clamping a flask or something if you're doing a chemical lab./nThat's the stuff that they were using to put the model together. And they published then a paper in Nature that told about this result. That's the entire paper reporting the structure of DNA. And maybe you can see there's a little hand-drawn double helix right there that captures the elements. That is the paper, and that was in the journal Nature. And it had in it, right near the end, one of the coyest sentences in the scientific literature./nThey didn't want to go into all the details that if you had an A paired with G and G paired with a C and you pulled them apart then you could replicate the molecule by redoing it. So all they said was, "It has not escaped our notice that the specific pairings we expostulated immediately suggests a copying mechanism for DNA." So this is a picture of Jim Watson wearing short pants at Cold Spring Harbor in 1953 reporting this structure of DNA./nCold Spring Harbor is on Long Island. It's been one of the Meccas for molecule biology since the 1940s. They have a famous symposium once a year. The topic changes every year and rarely repeats. And it was at one of those symposia -- This was the year that they discovered the structure of DNA. And there was Watson. So two years ago they had another meeting, a special meeting just exactly this time of year./nIt was in February within a couple of days of right now. So I gave this lecture and I showed the student in the class that this year, I said here's a picture of Jim Watson displaying the structure. They're having a meeting 50 years later in 2003. And I'm going down there. I'm asked to give a talk. And I'll come back and I'll tell you what it was like. So I gave my lecture. I dashed out to the airport./nI hoped on the plane. I went down and I registered. They gave me, you know, the stuff to get into my room, a little envelope with the key card and things. And I went up to my room. And I took out the key card. And what did I find myself looking at? The same picture I had shown to the class just a couple of hours earlier. Here's another picture of Jim the way he looked at the time when he made this amazing discovery. That's Salvador Luria who I mentioned./nI tell you about him in a subsequent lecture. I was at another meeting a few years earlier where some of the old-timers were razzing each other, and someone showed this picture. And then they got up and they gave it a title. And that was "Picture of a Man Picking His Own Pockets". So they would tease each other a lot. And I'm hoping maybe you'll get a chance to hear a little bit more about that soon./nThis is what Jim Watson looks like now. I asked to get a picture taken just so you could see he's still around and is very active and still very controversial. This doesn't make much of a difference. Here's a picture of Watson and Crick a little bit later just sitting out on a porch in Cold Spring Harbor. It's sort of right on the edge of a bay down there in a very relaxed kind of atmosphere that still permeates molecule biology research to this day./nFrancis Crick just died last July at the age of 88, so we've just lost the link to one of the two people who did this amazing experiment. OK. So I want to then set things up for the details of DNA replication. So there was a basic principle that came across from this that you could see how this could work, that DNA was sort of like having a photograph and a negative. And so the information is actually in there twice./nIt's just in different forms. And when I tell you about DNA repair in another lecture you can maybe see already how useful that is because if you damage one strand you're not really out of luck because you've still got the information in the other strand. And you could probably, on the basis of that, device a repair strategy if you thought about it. But more importantly for DNA replication finally gave an insight to this thing that had been vexing people forever./nIf you had to have all this information for making a cell, and every time a cell divided and you saw how it can happen pretty quickly with something like a bacterium of yeast, how could you accurately copy all that DNA, excuse me, all that genetic information? How is it stored? How could it be done? And once you saw ah, it's just a matter of separating the strands, and if there's an A there put a T there, if there's a C you put a G and so on, was a huge breakthrough./nBut that then didn't tell people how DNA replicated or even if this is the mechanism. You can actually come up with all kinds of models for how you could replicate things based on this principle, including crisscrossing between strands and all sorts of things. The predominant model and perhaps the simplest one was called semi-conservative. And it thought of the problem in this kind of way, that if you had two strands of the original DNA molecule and then you pulled them apart that one of the strands here would become one of the strands of the daughter, and then the new one would be here and the same thing would happen on the other side./nAnd then if you did it again this thing would happen again with a new strand. This time the skinny strand here would be like this, the skinny strand here would be like this, and then this one again. We'd have one that was nearly synthesized plus one of the originals. So this model was one of the simplest because it kept this strand intact throughout the whole process while some of the other models had them being patched back together, all based on the idea that A pairs with T and G pairs with C./nBut proving that this was the correct model was then another important advance. And that was done by Frank Stahl and Matt Messelson. Actually, I think I'll skip this for right now. Matt is a professor up at Harvard, just up at Harvard Square not very far from here, still very active. Frank Stahl is a professor out in Oregon. He's still active./nSo one of the differences about this course is a lot of the things I'm telling you about -- And this is pretty old stuff right now, right, molecule biology. The people who did these are still around and very active. This is most of modern biology is a pretty young science, and many of the major characters are still running around and with us today. So, anyway, what Matt and Frank were at Caltech. And they, with a bunch of other students had an apartment./nAnd they were sitting around trying to work out a way to figure out this model. And they came up with an idea, and that was to see if you could differentially label what we might call "old DNA" and the "new DNA" here. And since it's chemically the same stuff it's a bit of a trick. How do you tell old DNA from new DNA? So their idea was since nitrogen comes in two different isotopes, N14 which is the common one and N15 with is one mass heavier, that maybe you could start out with the DNA, for example, grown in N15./nAnd then when you started replication switch to N14. And then you'd be able to tell, if you could separate these molecules on the basis of their density since the one with the N15 would be heavier than the one with the N14, then maybe you could work this out. And the story goes, this has been written, they were sitting arguing about this, or talking about this idea at the table./nAnd it was a good idea but there was a problem. And that was how could you separate the two kinds of DNA based on their density? So they had a piece of fingernail and they were trying to see whether they could get it to float by dissolving more and more sugar. And they figured if they added more and more sugar the water would get denser and denser so the could float the fingernail./nAnd they weren't able to do it. But all chemists made a periodic, probably some places here at MIT, they had a periodic chart right in their living room. So they went and they looked. And then they looked at sodium. And they went down the periodic table and then they saw cesium. And thought maybe, you know, if you took a solution of cesium chloride and you put it a centrifuge and you spun really hard then you'd get a gradient of varying concentrations, of slightly different concentrations of cesium chloride./nAnd that they could tune that to a range that would discriminate between the heavy and the lighter forms of DNA. So the experiment they did is known as the Messelson-Stahl experiment. But, as I say, these are names that come from real people. And the idea was pretty simple. They grew the bacteria for many generations -- -- in N15 medium. This is the so-called heavy or H isotope -- -- of nitrogen./nAnd then at time equals zero in their experiment, when they're ready to start the experiment they switched to medium with N14, which we'll think of as the light or the L isotope. And then they isolated DNA -- -- after let's say increasing rounds of replication that you could tell simply by measuring how much DNA was in your bacterial culture when the bacteria had doubled their DNA. And this is the data they got which looks something like this./nIn fact, in this case the blackboard representation is pretty close. So this is cesium chloride. And it has been centrifuged very hard so that there's a gradient now that's light at the top and a little heavier at the bottom of the gradient. There's a little more cesium chloride per ml here then there is there in the tube. And I'll just give us three little sort of reference marks here./nSo what they found when they started was that all of the DNA was at that position down at the heavy end. And then this is after one generation. So the DNA has now doubled. What they found was that all the DNA was now at this intermediate position. And after two generations or two DNA replications they now found that some of the DNA was here, some of the DNA was there./nAnd if they went to three or more what they saw was they began to pile stuff up there. And I think most of you could probably make the connection between that data and that picture that I've got up there. This is the heavy-heavy DNA. This is the heavy-light. So this would be heavy-heavy, heavy-light, light-heavy. After one round it will all be here. After two we have heavy-light, but this one is light-light, light-light, light-heavy./nAnd so now we've got light-light, the heavy-light, no heavy-heavy is ever going to show up again. And the longer you do this the more you'll get the light accumulating. A very simple experiment done by real people but enormously powerful because now it showed that this basic idea, you have the photograph and negative, you pull them apart and copy them was right. So at this point you begin to see why data of Avery's that before people had trouble accepting, all of a sudden now it was really you needed a CYD and A was the genetic material./nAnd this is what sort of ushered in this great burst of molecular biology. So in the next lecture what we're going to start doing now is once you, this is all great, but once we start figuring out how to replicate it we're going to have to get down to enzymes and biochemical steps. And there are some formidable challenges to replicating DNA, and it's also awesome. I'll tell you at the beginning of next lecture how much DNA we have and just how accurate it is./nIt always blows me away. I'll see you then. Take care.
