 Hello. Welcome back. Hello. Quick question, is everybody able to download the PowerPoint? I just downloaded it on a Mac and a PC, but I guess some students are having problems. So, welcome back. So we're going to change gears. We're done with pathways. All right. Trust me, I'm happy too. Less to lecture about. All right. So I'm actually, I want to have a somewhat meaningful conversation about the exam, but to not rush that, I want to have it at the end of the lecture. So stick around if you want to be involved in that. So today we're going to be talking about DNA. We're going to be talking about the structures of DNA, how DNA, some a little bit about the stability of DNA, which is an important topic, how DNA is recognized by proteins, and then some of the higher order structures that DNA can take. Okay. So the last part of the class, this is a summary of what we're going to be doing until the end of the class. So today we're just looking at one of the molecules, the DNA molecule itself. But DNA molecules need to be copied, and there's a lot of co-biochemistry involved with that process of replicating the DNA. Now the DNA needs to be transcribed into RNA, and then that RNA is then translated into protein. And so we'll have a separate lecture, one on DNA today, and then another one on replication, one on transcription translation. And so this is an overall view of what we're doing, but ultimately DNA is a molecule that's not really an enzyme, right? So it's not catalyzing a chemical reaction, it's carrying information. And so the sequence of bases within the DNA molecules you're well aware carries our genetic information and allows us to synthesize ultimately new proteins, which are catalyzing the majority of the functions within your cells. And so these nucleotides, the order of nucleotides, translate eventually to amino acid sequences of proteins. But this needs to be highly regulated, as it turns out the protein coding parts of DNA is a very small, surprisingly small percentage of total DNA, and that there's a lot of structure in this DNA that affects which genes are transcribed and also gives signals about translation. But ultimately this DNA is just a polymer of deoxyribonucleotides. And we've already looked at how these molecules are made, the biosynthetic pathways. So today we're going to be using them. So DNA stores information, and the great thing about DNA is it can make a highly specific base pairing. And so you can make, for example, copies of DNA. So you take one DNA molecule and you make a copy. And so this is facilitated by the specificity of base pairing. And so these are the nucleotides that are involved in DNA. Remember these are the deoxy form. So the 2 prime position is a hydrogen atom, whereas an RNA would be a hydroxyl molecule or functionality. And so we have all of these, and you've already seen these molecules before. You've got the purines and the pyrimidines. Now today we're going to be bringing these together to make polymers. And we're going to be looking at the base pairing, the possibilities here. So this is a review slide. So this is how the polymer is assembled. So there's a polarity to it, a 5 prime end and a 3 prime end having to do with the numbering of the positions on the ribose sugars. So we know that these nucleotides are attached to ribose. And so you have phosphodiester bonds between neighboring nucleotides. And at the 5 prime position, at this methyl group of the ribose, you have a phosphate. And so that's called the 5 prime end. And at the 3 prime position of this polymer, you've got a hydroxyl group. And so that's the 3 prime end. So we can abbreviate the sequence. So this is sort of an abbreviation just showing us the connections between nucleotides. But you can also, there's lots of different ways you can abbreviate it that you might encounter. It's generally helpful to also specify the polarity. So this goes from the 5 prime to the 3 prime end here. So 5 prime to 3 prime. And so initially when this molecule was being discovered, it was unclear the relative position of the ribose relative to the bases. So these nucleotide bases could have been sort of on the inside of the outside. There could have been different structures, a helix or other sort of structures. And so what's really important here is the way in which these nucleotides interact with each other. They stack on top of each other. And that affects the structure. And also the lack of a 5 or a 2 prime hydroxyl also affects the structure that DNA can take. So the thing holding this together is similar to some of the forces we've seen that hold, for example, proteins together, predominantly the hydrogen bonds. And remember the linear form of hydrogen bonds where you have the two electronegative atoms in a linear orientation with the hydrogen atom. Those are the strongest hydrogen bonds. Whereas if there's a kink, then that would be a weaker hydrogen bond. So the base pairing within the nucleotides of DNA is achieving a relatively linear, strong hydrogen bond. So that's review. So here's an actual segment of DNA. So you have, again, the polarity. And the first thing you'll notice is the anti-parallel orientation. So one strand, that's what we call this segment of DNA on this side, would be from the 5 prime to 3 prime direction. So thinking about the ribose sugar is the way to number that. And then on the other strand you have the opposite orientation from 3 prime to 5 prime. And the base pairing that's observed here is between adenine, thymine, guanine, and cytosine. And you'll notice, and you should be able to, this is the thing of life, so you need to be able to take those nucleotides that you've already memorized and know how they hydrogen bond to each other. That's important. And you'll notice that these are linear hydrogen bonds here. And the AT base pairing has two hydrogen bonds, whereas the GC base pairing has three. So the base pairing between GC is a tighter binding. And one of the initial things that they observed before they even figured out the structure of DNA was that the amount of G was always equal to C and the amount of A was always equal to T. And so this gave them a hint that perhaps there's one way that this observation could be accommodated was if there was specific base pairing between those particular nucleotides. And so when you start thinking about structures, okay, if these are sort of equal, how can we accommodate that within a structure? And so they said, okay, it's probably more likely that these nucleoside bases are within the inner part of this structure so that they can actually touch each other. If the phosphate ribose was on the inside, then it would be hard to imagine how you could have base pairing. Okay, so this is the structure. And we're going to step back a little bit. And again, look at the structure or the types of interactions. So you'll see throughout this, we always have G base pairing with C and A base pairing with T. And again, remember the anti-parallel orientation of these strands within the duplex here. And so this complementarity in this base pairing allows us to very easily copy the structure. You think about proteins. That sort of complementarity doesn't exist. So we can't just photocopy proteins in the same way. It's a completely different process. So that's the structure of DNA. And then one of the most important things that was discovered by Rosalind Franklin was this X-ray crystal structure. And they got two important parameters from this structure. And those were that something was repeating at around 30 angstroms, and something else was repeating at around 3 angstroms, 3.4. And so they know that there was a periodicity to this. So the combination of the observation of the base pairing that A was always equal to T, B was always equal to C, and the periodicity of this led Rosalind first to propose and then Watson and Crick to sort of propose again and then get the prize. Rosalind did pass away before the prize was awarded, but it's a little bit of controversy there. She really was the one that discovered the structure of DNA and first published it. Okay, and so this periodicity combined with this base pairing, this equivalence of the amount of certain nucleotides led Rosalind to propose this structure. And here we see that this repeating unit was actually a turn of the helix of the DNA, whereas this smaller repeating unit was actually the space between nucleotides. And so this is the structure that was proposed. It is a right-handed helix. Some of the important features we'll be talking about today in the DNA structure are the presence of these so-called grooves. So we have a major one and a minor one. So the major one is wider. And it's actually wide enough to accommodate the alpha helix of a polypeptide. The minor groove is obviously much less wide, and it's not wide enough to accommodate an alpha helix. So you can actually, we'll see many structures today where an alpha helix is actually touching the sides of these bases within the duplex and allowing us to recognize them. Okay, and so look at the structure of the bases. Do you see how they're sort of perpendicular to the axis of the helix? And then there's this pitch to it. Okay, so there's about 10.5, as it turns out, in the type of DNA molecules that occurs predominantly itself, there's about 10.5 residues per turn. Okay, so this is the structure that was determined. Okay, and so here is a not turning duplex, but just so you can see it again. You can actually look on the web and see pictures of the DNA molecule rotating so you can get an orientation. And here, remember, so we have the ribose and the phosphates on the outside of this helix and the bases, base pairing on the inside. So there's a variety of different structures. So when people started modeling these, they started with sticks and balls and just like you would do in Orgo class and actually built up a variety of helices. And there's a lot of theoretical possibilities here. But as it turns out that in cells, this particular orientation of the helix is the one that predominates, while the A and the Z form are theoretically possible. The B form is the primary form that is observed within DNA. And so that in the B form, you have about 10.5 base pairs per turn. And you'll notice these are both, the A and the B form are right-handed helix. So again, if you put your thumb in the direction of the axis of the helix and turn it, you'll see that your fingers trace in a right-handed orientation. Whereas in the Z form, there's more of a squiggly line on the backbone here. And it's also in the left-handed orientation. So if you do the same thing, you'll see that the Z form is left-handed. Now the Z form really is predominantly an artifact of crystallization. Initially, people really didn't