Tags // Molecular Biology I Lecture
Added: April 2, 2009, 1:41 am
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Biochemistry VI Lecture Prof. Graham Walker
Biology > Introductory Biology/n * Email this page/nVideo Lectures - Lecture 7/nTopics covered: /nBiochemistry VI/nInstructor: /nProf. Graham Walker/nTranscript - Lecture 7/nJust very quickly, I mentioned to you the other day that article about Romney making a policy statement about embryonic stem cell research and said here were sort of examples of issues we were thinking about in this course that would come into your ordinary life. Here's on today's Globe there's, "Kennedy rips Romney over stem cell policy". This debate is continuing. It's on the front page of today's Boston Globe. We have two people neither of whom probably have the background in biology, that you guys are going to have by the time you finish this course, having to grapple with these very serious scientific issues that have all kinds of implications./nHere's another thing. This was in yesterday's newspaper. This was on, I think, the second page of the Boston Globe. It's about a curator over at Harvard at The Museum of Comparative Zoology that got all these different birds, some of whom are extinct and something. And now the curator, instead of just having them there as a sort of collection and that's all it is, is getting DNA out of these specimens and working back and looking at gaining new insights from animals that aren't found on this planet anymore./nAnd this was one was from, I didn't want to show you this on Valentine's Day particularly, but this is critical. I don't subscribe to The New York Times but for some reason one appeared on my porch on Saturday morning. I opened up the first page, "Rare and aggressive HIV reported in New York". I'll be talking to you about the HIV-1 virus which is the virus that ends up destroying some of your major defender cells in the immune system./nAnd then people die of AIDS which is sort of all the other stuff that then happens. And it was terribly scary when it first showed up. There was no treatment for it. It's like that in most of the Third World and people die if they get it. I lost a close friend to it after it first came up. Then they came up with these various cocktails of different inhibitors. And the idea was instead of just having a single drug target where it would be easy for the virus to mutate and, as I'll tell you, the HIV virus mutates like crazy./nInstead they'd use multiple targets and that's why they do it. And sort of then the chances of it getting a mutation, you multiply all these low probabilities of getting a mutation and would hope it would never happen. It's happened. There in New York is a version of HIV-1 that is resistant to everything. If that spreads, which it probably will, it will be just like what happened with all the bacteria and antibiotics./nIt works for a little while, natural mutation and natural selection, something will happen, the organism or the virus will change so that it's no longer resistant. So, you know, for the moment, as always, you know, make sure you never expose yourself to AIDS. This would take us back just like to before any of these things were inhibited. And the same thing goes right now./nThere are strains of mycobacterium tuberculosis that are resistant to every known antibiotic. If you get tuberculosis with that thing it's like you were living 200 years ago. They cannot do anything for you. They'd put you in a sanatorium but you cannot be cured. This is worse. You know, this is awful. This one kills you. So just be careful. This is just the tip, but what always happens when you have antibiotics or drug treatments eventually natural selection mutation will probably produce a variant./nAnd this looks like it is happening. I don't know, I hope not, but I'm afraid this won't be the end of this one. OK. So some of you, I've read some of the comments. I'm not terribly surprised that some of you think why is he telling me all this. God, there was a lot of work for tremendously confusing, there were all these chemical structures, I didn't really get it all. It seemed that somehow out of this the cell got to two molecules of ATP./nWhy are we wasting all the time? Why am I wasting all this time telling you about how to get two molecules of ATP? This is basically what we were doing. We were looking at glucose, which is C6H12O6, going through this process which pathway is of ten enzymatic steps. And what ends up coming out at the other end is two pyruvates./nAnd the net yield of that is two ATP plus two molecules of this weird thing called NADH. Now, I think most of you, although a few of you still don't seem to have gotten it, the reason you need to make ATP out of ADP and inorganic phosphate is it stores energy. It's, you know, like having electricity in your house or batteries in your flashlight or in your iPod or whatever it is. You've got to have energy in order to do stuff./nAnd cells need it. And the major sort of energy currency they use is ATP. And they put most of it by squeezing together a phosphate up to an ATP making this bond that's stable, unless there's an enzyme that'll break it open, but there's energy stored in it. And they'll drop downhill and you can use that energy to drive other reactions. So for life to happen it had to somehow figure out how to get energy in a form that it could use to do all the biosynthesis that's necessary for life to go on./nNADH, I showed you the molecule, you got a structure. The cell makes some number of molecules of that. It's around. And what happened, it comes in, it helped some of these processes go. It ends up, it's sort of like a banking system for electrons. If it gets NADH it's got electrons stuck on it. There are only a limited number of molecules of NADH in a cell, so if you make all the NAD molecules into NADH then you have nowhere else to put electrons and everything comes to a grinding halt./nBecause I was talking about redox reactions. If you're taking electrons off somewhere they've got to go somewhere else. And the cell uses NADH as kind of almost a currency for passing electrons around in just the same way it uses ATP as a currency for carrying energy around from one form or another. Now, the problem was and why this was so important is this apparently, as I said, we're basically looking at an evolutionary fossil of some sort because every organism on earth uses, or virtually every organism on earth uses glycolysis./nIt's in our cytoplasm of our cells. It's in the cytoplasm of bacteria. It's in the cytoplasm of archaea. It apparently evolved so long ago that every form of life on earth, you know, depends on this, anything that can metabolize a sugar molecule pretty much. And so it looks complicated. It is. Maybe you could have designed, I'd like to tell you that it's two reactions and you make two ATPs and it's all simple./nLife isn't often simple. It's often very complicated. It wasn't done by a team of design engineers in Building 10 designing it. It was done by something that happened with random selection. And when something finally worked apparently it got fixed in evolution even if it looks clunky. I think the other way to look at it is to say isn't that, I feel a sort of sense of wonder at this point, and granted I've had a lot of years working in the field, that something that complicated could work out and take this molecule of glucose and make it into two molecules of ATP that can then be used to allow the cell to do synthesis./nAs in I'll show you, there's a better way you could use this much more efficiently, 18 times more efficiently if we do respiration, which we'll talk about today, but that needs oxygen. And when life started 3.8 billion years ago or so there was no oxygen so it wasn't an option. The initial organisms had to make due with what they found and this is what developed./nSo ATP can go off and be used for lots of things. The problem, if there's no oxygen around, as I said there are a limited number of molecules of ATP. So to make that glycolysis go you're taking some electrons off and doing the oxidation steps, you're reducing NADH. And if you want that to go as a cycle you've got to somehow regenerate the NAD+ so you can go through another cycle and make more ATP./nSo there were two variants that I told you about. If you don't have oxygen around, one of these variants yields to lactate, and so the net yield of this is two ATP. Because what happens in this part is it uses up two molecules of NADH changing the two molecules of pyruvate to two molecules of lactate, so that gets rid of this, and the net yield of the whole thing is two molecules of ATP./nThe other way to do it is the way yeast does it. It makes two molecules of CO2 plus two molecules of ethanol. And I think when I wrote that on the board I showed you that there was acetaldehyde. We had two molecules of that going to two molecules of ethanol. And I think I forgot to write on the board that it used up two molecules of NADH because that's important. So that's what this whole thing is about./nIt achieves the same thing. And the net yield of this one is two ATPs as well. So I'm going to talk about photosynthesis at the latter part of the lecture, but if you maybe recall the initial sort of thing I called "release one" came up here about 3.4 billion years ago. Later on an improved version came along that started to generate oxygen. Now, it took billions of years for us to get to our present level of oxygen, but when oxygen became available in the atmosphere then there was a new option for making energy./nAnd that was instead of taking this NADH and, remember, there are about 50 kcals per mole of energy in there, it's just being thrown away. It's not being used at all. But if there's oxygen around there's a new and better system, which you know as respiration, it's a word you've heard. You know we breathe oxygen. We need to breathe oxygen because we're using the oxygen to make energy./nAnd I'll tell you again. You don't have to know all the details. But there's a biochemical cycle called a citric acid cycle. And it goes together with another process that's known as oxidative phosphorylation. And so this is if we have oxygen what we end up with at the end of the day instead of these products is six molecules of CO2 plus six molecules of water. But more importantly 36 molecules of ATP. So respiration is tremendously better at capturing energy from a glucose molecule./nBut this, I'll show you, is a later arriving development in evolution. It had to be because it required oxygen in the atmosphere. And so even though we only get two molecules out of ATP. We all do it. And the other thing it generates is pyruvate. And, as