find any evidence for Z form of DNA within cells. But then later on, it was found that it can occur close to areas that are being transcribed. And it's a way for the DNA to contort and adapt as the transcription machinery is passing through the DNA. And so there's actually a protein that can bind it. But it's very, very uncommon. And the B form predominates. The A form has a slightly different pitch to it. It's about 11 base pairs per term. But it's also right-handed. This A form is found in a solution. But the only place we've really seen it was in a DNA-RNA hybrid duplex or in an RNA-RNA duplex. So DNA-DNA molecules, we really don't see much of the A form. And there's actually a reason behind that. So we know that really the major difference between DNA and RNA is that 2-prime hydroxyl. And so DNA doesn't have that, whereas RNA does. And so if you look at the next slide, it might be a little bit hard to see. But here are the two different forms. So now this is actually looking at the ribose ring. So we have one through five. Remember there's five carbons in the ribose. And you can see that this five-membered ring is not flat completely. It's got a bit of a pucker to it. So it's got a bend in it here. And we can have two different possibilities. And the position of this pucker affects whether you're going to have A or B DNA. In this form, you have these two groups, the 2-prime hydroxyl and the 3-prime phosphoester linkage. These are actually too close together. And so RNA can easily accommodate this configuration because of steric clash. Whereas here, you'll notice that the 3-prime phosphoester bond is way over here in relation to the hydroxyl group. So that's what actually drives this tendency of RNA to fit in here. But DNA, you could think of it, DNA is a little bit more jiggly, right? So it has a hydrogen atom here. So it's predominantly in the B form, but when you see animations of what people think DNA is looking like, it's a little bit of a jiggliness to it. But the 2-prime hydroxyl makes the RNA molecule a little bit unstable because it can react with alkali conditions. And that causes a cyclization and degradation of the molecule. And also, RNA molecules oftentimes occur in segments of single-strandedness, whereas DNA is somewhat protected by being in a duplex. Okay. And so these are the actual chemical foundations of why certain forms are preferred. So if you think about the structure of RNA, remember it's an A-form helix because that's the form that can accommodate that 2-prime hydroxyl. And so if you have just segments of single-stranded RNA, those can adapt to helical configuration. And that's held together both by the structure of the ribo string, but also by pi-pi interactions of the nucleoside bases on neighboring nucleosides. So that helps this to adopt this particular type of configuration. Now RNA molecules can be very much more complicated in their secondary, and what you could think of is their tertiary structure so they can have regions of base pairing. The base pairing in RNA is always anti-parallel, just like in DNA. But you can have these sort of odd base pairings. You can have segments that are not base paired in RNA molecules. And you can have hairpin loops. And look at the ends of these hairpin loops. You've got bases flying on the outside of the hairpin. The other thing that you can see here is this sort of weird, not-so-good base pair. So typically, you know, GU, I mean, that sounds weird. How could that even happen? So typically what you see in RNA is AU and GC, right? But you can also get GU, weaker, non-linear base pairing. So this is a structure of RNA. Any questions so far? I think a lot of people have seen much of this, yes. Well, so the double-strandedness often comes because complementarity exists within the primary sequence of the polymer. And so when you have base pairing possibilities, these things can come together. But that brings up a really good question. How does it fold? Is that folding assisted? In general, these things tend to fold on their own, right? So I'm not aware of a chaperonin, for example, that helps in this process. But the thing that holds this secondary structure together is the complementarity, the hydrogen bonds between nucleotides. Any other questions? Someone from the audience would like you to explain the difference between the major and the minor grooves once again. Yes, so the major groove is wider and it's actually a little shallower. And the minor groove is narrower and a little bit deeper. The major groove is wide enough to accommodate an alpha helix, whereas the minor groove is not wide enough to accommodate an alpha helix. Yes. We just got one more. What kind of bond is there between the G and U? Single or double? So these are double. And we'll see that on the next slide. Wow, this is good. We're in real time here. And so this is the general structures you can get from RNA molecules. So here is the GU base pairing. And you can see it's not really ideal. It's not linear. So this is not going to be anywhere as strong as an AU base pair, which has linear hydrogen bonds. And this just has two hydrogen bonds. And when you see it in the structure, they put a little dot to remind you, okay, this is a little bit weaker. And so perhaps there's a slight bulge any time you have that non-traditional base pairing. So here's a structure of a RNA molecule. And unlike DNA, RNA molecules can carry out enzymatic reactions. They can act as catalysts. So this is RNA-SP. This cleaves other RNA molecules. And the RNA molecule alone has this catalytic chemical activity. And you can see the structure here is very, very complex. So you've got all kinds of stem loops. You've got massive bulge here. You even have this insanity where you have base pairing from all the way down here to up here. So the true structure of this is three-dimensional. It's not. Here we've laid it flat. But in reality, these nucleotides are right next to these nucleotides that are base pairing together. And so this is called a ribozyme. And we'll discuss more later in the class this idea that perhaps RNA came before DNA and RNA helped to make DNA. But we'll get to that later. So this is the structure of the 5S ribosomal RNA. And you can see its structure as well. It has a lot of base pairing and perhaps more or less of these stem loops. Looks like the movie is advancing, but not showing you. But it's just rotation, so it's not really adding much. So there's many very ornate and beautiful structures that can be made by RNA molecules. In other words, DNA is boring. You don't see anything like this for DNA. You just see the plain duplex, the helical DNA. Any questions so far? So that pretty much covers up our discussion of structure of DNA, perhaps a recap for many of you. Let's look at the stability. So DNA duplex can be denatured. And you can do this by high temperature or pH. And so these change in conditions cause the unwinding or the release of all of these base pairing that originally holds the molecule together. So over time, you eventually completely denature the molecule. Now a partially denatured DNA molecule is much easier to reform the original full duplex in a process called annealing because it sort of started in the right position. But once the two molecules are separated, it's extremely difficult, if not impossible, for the molecules to find each other and rehybridize. For shorter segments, yes. So many of you have heard of PCR. In PCR, we have the hybridization of very small primers. If you have massive pieces of DNA, it's a lot more difficult to find the exact correct registry of the base pairing. And so the stability of the DNA can be measured in terms of its melting point. So the stronger the interactions between the two pieces or the two polymers, the higher the melting point. And so we can plot this in terms of a denaturation curve. Now where we get this data is from looking at the UV absorbance. As it turns out in duplex DNA, you have some of these pi-stacking interactions. And that tends to suppress this UV absorbance. So once you've denatured the DNA, the UV absorbance goes up because there's no longer these pi-stacking interactions. So you can adjust the temperature, titrate in higher and higher temperature, and measure the amount of UV absorbance. And you'll get this type of curve like here. And you can measure different DNA molecules. So here's one DNA molecule that is half denatured at this particular temperature, around 83 degrees. Whereas this other DNA duplex is denatured at a higher temperature. And when you think about what you've seen so far in terms of the structure, can you guys guess what is causing these differences or what kind of trends you might see? So here I've shown that this one that has the higher melting point tends to have a higher percentage of GC in its sequence. Any ideas why it might be higher based on what we've seen? Absolutely. So it has three hydrogen bonds instead of two. The more of the GC, the more you'll have triply hydrogen bonded nucleotides. And so that will tend to hold this thing together. And so this effect, the disruption of the UV absorbance is called the hyperchromic effect. And so you can actually plot this as a function of GC content, percent of total content. As GC content increases, this is a beautiful, nearly perfectly linear relationship between that content and the melting temperature, the temperature at which half the molecule is melted. And so this gets at the stability of the DNA molecule. Okay. All right. Has everybody had a chance to think about the question? Any other votes? One more? Any other? So the correct answer is what? A. All right. Different strands, not the same strands. That's tricky. All right. So the answer is A. They're definitely perpendicular. Okay. So so far we've looked at the structure. We've looked at the base pairing, which adds the stabilization and how we can disrupt that by high temperature or pH. Now we're going to think about the interaction of DNA and protein. So the remaining lectures in the class, a lot of these lectures are about the ways in which DNA is copied, DNA is converted into RNA, and this involves the action of proteins being able to sense particular sequences in the DNA. And so what's important in this process is hydrogen bonding between amino acid side chains and the bases within the DNA. And the binding and the interaction of the proteins in the DNA occurs almost exclusively in the major groove, because that's large enough to get in there and to make good hydrogen bonds with amino acid side chains. And so we'll look at some of these structures. But first, this slide is really confusing, and it's totally okay if we dwell on this for a while. So it's important for you to recognize that you can specifically be able