you'll see, this process takes those pyruvates. That's the starting material for this part over here./nOK? I hope this is making a bit more sense as to why you've got to keep your eye on the big picture or all you're going to see is a whole lot of structures and chemical transformations that don't make any sense. This is all about making ATP and energy. And, as we'll see later on, photosynthesis. In cases it can be something to do with making reducing power for biosynthesis. So in order to understand this, though, I have to introduce you to another way of thinking about energy./nSome of you still seem to be struggling with the idea that you can store energy in a chemical bond, but I think from the comments the majority of you have that. If you're having trouble, ask your TA or look in sections and stuff. But the insight to how this part came, even though this process, what underlies this was invented about roughly 3./n4 billion years ago, scientists didn't begin to even get a glimmer of how this worked until about 1961 when there was a scientist named Peter Mitchell who got a Nobel Prize for the insight that he had. And what he recognized was there were sort of three forms of energy that are interconvertable. The energy of a chemical bond. And I keep telling you this, that ATP, if we hydrolyze that it goes to ATP plus inorganic phosphate./nThe delta G prime zero is about minus 7 kilocalories per mole, but under physiological conditions it works out that each ATP gets you about 12 kilocalories per mole because life doesn't happen under these standard conditions. So that's one form of energy we've been talking a lot about. There's another form of energy, which you probably know intuitively, and that is if you have a high concentration of some compound on a side of a sort of impermeable barrier and a low concentration on the other side, high concentration of sugar, low concentration of sugar./nIf you give the system a chance it will come to equilibrium. So the concentrated stuff will flow downhill until you're concentrated on both sides. There's energy that you could get out of that. So there's energy basically stored in a concentration gradient. There's another form which should probably be familiar certainly to some of you, and that's the idea you can store energy in an electrical gradient./nIf you have some kind of impermeable barrier and we have a lot of charges on one side and less charges on the other side then there's a polarity, there's an electrical gradient, and that's a form of stored energy. Now, it happens that a membrane is impermeable, as I've told you, to most things. So you can get a high concentration of something on one side and a low concentration on the other and controlled by a protein imbedded in it, whether those ever get a chance to come across./nThe same idea applies for an electrical gradient. And in particular of interest to biology are hydrogen ions. So if we have a situation like this where we have more hydrogen ions on one side of a membrane than another then we have an electrical gradient, we have a polarity. And so this is the membrane. And so all cells then have -- And the way it works is this is the outside of the cell and this is the inside of the cell, so more pluses on the outside, hydrogen ions on the outside than the inside./nAnd it's about 70 millivolts. It may not sound that impressive, right? Another way to look at it, though, is the membrane is about three nanometers. So if you say, well, OK, what's the electrical gradient across that? It's about 200,000 volts per centimeter. And high tension lines are, you know, more like that per mile or something like that. So although it seems modest because the membranes, you've got have a very, very powerful electrical gradient in all cells./nAnd so this is a form of energy. And I think this was proposed in 1961. Textbooks often say it was adopted in the early 70s. I went to a post-doc at Berkley in 1975 and people were still having arguments about whether this really was the way. Was this really something that was used in nature? It is. And I think one of the most dramatic demonstrations that a proton gradient can be a source of energy comes from this sort of thing./nI'd showed you how this bacteria, these are E. coli swimming around with these rotary motors that spin 10,000 to 100,000 RPM driving those flagella. I showed you the picture of the inner membrane of E. coli. And I mentioned it has a double membrane. This is sort of a protective layer. You'll see, a little bit later in the lecture, some double membranes again./nAnd here's the motor with the big propeller, the flagella sticking out from it. And the way this thing, and I showed you, I guess, this picture and then this textbook diagram. What drives this motor are protons flowing from the outside of the cell through here, through the proteins in here, through channels in them. And that's what provides the torque for the motor. That's what drives it./nIt's not ATP or anything. It's protons on the outside and inside. And there's a very dramatic demonstration that's sort of like Friday night horror films, where people found a way of popping an E. coli open so that all of the cytoplasm ran out and then it would reseal. So what you've got is sort of an E. coli that is just a shell, just the membranes and the proteins imbedded in it, and it just sits there at neutral pH./nIf you now acidify the medium what's happened is you've created a proton gradient because there are now more protons on the outside. And this is just the same picture I showed you before. But what happens if you do that experiment is the bacteria start swimming. They don't have any insides or anything but you have created artificially a proton gradient and it drives the motor and they start swimming./nIt's sort of like "dead man walking" or something at a bacteria level. I think it's a really dramatic demonstration of how you can use the energy of a proton gradient to make energy. So the principle that underlies how respiration works and photosynthesis works is that you take advantage of this combination of concentration and electrical gradient./nAnd it's known as the chemi -- -- osmotic hypothesis. Because here you have an electrical gradient because of the charges. You also have a concentration gradient because you've got more hydrogen ions on this side. So you cannot really separate them. They're kind of coupled. But the idea is that life uses this proton gradient in order to make energy and do some of these energy transactions. So here's the sort of principle of how it's done./nYou have a membrane like this, then we have a protein, and now we're not going to see all the alpha helices and beta sheets. It'll be one of those things we talked about that spans the membrane. The membrane, as you might guess from what you know about it, is impermeable to hydrogen ions. So what this protein does that's imbedded in the membrane, it's a proton pump./nAnd if you provide it with energy in some form what it does is it takes a hydrogen ion that's on the inside and it makes it into a hydrogen ion on the outside. There's no chemical transformation of the proton. It's just gone from one side of the membrane to the other. It's almost like recharging a battery or something if you want to think about it perhaps in that kind of way. And then the second stage is once the proton gradient is established and you have now many more H+s on the outside than on the inside then there is another protein that lets the proton flow./nThe proton flows down to gradient and therefore is able to come back inside the cell. Now, if that's all there was to it we wouldn't have achieved anything. We would have wasted energy, pump something out, pump something back. But this molecule has an interesting property, and that is the ability of the proton to flow down the gradient obligatorily requires ADP plus inorganic phosphate./nAnd as the proton comes down this energy gradient there's enough energy given off by that, that the cell is able to capture it and use that energy to synthesize a molecule of ATP. Got it? So you produce some energy like from the light in photosynthesis that we'll see in a minute, other ways of doing it, and then get it outside. Once you've got the gradient now you can make ATP./nAnd, actually, one of the completely remarkable discoveries of structural biology, this is known as an ATP synthase. It's a protein. It's an enzyme. These are the kinds of things we've been talking about. This is also a protein. You see all the different things proteins do. So this ATP synthase, which uses this energy of the proton gradient to make ATP, its structure has been worked out and at a level, here's part of the crystal structure./nYou can probably see some alpha helices beta sheets. Here's a textbook diagram of it showing, it's upside down I'm showing, but here's a proton on the inside flowing across. And what's remarkable is it turns out that this ATP synthase is structurally related to the protein that's at the heart of that rotary motor that drives the flagella. And, in fact, remember, I think I showed you where you could stick the flagella to a cover slip and the bacteria twirled around so you could actually see they were rotating? So people did sort of the equivalent thing, they managed to stick this ATP synthase./nAnd you couldn't see that the top part was turning, but they used some tricky stuff we'll talk about with antibodies and there's something to attach a long filamentous molecule. It's the polymer of proton called actin. That's the same stuff we find in our muscles. And it made it long enough. You could see that when this thing was working that it was twirling around./nAnd so evolution took this same basic sort of piece of protein machinery. And in one case it used it to capture the energy of a proton gradient and make ATP. And in another case it used it to drive this propeller, if you will. And here's sort of a simple diagram. So as the proton flows kind of what happens is the inner part of this thing sort of goes click, click. And every time it does it synthesizes an ATP./nAnd it sort of takes that energy to push together the ADP and the inorganic phosphate overcoming that activation energy and getting them close enough that you're able to get that bond. It's a truly remarkable thing. This is a somewhat hard concept to grasp, I understand, but if you can understand this inter- convertibility between energy in the form of a combined concentration of electrical gradient and ATP, and nature goes back and back and forth, it's absolutely fundamental to life./nIf we didn't have this stuff going on we couldn't do it. You know, as I say with glycolysis, I wish it was simpler, but this is the