to discriminate all of the four bases in the major groove, but not the minor groove, if you're only able to perceive hydrogen bonding patterns. Okay, so look at this. So I've sort of color coded things. And so in the major group, we have exposed a variety of groups. We have hydrogen bond acceptors marked in blue. Orange-ish, reddish color is the hydrogen bond donors. Then methyl groups, which can affect the sterics of an interaction, are highlighted in yellow. So we have adenine here in base pairing with thymine, and then we have thymine base pairing with adenine. And you can see that these are similar, but they're mirror images, right? So if a protein is facing a DNA molecule and at one level they see AT, the other level they see TA, it's completely different surface exposed to the protein. Do you see that? And the same thing happens for GC and CG base pairing. Here you have blue, blue, red, purple, and then purple, red, blue, blue. Do you see that? They're absolutely opposite. But if you're a protein interacting with those, you can totally discriminate them because they're upside down. And when the proteins that do interact with DNA, they don't just interact with a single base, hydrogen bond with a single base, they're hydrogen bond with lots of bases. Whereas in the minor group, we have a little bit of a problem to discriminate an AT base pair versus a TA base pair, because from the perspective of a protein, it's there about the same. So you've got this blue, purple, blue, blue, purple, blue. They're slightly shifted, but that's not enough for amino acid to discriminate these two possibilities. And the same thing is happening in the minor group of a GC or a CG base pair. You've got blue, orange, blue, blue, orange, blue. It's the same. The protein can't really tell those apart. So that's one of the big reasons that proteins interact in the major group besides the accessibility is because you can have finer sensing of the actual sequence of the DNA. So we want to have certain stretches of DNA sequence that are specifically recognized so that those proteins that are interacting with the DNA start at the right position in the DNA molecule, whatever they're about to do. Does this make sense? Do you see that? How they're different surfaces? Any questions? Yes. Well, that's just how this is drawn. So yeah, it's a little bit hard to see. So here's where you're going to be attached to the ribose here with these dashed lines. So that's where we've sort of removed the details so you can look at this at its most simplest part. So the dashed lines are where you're attaching to the ribose. Good question. Yep. Yep, that's right. So when you think back to the structure of DNA, so what's your sensing? So imagine, see how there's a wider set in the B form. There's a wider segment than a narrow segment. So the protein is going to come in and be able to make hydrogen bonds with those bases that are sort of perpendicular to the axis of the helix. And so this is what determines a face, right? So if we had, for example, ATTA, that's going to be completely different than ATAT. It's a different distribution that the amino acids can sense, can bind. It's sort of like a rotating line. That's right. So the, I wish I could point over there, but the interactions are actually in the, the hydrogen bonds are coming out at you. See the way the bases are oriented. And so, yeah, that's the orientation. So the protein is going to actually assert a helix in that sort of 45-degree angle into there. Get those amino acid side chains right in amongst those bases and make hydrogen bonds. And we'll see how the hydrogen bonding works in a moment. That might help to, yeah. So this is just looking at two nucleotides base pairing, right? And so you can see the point I'm trying to make is that those are different only in the major group between ATTA and GC and CG. Okay. So two more questions from the audience. Yep. One, can you explain again what pi-stacking interactions are? Right. So this is just a fundamental type of bonding interaction in organic chemistry where you have, so notice, like here you can see it, these are aromatic, these nucleoside bases. And so these bases, that aromaticity, and look at the way they're oriented in that molecule, flat, flat, flat. And so the faces are actually close to each other and that provides an attractive force. So a pi-pi interaction provides attractive force, attractive force between two aromatic rings that are lied on top of each other in that manner. And the other question, why do the major and minor groups arise? It has to do, so the orientation of the major and minor groups has to do with the ribose and that is the actual structure of the ribose. And so, yeah, so it's just a fundamental property. So you'll notice, for example over here, the a-form, the major and minor groups are slightly different in the a-form than the b-form. So the b-form has a wider but narrower, or a wider but shallower major group than the a-form, which is sort of a little bit more narrower in its major group. But still, in the a-form, one of the areas is wider than the other. So you still have a major and a minor group. And that difference is solely caused by a little bit of pucker in the ribose, and that pucker, the position of the atom that's actually moved out of plane. That's a great question. All right, so the important thing here is what exactly