way nature did it, this is the way we are, and from what I know about biology, this is how it goes. And I wouldn't be doing it justice if I didn't tell you some of the details. You're MIT students. You should be able to, I hope, some of how the world actually works at this kind of level./nOK. Now, with that kind of background, I think we can kind of -- Pretty quickly I can help you begin to see what happens here. Now, remember the problem up there with glycolysis? It started when there was no oxygen, and therefore it generated these NADHs. They weren't any good. You had to just get rid of them so you took the pyruvate, organisms learned how to put them to make lactate or ethanol and carbon dioxide, but if there's oxygen around then there's another possibility./nAnd that is you can combine these molecules with oxygen. So if we take two NADH plus two hydrogen ions plus a molecule of oxygen what we get is two waters. And I think I can show you what's going on there kind of simply. What NADH was, we got a pair of electrons here and a hydrogen ion. Well, if we took that, what's that? I think you'd recognize it as a molecule of hydrogen gas. So if you take NADH and oxygen, what the cell is really essentially doing, it's taking hydrogen gas plus oxygen and it's giving two waters./nSo it's basically burning hydrogen. And I think most of you know what would happen if I had a mixture of hydrogen and oxygen up here and chucked a match at it or something. We'd have a massive explosion. And, in fact, that's why there are 50 kilocalories of energy released when that happens. And so on an energy sort of diagram, these free energy diagrams where we had the two NADH here plus the molecule of oxygen, and down here we have the two molecules of water, there is this./nIf that happened in one step it would be a huge amount of energy. No cell or organism on earth has figured out how to do that in one step. And I think some of the textbooks liken it to sort of say it would be like setting a stick of dynamite off in the cell. And this is where one of these things that seemed probably like a kind of uninteresting thermodynamic property, to some extent, becomes to be really important in understanding biology./nAnd that is the fact that remember I said this drop in energy from reactants to products was a thermodynamic property? It didn't matter whether like you skied straight down the hill or you came down in a series of little things, you still got the same amount of energy release going from here down to there. So that's, in essence, what the cell does, is it takes this energy and it breaks it into little packages that it's able to manage in a chemical way./nAnd so instead of coming down, and once it comes down in a series of steps, and every time it flows partway down hill it does something. And what it does is it passes two electrons to some kind of carrier. And then the two electrons flow downhill a little bit to another one and then to another one. And what happens when these electrons are flowing downhill, though, is that they drive that proton pump./nSo a proton pump takes the proton from being on the outside to the inside. And when the two electrons drop down to the next level another H+ goes from out to H+ in, again here. And if you understood what I said before, what the cell can now do is it can make three ATPs by using that ATP synthesis, and now taking advantage of those three pump proteins and making them into three ATPs. And then at the end of the day what happens then is these two electrons get together with two hydrogen ions plus I'll show it as a half of an oxygen here to give you water./nAnd what's happening up here is, in essence, the molecule of sugar, C6H1206 is being burned with six molecules of water plus this to give you six molecules of C02 plus six molecules of ATP so that the cell is essentially achieving the same kind of thing by this process as if it had burned it in oxygen. There's that much potential energy released. And the total amount of this change in the G prime zero is something out of the order of I think it's minus 686 kilocalories per mole./nIt's able to capture respiration. It captures about 60% of that energy as ATP. This process of fermentation, this is what these processes that happen in the absence of oxygen are known as fermentation. As you can see, they're much less efficient. It gets more like 3.3% of the energy captured as ATP. So you can see when oxygen arose in the environment what a huge boom it was to life because you could get a lot more energy for the same amount of starting material./nThere is one thing, though, that the cell has to do. In order to do this it has to do some more chemistry because it has to take those pyruvates and it has to somehow run them through this thing that I've called the citric acid cycle and oxidative phosphorylation. Well, the oxidative phosphorylation is basically this chain I've diagramed for you here in a schematic way./nAnd that's about the level you'll have to understand it. I mean physically it's going to be a bunch of proteins stuck in the membrane, and as the electrons get passed along they pump a proton as this pathway occurs. But the other thing the cells have to do is they have to take those two pyruvates and they have to burn them all the way down to C02 and water. And what's happening, if you remember that chain of oxidations, the carbons are being successfully oxidized all the way up to carbon dioxide./nYou cannot be anymore oxidized than that if you're carbon. What that must mean is something else is getting reduced. And what gets reduced, where the electrons go is NADHs. So once oxygen became available in the atmosphere then the name of the game was to take those two pyruvates and to somehow burn them all the way down to here, and therefore generate as much NADH as possible. And if you can make some ATPs along the way well and good./nSo there had to be a whole other set of chemical reactions that's every bit as complicated as glycolysis that emerged in nature, that carried out that job. And this time I'm not going to take you through all of the chemical structures. You can look at it in your textbook and stuff, but what I really want you to kind of take is to keep your eye on the number of carbons./nIf you look at the structure of pyruvate you'll see that it's three carbons, and it was this. Here's the keto group and an acidic group. So this is almost carbon dioxide. It's just one oxidative step away. So what happens in this cycle is that first the carbon dioxide is removed and this makes an acetate. In essence it's actually joined to something right here. And then this feeds into this thing called the citric acid cycle./nAnd what it is, as I said, is it's a set of chemical reactions that are designed to take this pyruvate and burn it all the way to carbon dioxide and water, and to generate as many NADHs and ATPs as it can along the way. And, again, I wish it were simpler. It would be really nice if it just did it straight from acetate, but instead you'll see, if you look at the TCA cycle, the C4 compound. And this gets joined together to give us a C6 compound./nAnd then it oxidizes one of these carbons to give a carbon dioxide. Now you're down to the C5. It does it again, another molecule of C02, you're at C4. And then it takes this C4 carbon skeleton, puts it through some transformations that enables this cycle to go all over again. So basically the C4 thing just goes through the cycle. It's a carrier that takes this C2 right here, lets it be processed to give two molecules of C02./nIn that process it generates more NADH, a bit of ATP, and a kind of reduced carrier that you can think of for the moment largely equivalently to NADH. OK. Now, the entity in our cells that carries out this process of respiration -- Of respiration. So the citric acid cycle and the mitochondria is not actually in our cytoplasm, which is perhaps surprising, but all the enzymes for glycolysis are. So remember my simple diagram of the eukaryotic cell one the first day? We had the nucleus which is a membrane compartment that has the DNA inside it, and we'll have more to say about that in the next lecture of so, but then there were some organelles./nAnd one of them I said was the mitochondrion. And I also said in the first lecture that there's pretty good evidence now that the mitochondrion arose by some earlier progenitor or ancestor of a eukaryotic cell capturing some kind of bacterium. It's actually one that's sort of kind of related to E. coli. It had double membranes. And the mitochondria actually still have some of their own DNA, but this is where all the energy, all the enzymes for the citric acid cycle -- -- and oxidative phosphorylation are found in the inside of the mitochondrion, instead of something that probably used to be a free-living bacterium./nIn contrast, the enzymes for glycolysis are in what's called the cytoplasm of the cell, which is sort of the main part of the insides of the cell. So you see even here this sort of evolutionary history that I kind of put out across the board. The enzymes for glycolysis arose so early in evolution they're in the cytoplasm of virtually every organism on earth. Eukaryotic cells manage to figure out how to get 18 times more energy for a molecule of sugar, but in order to do it they kind of had to cheat./nThey took a bacterium that figured out how to do it, put it inside our own cells, and then it's able to run now, it's become a part of our cell and it's where all the process takes place. And if you look at a structure of a mitochondrion it's actually got two membranes. You just saw a picture of that, an E. coli. And it's sort of involuted a little bit like this. And so this is an outer membrane of a mitochondrion./nSo I'm basically taking this and blowing it up. So this is a mitochondrion. And the in and the out, in this case there's an inner membrane. This is where the proton pump is located. This is where the ATP synthesis is located. And when a mitochondrion is working what it's doing is it's pumping hydrogen ions from its inside, which is more or less the equivalent of its cytoplasm, it's whatever used to be the cytoplasm of the original bacterium, outside into the space between the inner and outer membranes./nAnd then it generates ATP by flowing back down. OK. So there are a couple of sort of things that affect your life that come out of this. One of them is understanding the "freshman five" or "freshman fifteen" or whatever it is. You come to college and all of a sudden you put on a lot of weight. Sometimes you can figure it out because you ate a lot of Ben & Jerry's or a lot of chocolate bars or something and didn't do as much exercise./nAnother one of you said, gee, now I understand why I'm not putting on any weight. I do water polo, and I guess I must be burning up everything I eat as energy. And you're right, but now I can show you at a molecular level what's happening. So what happens, your body, everything is regulated, and it can tell if it needs, cells in your body can tell if it needs energy./nIf it needs energy and you eat sugar it runs right through here and it makes ATP. But if you've eaten enough and the things that monitor your body say that you've got enough ATP then it doesn't keep this process going. Instead what it does is it stops it and says instead we're going to put something away for a rainy day, which you could see makes a lot of sense in evolution. You know, if things are good you just kill the mammoth and you can all eat well for a while or something./nIt would make sense for your body to be able to pack stuff away. So what it does is it intercepts the process at this level, at this C2 level. The pyruvate gets to here, and instead of being run through this in ATP this acetate or acetyl moiety which is a C2 gets run through a cycle of successive two carbon additions. And what comes out of that are fatty acids. It also takes three phosphor -- -- glyceraldehyde./nYou might remember that. That's one of the products stuck in the middle of that glycolytic pathway. So this is a three carbon compound. It makes it into glycerol which is a three carbon compound. And if you look back when we talked about lipids a couple of lectures ago you'll realize what happens if you combine fatty acids with glycerol then you've got fats. And, in essence, I mean that's what happens./nWe put on weight if we eat more than we're burning. Our bodies say, OK, I've got as much ATP as we need. I'm going to take some of that ATP and use it in this kind of way. Just a second here. I'm going to see -- So there's another thing that happens, a physiological thing that we experience. Well, we seem to be stuck at the moment. I won't worry about it./nI've run some marathons. I don't know. A few of you may have. If not almost all of you have heard about "hitting the wall". And it happens generally around 21, 22, 23 miles. It depends on the condition you're doing. And physiologically I have experienced, this is amazing, I understand why they call it hitting the wall. You're running along and you think, boy, I'm tired but I'm doing OK./nAnd in a space of a quarter of mile it's like you banged into a brick wall. And you can sort of keep yourself going but it's a profound physiological change. And what has happened at that point is you've run out of sugar to burn. Now, I've told you, you can take sugars and you can polymerize them into glycogen. That's one of those polymers with alpha 1,4 linkages. That's a sugar storage molecule./nSo if you start running, you carbo-load, you try and get as much sugar into glycogen as you possibly can. You start running the marathon. Your body starts taking that glycogen and breaking it down into sugars. It runs it through that process. What happens when you hit the wall as a human body is most human bodies are designed so that you run out of sugar. You just cannot carbo-load enough to do 26 miles./nYou can get through 20 or something there. And when you run out of that what happens then is your body can no longer burn sugar so it switched to burning fatty acids. And that's less efficient and you really feel it. If any of you have done long endurance things or triathlons or anything you've experienced that change. The final thing, just to close for the lecture for today, is one thing you can appreciate./nYeast is somebody that can do both of these things, right? It can either grow anaeorbically. And it makes C02 plus ethanol. Or it can grow aerobically. And this gives it two ATPs per glucose and this gives it 36 ATPs per glucose. So if you were a yeast, you would have to somehow regulate the rate of glycolysis depending on whether there was oxygen around or not. And it's very tricky and neat way it happens./nThere's an enzyme, that wouldn't surprise you, that has an active site for one of the key steps in the pathway where it's going from fructose-1-phosphate, fructose-6-phosphate to fructose-1,6-diphosphate. That's one of the intermediate steps in glycolysis. It's the enzyme that turns out to be rate-limiting. You can control passage of something through a pathway by just making one step rate-limiting./nSo this has a place for the fructose 6 phosphate plus ATP to bind, and it catalyzes the formation of the fructose-1,6-diphosphate. But that enzyme has to work at different rates depending on whether the cell is aerobic or anaerobic. So what it does is there's a second binding site over here. And if it binds ATP, this speeds up, excuse me, slows down the rate that's catalyzed over here./nAnd that's what you'd expect. If it's got enough ATP it doesn't need to run glycolysis as fast. And it also binds AMP or ADP, and that speeds up the rate. So the yeast are able to monitor, to sort of have a control on how fast sugar flows through this glycolytic pathway. And what they're really doing is they're monitoring do I have enough ATP or not? And so if they're running with respiration, making lots of stuff, they don't need to do glycolysis as fast./nIf they're anaerobic they have to run it 18 times as fast to get the same amount of energy. OK? We'll begin the next lecture with photosynthesis and then we're going to get into a bunch of molecular biology. OK?
Tags // Biochemistry VI Lecture Graham Walker
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Biochemistry III Lecture Prof. Graham Walker
Biology > Introductory Biology/n * Email this page/nVideo Lectures - Lecture 4/nTopics covered: /nBiochemistry III/nInstructor: /nProf. Graham Walker/nTranscript - Lecture 4/nSo the big issue that I was trying to take on yesterday, and this is of really fundamental importance to biology, is that you saw from that molecular composition of cells 80% water. Of the rest of it about 50% of what's there by mass is protein. Proteins do most of the really interesting stuff in the cell. They're the ones that are able to catalyze specific chemical reactions with all this amazing chemistry that's needed for life to take place at physiological conditions./nThey are structural components of the cells. They are all kinds of amazing machines. I showed you the little flagellar motor that turns it, but that's just one of many, many nano machines that are necessary for life. They have exquisite specificity when you get sick and you get an immune response. You develop antibodies and other cells that are able to recognize exactly some piece of that virus or bacterium that has infected you and mount an immune response./nBut all of the things that are doing that are proteins. And the sort of, most of you I think know that, as we've sort of said that amino acids are just a chain, one amino acid joined to another amino acid to another amino acid and so on. And so the backbone, that peptide bond that I showed the other day is absolutely regular piece of backbone./nAnd what gives the amino acids their character is the side chain that hangs off. And you'll have different side chains hanging off depending on what the amino acid is. And you will not have to memorize all of those structures. But the important thing is that these various amino acids fit into chemical categories that give them properties. They either have a plus charge and a negative charge, they're hydrophobic, they don't like to go with water, they are polar, they cannot interact with water and so on./nAnd it's clear from a couple of your comments, some of you are why are we going through all of this? Well, the reason we're going through all of this is all amino acids look like this. It doesn't matter. They're going to be an enzyme, a part of a motor, a structural part of yourselves. They all consist of the same backbone made up of those 20 amino acids. And what gives these, makes the proteins so important is the ultimate 3-dimensional structure./nI'm not sure what this sound is. OK. Let's try standing back here. What gives all of these proteins their individual character is how this chain of amino acids, you could just think of them like this, folds up into some 3-dimensional structure that ultimately is able to do the biological function that we're trying to understand. And one of the real holy grails still in biology is how to look at the sequence of amino acids that constitute a protein and figure out what the 3-dimensional structure is./nIt's one of the holy grails that hasn't been solved. One of you may have a key insight that will solve this. If we could do that it would really be a huge advance, because what you can get out of the genome sequences is you can read the sequence of every gene and you can predict the sequence of amino acids in the protein. But all it tells you is the linear sequence of the amino acids./nIt doesn't tell you what the 3-dimensional structure is. And how an amino acid gets from the sort of floppy chain linear structure to the 3-dimensional thing is complicated. And you have to understand several kinds of forces. And so I introduced a few terms. When we think about protein structure, the primary structure, that's just the linear sequence -- -- of amino acids. So valine followed by a tryptophan, followed by a proline, followed a threonine, whatever it would be./nThat doesn't tell you very much. Then the first key part of understanding how proteins get to a 3-dimensional structure was the discovery of what's termed secondary structure. And these are the thing I introduced to you to the other day. There are two important ones. An alpha helix and a beta sheet. And this is a propensity of a certain string of amino acids in this linear sequence to adopt one of two very common protein structures./nAnd the important thing about these elements, the alpha helices and the beta sheets is they are not dependent on the side chain. So they are not. They are instead dependent on hydrogen bonds -- -- between N-H and the carbon double bond oxygen in the backbone. And that was how Linus Pauling figured out originally the alpha helix. He decided to ignore all the side chains. And he worked out that you could arrange the backbone of a protein, the peptide bonds into these repeating structures that would account for the reflections he'd seen./nWhen we get to talking about how Watson and Crick worked out the structure, that's how they started out, too. They decided to ignore, if you will, the side chains, which are the As and Gs and Cs and Ts, which turned out to be not a productive way to go after the structure of DNA. But, in any case, that was part of what Linus Pauling did in working these