is going on in the interaction of amino acids with these thymine, anine, cytosine, guanine? Well, it's all about hydrogen bonding. And so there's a variety of different amino acid side chains that can be involved in hydrogen bonding. And here we've drawn some of these possibilities. Now some of these, as drawn, would be slightly weaker hydrogen bonding interactions. So here we have electronegative atom, hydrogen electronegative atom. It's not quite linear. It's a bit of a bend. So this would be a little bit weaker if this amino acid was positioned in this precise location compared to here the arginine, where we have a more linear orientation. But remember the proteins can very precisely position these according to where they occur on the secondary and tertiary structure of the protein that's facing the DNA molecule. Okay? Something's stirring. We're being stirred. Okay, does this make sense so far? So this is the fundamental thing. Now for the rest of the class, we're going to step back and we're going to have little blobsicles sitting on the DNA. But this is what's happening in that interface, a bunch of hydrogen bonding of a variety of amino acid side chains. With the DNA molecule. Okay. And so these DNA binding proteins are what actually are going to bind the DNA. That's pretty obvious. Okay. So they bind DNA in the major groove. In general, the actual secondary structure of the protein that's touching the DNA molecule is a helix. And the reason is, in the B form, the predominant form of DNA in cells, that major groove is wide enough to accommodate a helix being laid into it. Okay? So you can get the closest interaction of amino acid side chains with those bases. And there's a variety of different DNA binding motifs. So these are, remember what a motif is? Sort of a short, we had a very long conversation about what a motif is. But you can also call them domains. So they're short segments or subsections of a protein that adopt a particular fold. And so these DNA binding motifs, these are the variety of motifs that can actually interact with the DNA. But every single one of these motifs, you have an alpha helix placed into the major groove. You might have other stuff going on, but they all have that in common. Okay, so let's look at some of these. So we have the helix-turn helix. So the helix-turn helix, you see this helix here sort of going in? So we have that actually, here's the, if you look at the plane of the DNA helix, this thing is actually inserted inside of the DNA molecule there. And that's making hydrogen bonds with the sides of these bases. Okay, and so that's one, but now of course, these folds or these motifs, these don't mean that there's a particular set of amino acids that necessarily has to be there. We can put any amino acids, or there's some amino acids that are not favored in an alpha helix. But generally, we have a lot of possibilities. The fold just tells us the secondary structure, the arrangement of different segments of secondary structure relative to each other. But please realize, this is not the structure of the entire protein. It could go way out over here. This is just a domain or short subsection. Okay, so here's a helix, and the helix-turn helix, it's actually inserted in the major groove. Another common one, this picture is a little bit hard to see, but I hope you can see that there's, again, a helix in this zinc finger that's actually inside of this major groove here. And the zinc ion is helping to hold things together. Okay, and so that's holding the tertiary structure of the protein together. But again, it's the same thing. You've got a helix, and here you can see one here. Here's another zinc finger motif. And you can see it maybe a little better. Here's your major groove. And you're looking down the axis of the helix. You have amino acid side chains. Remember going out tangentially to the axis of the helix, making the interactions. Here maybe you have, oh, I don't know, about three or four bases that you could potentially hedge and bond to. And remember that this is a corkscrew. So these are not necessarily, these helices are not perpendicular to the axis of the DNA helix. So there's a bit of a pitch to it. So they need to be coming in, making interactions at an angle. Another motif that binds DNA is called a homeo-domain. And so this is very similar to the others we've seen. Again, we have a helix inserted in the major groove where those amino acids can make hydrogen-binding interactions with the bases. Okay, so that's a homeo-domain. And next, leucine zipper. So a leucine zipper is zipped by, the zipping part of it is the part that doesn't touch the DNA. That's the part that holds these two helices together. So in a leucine zipper, you have two long helices, and they're held together when they're away from the DNA by hydrophobic interactions, leucine-leucine interactions. Okay, and so this gives this protein some tertiary structure. And oftentimes these can be heterodimers. I can't think of a reason why it couldn't be a homodimer. But in general, they're heterodimers. So like FOS in June, some of you might know those transcription factors. That's a leucine zipper that is a heterodimer of one FOS protein and one June protein. But again, you're just inserting these into that major groove. Here you can even see some of the amino acid side chains, perhaps not too easy to see the precise hydrogen bonding. So in leucine zipper, it's actually very similar to the next structure that we're going to look at, which is this basic helix loop helix. So the part over here, you have two fingers again intercalating into the major groove of the DNA. But here it's a little different on this side of things. So you have some other two alpha helices that are not necessarily held together by leucine amino acid hydrophobic interactions. It could be other types of interactions. But here you have total of four helices, two that are inserting into the DNA and other two that are holding the ends of these in a stable configuration and being binding to each other. So these are all the different types of general protein structures and motifs that can interact with DNA. It's once you've seen one, it's easy. The plan here is put an alpha helix in the major groove where you can have hydrogen bonds. Any questions on that so far? So we've looked at the interactions of the actual molecular interactions, proteins and DNA. So next we're going to think about the necessity to compact DNA. So you might not be aware, but actually every one of your cells has about approximately 1.7 meters of DNA inside of them. And you have a lot of cells in you. So the amount of DNA, and the size of a cell is what? On the order of micrometers, not meters, that would be a scary monster if you had a meter long cell. So we need to compact this DNA. So look at some of these comparisons. So there's a variety of viruses and you'll see the size of the viral particles is about 100 fold. There's two orders of magnitude smaller than the width of those viral particles is 100 fold smaller the actual length of a DNA molecule. So we can't just have DNA spewed out everywhere. We need to compact that down. We need some biochemistry to help in that process. So if you look at your DNA, if you sum up the lengths of all the DNA in all your cells, that distance would be equivalent to 500 round trips to the sun. Just every one of us. Right? Humans haven't been to the sun yet. It's a little toasty up there. So it's a 500 round trips every one of you. Just the DNA inside of you. That's how long the DNA is. So we don't need hundreds of fold compaction. We need thousands of fold compaction. So there's all kinds of ways in which the cells come up to deal with this. Looking at this problem in a slightly different way. Here's a artistic rendition of a DNA molecule. And you'll see here under the letter O is a yellow dot. That's the size of the cell in relation to the DNA that's inside of it. So you can't, it's like a spec. No, that's not an insect running on the lens. It's the size of a cell. We've got to get all this DNA inside of there. So you can take, for example, an E. coli cell and just release treated under conditions that will release the DNA and cause it to become uncompacted. There's just like this huge knots of DNA coming out of the cell. So that's the problem we're facing. Now how are we going to deal with this? Well, some of you might recall this device. This is called a telephone with a cord. So it's different than the iPhone. I've got one in my office so I don't know about you. Yes, this illustration will be invalidated eventually. But when you think about it, so this is a helix. So you could think of the access here as, you know, so the DNA is a helix. And if you remember, you don't remember. Imagine if you turn this cord, you take your phone, you whip it around, you hit your sister with it, whatever you're going to do. Eventually, the cord gets all coiled up. So this sort of excessive coiling, if you don't got one of those gizmos over here that relieves that, that's called supercoiling. This is the strategy, the same strategy used on this primitive communication device is used by your cells to compact the DNA. And so there's the presence of supercoil. So as you wind this thing up, you could either wind it up, make it tighter, or if you made it looser. It will also do this. So when you make it tighter, those are called positive supercoils. And if you make it looser, you turn it in sort of the direction of the pitch of the helix, those would be negative supercoils. You could have both the possibility of positive or negative supercoils. Okay, and so when you think of a molecule of DNA, so here we have a DNA and in bacterial cells, the DNA molecules are in what's called plasmids. So these are covalently linked circles of DNA. And within this, you could have some supercoiling of this plasmid such that it folds up on itself like this. And so this would be a right-handed helix. Do you see it? Here? So you have a twist to this. And if there's a tightening of the helix, so if you turn the helix within this molecule, it can accommodate that force by twisting in this large sort of way. So this is really hard to see. There's a whole bunch of corny movies that you can look at to help you understand this idea of supercoiling. So here you could take one hand, hold this rubber band and then take your other two hands and pull these things apart. Or isn't that disturbing? I had a dream last night. Okay, no. This is disturbing. So if you had three hands, you could actually have this beautiful illustration of supercoiling. You'll hold with one hand, one end, don't let it rotate. If you pull these things apart, it'll tend to kink up like this. And here, this is perhaps easier to see than telephone