out. And so those little movies I showed you, this is an alpha helix./nNow, what's been done in this picture is all the side chains have been taken off. And so you can look at this in your textbook, you'll see pictures, but these hydrogen bonds are -- The amino acid is just in this helix. It's coiling around. And at regular intervals there's the opportunity for forming a hydrogen bond. And we can, with some success but certainly not certainty, predict that a particular sequence of amino acids is going to form an alpha helix./nAnd part of what that's based on is there are some amino acids that don't fit easily into an alpha helix, so they'll disrupt one if it ever tried to form. So that's one of the elements of protein structure. So what you might get from this is the idea that somewhere along here this little piece of the linear sequence is apt to be an alpha helix. And you can represent that as this little sort of coil that you see in these 3-dimensional structures./nThe other one, which is the beta sheet, now that involves interactions between two pieces of, two stretches of amino acids. Maybe there was a loop in between. And then you can get interactions between them. And that, oops. Let me just go, there's the beta sheet interaction. Now, those are represented as arrows. You know, it takes two of them to go. So you've got to have, to represent a beta sheet in a 3-dimensional structure you have to have two of those broad arrows./nAnd there was a question why were there arrows on them? Well, one of the things, I think you can see if you look at those backbones, is that both nucleic acid and protein backbones, there's a polarity. If you start in this direction, the amino terminus, it's got a particular direction. It's not symmetric. If you come back the other way you find carboxyl, amino, the alpha carbon./nAnd you'll find it in the opposite order if you come back the other way. So there's an inherent polarity. The arrows aren't represented on here, but they are when you look at it in 3-dimensions. And you can either form beta sheets where the two strands have the same polarity or, if in a case like that where they loop back, then of course if this one was pointing in this direction as it goes through the loop then the opposite strand will be pointing in the other polarity, one going this way and one going that way./nSo this part is sort of helpful. You can make guesses that maybe this part has a tendency over here to form a beta sheet, but you still haven't gotten very far towards understanding how you get to the 3-dimensional structure. And just by putting on and superimposing some amino acids onto that alpha helix then you can see what happens, that if you form an alpha helix what happens is all the side chains stick out./nAnd now I think you can see, those of you who are engineers anyway, if you wanted to build something you have a cylinder and you can stick amino acids out that have particular chemical characteristics. And depending on the characteristics of those amino acids, whether they have charges on them or if they hate water or something, that will influence what happens to that component of the protein structure when it gets into a 3-dimensional thing./nAnd, as I think I showed you the other day, when we caught it looking down the end on this particular example, here are a couple of aromatic amino acids right here, they're on the same side of the helix and they would hate water. Whereas, some of the other amino acids up here are ones that have charges so those would love water. So what this would look like is a cylinder part of which hated water and part of which loved water./nAnd you might guess it folded up in 3-dimensional space. The part that hated water might fold towards the inside of the protein. And the part of the cylinder that loved water would face to the outside. So that's sort of the underlying principle. So the rest of the other forces that we had to understand in order to get to what's called the tertiary structure, this is the full 3D structure, which we can now determine by a variety of methods./nX-ray crystallography of proteins is probably the most common. The NMR, for example, can be used to derive a 3-dimensional structure as well. And the other forces then that go into this are ionic forces. Someone seemed confused by this, but if you have a plus charge on this part of an amino acid and a minus charge here, if in 3-dimensional space the plus charge got somewhere near the minus charge then that would form an ionic bond./nAnd I think most of you know enough electricity and magnetism that wouldn't surprise you that those two would be attracted. The one that I think that has been harder to understand is van der Waals interactions that we talked about the other day, which is tricky in the sense that for this course you don't particularly really need to understand the underlying chemistry./nBut the principal of it is that if you have a nonpolar bond, one that hasn't got any particular attraction to it, gets very, very close to another one, then the transient fluctuations in one induce something in the other one that makes them stick together. And the whole point about this is if you get two molecular surfaces that are very, very close together, about, you know, many two times the length of a covalent bond or something, then you can generate very powerful forces./nBecause even though each individual interaction is weak, about a quarter or a third of a hydrogen bond, summing them up can make them very, very strong. And so that's another kind of force that's important when a linear molecule is trying to figure out how in space it's going to fold up. The point of the gecko thing was it's only relatively recently been discovered that the reason those lizards can stick to walls is they have sort of incredible split ends./nI noticed a couple of you came to Bob Full's talk the other day and you got to hear the full treatment. But because the hairs on their feet are so split they're very fine and the molecules are able to make very close interactions, van der Waals interactions with the surface. And that's what's holding the gecko to the wall. And there are just so many of them that it can support a whole gecko./nAnd that's what could be the basis of, he said to me, a $30 to $50 billion adhesive industry, a self-cleaning dry adhesive. And it's not something magic only the gecko hair will do. You can design synthetic molecules that have the same property and are able to make these millions of van der Waals interactions. So that's two of the other things./nAnd the final thing, which isn't really a force but goes into this, is this hydrophobic effect. And that is that if we have things, amino acids such as valine or something that doesn't like to mix with water, then when the protein folds up, the things that don't like to interact with water will kind of go together just the way if you put a lot of oil in the water it will sort of pull together./nBecause any time you have something that gets stuck in water, it disrupts hydrogen bonds and that's energetically unfavorable. So the things that hate water will tend to lump together. And you're all used to seeing little drops of oil and stuff floating around. And let me switch over to this other thing now. So this was just showing you one of these protein chains that's folded up into a 3-dimensional structure./nThis happens to be something with an enzymatic activity. But the important thing for right now is what's been colored in here are the amino acids that if you look back on the list of amino acids and what categories are, you'd see some of them are said to have hydrophobic side chains. You can see quite strikingly how the amino acids in the interior part of this protein have clustered together./nThey don't like to interact with water. They interact very well with each other. Just like you can mix butter and oil, they mix together very well. And so that is another factor that contributes to the 3-dimensional structure of these proteins. So understanding what proteins are all about means ultimately understanding their 3-dimensional structure. And, as I say, a big unsolved problem right at the moment is how do you get from a linear chain of amino acids to one of these 3-dimensional structures? And you can imagine with 20 different side chains there's an unbelievable number of combinations that you can make./nYet almost every protein in nature has one unique or one or two or something confirmations that takes out, out of all the kinds of things that you could do. And it's this combination of forces that does that. You know, let me just go back for one second. So the final thing that ones talks about when you're talking about proteins are quaternary structure. And what that means is when you have more than one polypeptide chain, so if we have two different proteins that interact, this is protein number one and this is protein number two, then there has to be some sort of interaction between each of these three dimensional structures in order for the proteins to stick together./nSomething like that flagellar motor that let's the bacteria swim, has many, many parts, all of which have to fit together just the same way all the different parts of an engine have to fit together. Now this next little movie is just a dimer. It's actually a heterodimer so it's made of two proteins. They're different but they've come together and they're interaction./nSo what you will first see is the 3-dimensional structure of each protein showing the alpha helices, the beta sheets, and nothing else is shown. The side chains aren't shown. The molecular surfaces aren't shown. You can just see the backbone. You'll see alpha helix a turn, some beta sheets, just the kind of stuff you were seeing the other day. But you'll see that the two proteins are together./nAnd this is actually a movie made by Tom Schwartz who's a crystallogram who just started on our facility this fall in the biology department. It's one of the proteins he studied. And then after that he rotates it around so you can see it. After that he then puts on the side chains and then traces the surface. So this is what they call the van der Waals surface. So this is what the protein would actually look like./nAnd what I think you'll see from this is how incredibly well the proteins fit together. The theme I'll probably keep saying all the way through the course is biology works from fitting shapes. And things have to work incredibly well, and that's also why these van der Waals forces become so important. Because evolution has ended up making things that just go together just like a hand in a perfectly fit glove./nThat's the way most of these interactions are. So watch this little movie. So the light blue is one of the proteins. There's an alpha helix. There are a lot of alpha helices in this one. And the purple one is the other protein. And you can see that in between them there's an interface. And so those must be interacting. But when he superimposes now all the amino acids in the surface, now what he's going to do, he's going to pull those apart so you can see where they were interacting./nDo you get the idea now of how beautifully these things have folded up in 3-dimensional space in positions so that they can fit together and work together as a machine? Just the same way if you were building a machine and you needed to have two parts that you had to join together you've got a tool thing so that the surfaces go exactly together. That's what nature does./nThat's also why I'm making such a deal out of this 3-dimensioanl structure of proteins and how it got there. I could just say it gets there by magic, but it doesn't. It's determined by this set of forces. And one of the things we cannot do at this point is predict. Here's a linear sequence of amino acids. Here's the 3D structure. That would be a huge advance in biology if one of you guys could figure out how to do that during your career./nOK. So I just want to reiterate some of the things that proteins do because we'll be talking about them as we go along. One thing they do, they act as enzymes which are catalysts for biological reactions that take place under physiological conditions. And we'll give you a lot of examples of those enzymes starting very soon. They play structural roles./nOur hair, our fingernails are made of protein. The hairs on the gecko's feet are made of keratin which is the same stuff our hair is made of, except they basically got a whole lot of split ends. They're finer hairs to begin with and a lot of split ends. And that's what makes these very, very fine things that can make van der Waals interactions with surfaces. They play roles in specificity./nFor example, I mentioned the antibodies. And we'll talk about the immune response in some detail at the end of the course. And one of the really magic things that we've come to understand in biology recently is how it is that your body has this immune system that's able to recognize literally any molecule, any molecule. It doesn't matter whether it is existed or you and your PhD thesis in chemistry synthesize something the world has never seen before, your immune system can create an antibody or something that will very, very specifically recognize that particular shape in just the same sort of way that you saw the shape on that movie./nAnd you might think you'd need to code a zillion millions of DNA in order to do that. But there's a trick using combinatorial functions and mutation that lets your body do that. Another example, which I was showing you, that can do all sorts of little motors and machines, I showed you the bacteria swimming around. These are just E. coli. And you cannot see the flagellar motors, but you can see them buzzing around just under a cover slip./nSome of them are stuck to the cover slip. There we go. In this one, which was taken by Howard Burge who is a professor at Harvard, I just took this off his website, you can see the bacteria swimming by having these flagella which are basically like sort of propellers more or less that they turn. Here's one where he used the strobe so you can get a little bit better view of it. And I showed you this picture in the first lecture./nSo that's the machine, but every single part of that machine is made of a protein that's got a very certain 3-dimensional space. And we're going to start talking about energetics, how does this cell get energy? And one of the things you might wonder is if you were to design such a nano machine how would you power it? They exist. I mean it's here./nBut that's why in part I'm going to start talking about energy and how cells make energy, because this is one of the things they have to do. And that was, as I said, was an average electron micrograph of a lot of those motors. So you can see that although, you know, that's the textbook thing, the actual thing is pretty much the same shape. This is not at a resolution where you can make out the individual proteins that put it together, but some of those are starting to be known in 3-dimensional detail./nAnd I thought you might enjoy seeing this just to convince you it's a motor. In this thing, what Howard Burge did was he stuck the propeller, if you will, the flagellar to a cover slip using an antibody. And then he let them do their thing. And normally they would be turning the propeller and swimming, but if the propeller is attached the same thing would happen if you held onto the propeller of a boat and turned on the motor the boat would start twirling around./nAnd what you're seeing here is bacterial that are twirling around because their flagellar are stuck on. And those of you who are observant will notice even that they change direction. And that's part of the system that bacteria use so they can swim towards a food source or away from another one. OK. Here's just something to let you think about it. If anybody can figure this out send me an email./nHere's something else. The phenomenon I'm going to show you is due to a protein made by a soil bacterium called Pseudomonas syringae. You don't need to know that. It associated with plants. And what you're going to see is a little movie made by a couple of post-docs in my lab where they took pure water. And if it's pure water you can cool it below freezing./nYou can get it down to, I don't know, minus eight degrees centigrade and it still will be a liquid, even though you know water freezes at zero degrees centigrade. And what it has to do in order to turn into ice, somewhere you have to nucleate the formation of an ice crystal. And once it goes, going. So, anyway, what you're going to see is some super- cooled water they've made./nYou can see there is zero degrees there. And this is Metchitaga, one of the post-docs in my lab. That's the super-cooled water. She's taking a little bit of culture of this Pseudomonas syringae, and she's just going to put it in, give a little tiny squirt, a few micro-liters into that water. Now it's going all cloudy. And now you might wonder what's happening there. But, as you'll discovery, what happened is that what was liquid water is now ice./nThat is due to one protein that this bacterium makes and displays on its surface. And here's a controlled experiment. This is putting in a little bit of rhizobium meliloti, another soil organism, and the same amount of bacteria. It didn't happen. OK. So that's due to a protein that was on the surface of that bacterium. Anybody have any idea what that could do? Send me an email. OK. There's one last class of biological macromolecule./nThose are lipids. These are a little different in the sense that this is not know a long chain made by joining together subunits as you see with the proteins and nucleic acids, but putting together the parts necessary to make a lipid involve the same principle, that you end up splitting out water molecules. That's a theme you've heard over and over again. And if we take three long chain, three fatty acids, some number of carbons -- Some number of carbons./nJust some arbitrary number here. And then we take a three carbon compound that has three hydroxyl groups. Now, this is not a carbohydrate because you'll notice there's not a double bond oxygen as you saw in the carbohydrates. It's actually an alcohol that's known as glycerol. And if we split out water like this what you get is a fat, something you're familiar with from beef fat. Or if you get something like olive oil, you've heard the term unsaturated fats./nMaybe you'll recall from the second lecture that if we had a single covalent bond, or if we had a double or a triple bond that was called an unsaturated bond. So if you have an unsaturated bond in here somewhere then you end up with an unsaturated fat. And most of you know probably something like beef fat is solid. If you put it in a refrigerator something like peanut oil will stay liquid./nAnd that's because if you have just saturated side chains from this then they pack together very tightly and they will form a solid. If you put a double bond in then there's a kink in the backbone and it's hard for these things to pack together. And that's why they're called unsaturated fats. Now, there's a very particular kind of lipid that's of unbelievable importance in biology known as a phospholipid./nAnd the reason that's so important is that that is the boundary that determines the outside of a cell. So every cell, every organism either is a single cell or is made up of multiple cells. And, as I said in the first lecture, that one of the secrets to life is having a boundary that goes around your insides it separates your insides from all the rest of the universe./nAnd the way these membranes, as they're called, are made of is what's known as a phospholipid bilayer. And it's the same principle as before. It uses a glycerol, except that one of the fatty acids is replaced by a phosphate group that will have some kind decoration added onto it. And the other will have fatty acids at the other two positions. Now, you'll notice by splitting out water here, the kind of bond that we have created, the chemical name of this is an ester bond./nAnd if we wanted to break it we could add water back across it. So what's important about this molecule is this part of the molecule, if you will, is water-loving because the very polar bonds here, the oxygen here would have a negative charge under physiological conditions. And this part is, if you will, water-hating. So phospholipids are often represented in the following way where this is the water-loving and then this would be the water-hating part here./nAnd so if you take phospholipids and you just try and disperse them in water, they spontaneously self-assemble into structures that bring the water-loving parts together and the water-hating parts together. And by so doing this they form what's known as a phospholipid bilayer. And that's what this membrane is made of. Membrane of bacterial cell, membrane of our cells virtually the same thing. It has the property that is not permeable to very much./nWater can get across a very limited number of other chemical compounds, but most things cannot. And so, by having this membrane, what the cell is able to do then is control who comes in and who comes out. And the way it does that is it has to put particular importers or exporters imbedded in the membrane that can carry out those functions. Because, as you would guess, any system would have to bring stuff in, get rid of waste, you'd have to be able to go back and forth./nThe things that do all those transports across the membrane are, what kind of molecule do you think it likely to be? If nature was going to design something that was a pump to get something in or something that would get something out, any idea what kind of molecule? Take a guess given what I've said so far. Protein. Yeah. Absolutely. And let me just sort of show you a couple little pictures here./nSo here's a representation of this phospholipid bilayer. This is pretty standard stuff. This is what you'd put on a blackboard. Here's in gray now the phospholipid. And here's one of these proteins, a picture of one of these proteins that functions to get things across the membrane. And hopefully what you can see now is that it's made up of a whole lot of alpha helices, and they pack together to give sort of a cylinder made up of different alpha helices that weave in out like this./nAnd then by this sort of trick the protein is able to create a channel that runs up and down the middle of this protein that's imbedded in the membrane. And then, depending on the characteristics that channel, it can either be used to bring stuff in or get rid of it. There is a more fanciful depiction of it. This is not reality, but there you are with the water-loving parts./nHere are the fatty acids going in. And this is supposed to be one of these membrane proteins. Now, this next movie is trying to pretend here that it's looking at one of these membrane proteins colored here in red as it spans the membrane. So here's looking from the membrane surface on. And now it's going to dive into this thing as it crossed the membrane./nAnd basically what this movie is letting you do is feel like if you were the molecule that's being transported across the membrane you'd see how you'd go right down through a channel in the middle of the protein. So that's one of the underlying principals then, is that you have a phospholipid boundary that's critical for life. But then to have everything else that needs to happen the cell makes a series of proteins that function either to bring stuff in or to bring it out./nOr in the case of something like the flagellar motor we talked about it has to imbed a part of the machinery right in the membrane. And one last picture I just want to show you. Usually, even on that movie, you tend to see the cell represented something like this with a membrane and every once in a while there's a protein. This is a cartoon but it is much closer to a to-scale drawing./nThis is an E. coli cell. Now, they have an extra membrane that we won't worry about right for the moment. But right there, this little piece that we can see little bits of, is the cell's membrane. And what this picture is showing is that this membrane is just absolutely studded with membrane proteins that are going to carry out various functions. And here actually we're seeing that motor which is imbedded both in the inner and outer membrane./nAnd there's the motor going off. But a couple of things maybe you can take home from this is there are a lot of proteins stuck in those membranes that control what goes in and out. You also get a sense in here of how crowded the cytoplasm is. The proteins are really at amazingly high concentrations when they're inside the cytoplasm. OK. So that's sort of a quick survey./nIt's nothing more that a really superficial introduction to the four classes of biomolecules. But to go any farther we're going to have to think a little bit now about of the characteristics of living cells. I don't know if any of you know if any of you know what this is, but this is bakers yeast. If you were making bread you know you put some yeast in it and it divides and it gives off carbon dioxide as a waste product and makes the bread rise./nAnd what's happened in that little movie you just saw were two cells dividing to give four, and four dividing to get eight, and I don't know what we're up to here, but you can see a cell grow. That's sped up. It takes probably something closer to an hour for a cell division to take place. But this is the kind of thing that microorganisms do when they grow, is you can start with a single cell and it will make two cells that are identical to itself and those will make four./nAnd what happens when we start out as a single cell, we start out initially like this. And we make cells that are identical at the beginning. And those are the famous embryonic stem cells, because at this point they can become any cell in your body. And if you're a yeast it doesn't matter. Everything you make is the same. If it's a human, once you start dividing at some point cells are going to have to start making decisions and the progeny will have to start to be different of each other so that you can have something that's an eye and another cell that's in the liver and so on./nAnd we'll talk a little bit about that as we go on. But the major point, right at this point, is that all of life involves one cell dividing and giving a couple of other cells, and then those going on. So these cells, as we've said, characteristics of organisms which are to be true at their cellular level as well is that they carry out metabolism, they undergo regulated growth./nAnd you have a nice example of yeast undergoing regulated growth and they reproduce, which in the case of a single-celled organism is the same as cell division. For us reproducing is a lot more complicated because we have to make a whole other multicellular organism where the cells have differentiated functions, but the point about that is there has to be an unbelievable amount of synthesis./nThe DNA in our body, we start out with two meters in a fertilized cell, and we have ten to the fourteenth cells by the time we're an adult. So we've had to make a tremendous amount of DNA let alone protein and everything else. And something almost all of you know from your engineering background from this place is that you need energy in order to synthesize material./nAnd what we'll start to talk about in the next phase of this course then is how do cells make energy and how do they carry out metabolism. So I'm going to, just before we do that, introduce to you very quickly, to close out here, two classes of organisms that we find in nature. We find organisms that are known as autotrophs. These are certain bacteria, and they're able to make everything they need starting with CO2, ammonia, phosphate, water, a few things, but that's all they need./nSo, for example, an organism that lives, a bacterium that lives out in the open ocean is able to make everything from those very, very simple basic building blocks. Heterotrophs need to eat -- -- some things made by other organisms. An example of a heterotroph that you're familiar with, that I'm familiar with is us. You probably remember your mother reminding you, as you're about to have yet another hotdog, that it was important to eat your vitamins./nThe reason you need to eat vitamins, those are things we absolutely need for our life but we cannot make them ourselves. Vitamin C is one you probably know. It has an interesting history how people figured this out. It was sailors at sea got really, really sick. Their teeth would start to loosen, they would start to bleed and they would die. Some of the famous sea voyages you heard about in high school, I think the Cape of Good Hope, on that trip where that was discovered 100 out of the 160 sailors died at sea because of scurvy./nNow, scurvy turns out to be due to not having vitamin C. And there was finally a guy, Lind, I'm just blanking on his first name at the moment, in about the 1700s who was a naval surgeon in the British Navy who actually figured out that if you gave sailors lemon juice that they didn't get scurvy. It was a controlled experiment. It took about 50 years. I think it was 1795 when they started to finally give the sailors lemon juice and stopped having this terrible sickness amongst their sailors./nAnd then in about 1950 they substituted lime juice. And some of you may still know the British sailors are called "limeys". And that was because of this solution they found to avoiding scurvy. And what was really happening was they were finding a way to provide vitamin C which is in fresh fruits and vegetables which wasn't part of the classic sailor diet which was sort of biscuits and dried meat during long voyages at sea./nAnd there are several other vitamins, but the reason they're called vitamins is they're things that you body cannot make but other organisms can. The other thing that we cannot make, we can make some of our 20 amino acids, but there are eight amino acids that we cannot make, lysine, methionine, lucien, isoleucine, valine, threonine, phenylalanine and tryptophan. And this actually has consequences for us because those of you who are vegetarians probably know you have to be kind of careful about your diet./nIf you're eating animal protein you're getting essentially all the different amino acids, but if you're a vegetarian you have to be careful because the major food crops such as wheat and rice, for example, are very low in lysine. So if you just eat those you end up with a lysine deficiency that's not good. But, on the other hand, beans, lentils, the various leguminous plants, which also are those ones that form the special associations with bacteria that let them convert atmospheric nitrogen into ammonia, legumes are high in lysine but low in methionine./nSo peoples all over the world figured this out by trial and error. So the Mexican diet is rice and beans. There's a reason for it. What's happening actually is just in the rice you're low in lysine, but by having beans at the same time you're balancing out the two. Or the Native Americans in this pat of the country had "the three sisters" with the corn, the squash and the beans./nAnd again they were balancing out the diet by making sure that they got the various amino acids, a balance of all the amino acids that were necessary for life. It also actually was really good gardening practice because the beans were able to convert atmospheric nitrogen into ammonia, which was fertilizer, and the squash leaves shaded the ground so that the ground didn't dry out, and the corn could grow even when it was short on water./nBut what was really happening, as people grew without even understanding about chemistry, they were compensating for the fact that we're heterotrophs and needed to do this. So we'll start in the next lecture on trying to talk about how cells make energy and how it makes some of this amazing stuff happen.
Tags // Biochemistry III Lecture Graham Walker
Added: April 2, 2009, 1:29 am
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