cord because you actually have the two strands. So as you tighten this up, you're going to have this supercoiling going on. Okay, so no more disturbing pictures. Here's what cells do. So here's some plasma DNA from bacterial cells and you can see this supercoiling here. And this supercoiling could be completely relaxed or it can have an increasing amount of this supercoiling and its tertiary structure. And this process needs to be precisely regulated. So there's a set of enzymes that aid in the stable formation of these supercoils. So the way this goes, so let's think about this possibility. So here you have remember our B form of DNA has about 10.5 base pairs per turn. But what if, see we're holding a segment of DNA that has 8 total revolutions, 8 turns, right? And then we relax that, removing a turn. Now you would have 7 turns, you have the same number of bases but less turns, right? So you would have more bases per turn, right? And so that would cause this molecule to be strained. And the response of the molecule is that it can do this supercoiling which would return it back to 8 turns of DNA, right? So you'd unwind it by 1 turn, it does 1 supercoil, then it's back to 10.5 bases per turn. Because some of that excess strain has been relieved by the coiling. So B form DNA is a right handed helix and so in general in cells the DNA is under wound and so this supercoiling should occur in a left handed orientation. Here, so this is not drawing, this is drawing actually a right handed supercoil. But in your cells it generally would be the opposite orientation. And why this is important is because as you read through the DNA you want to make a copy of your DNA molecule. To do that you have to pry the two strands apart and start making copies of both strands. So that prying of the strands apart causes strain on the DNA that's either upstream or downstream of that bubble, right? And so you can have for example if you had a strain, if you have this strand separation so you can, if you were in a strained form, so say you were in a relaxed or this strain formed here, one way you can release that strain is by pulling these two strands apart. And so that would return these bases to about a periodicity of about 10.5 bases per turn. So the cell is very actively monitoring the amount of forces on the DNA duplex and it's adjusting them, changing the strain in the molecule. And so to describe this in a little bit more practical way we can think of a linking number. So these are topoisomers, right? So we have different molecules and we can have a different number of intertwinedness, if that's a word, right? Of these two molecules. So you can have for example one ring here, a second ring, so when you think of a plasmid there's two strands. If we sort of whip them all apart and remove all that strain, then we would have two circles connected to each other, right? So here's one DNA strand, here's the other. And we can increase this linking number. So the linking number is defined, it's a mathematical concept. We didn't come up with it in biochemistry. And it's defined as the number of times this second strand breaks the plane of the circle, right? So you have one, two, three, four, five, six, seven. So this is six, I should say. We started here. And so that's a linking number of six. So this second strand has broken that plane six times, okay? And so let's think about this a little bit further. So let's consider a segment of DNA that's 2100 base pairs long by side notes. So we have base pairs but there's a KB so kilobase pairs or megabase pairs. These are ways to specify the number of nucleotides. So here we have 2100 base pairs. We know totally relaxed DNA would have 10.5 base pairs per turn, right? And so we can calculate this linking number. It would be simply the number of base pairs divided by the number of base pairs per turn. So this linking number of this 2100 base pair plasmid DNA would be 200. So let's take that and consider two possibilities. So this is our fully relaxed 200 linking number DNA strand. And so let's think about what if we introduce a nick? Well as it turns out when you have a nick the linking number in a orthodox sense becomes undefined because if you have a nick you could just pull the two strands apart no problem, right? You're not intertwined with each other. But then let's consider the possibility of underwinding the DNA. So let's say let's change this linking number and decrease it by two. So if you count the number of times that second strand breaks the plane of the circle. So imagine you have this second strand going around this first strand 200 times. Now it's going to be going around 198 times. And so we can define something called a super-hilo-code density which is the change in the linking number divided by the number of times or the number of times that length of DNA would cross this plane in a relaxed DNA molecule. So that's 200. So here we have for example a change, a decrease a negative sign, decrease of the linking number of two. We divide that by the relaxed linking number and you get a super-hilo-code density of negative .01 or 1% decrease. So you can think of this because this particular molecule would be 1% underwound. So there's this concept. So in human cells to achieve the compaction necessary so we don't have to go back and forth to the sun the super-hilo-code density is about negative .05 to .07. So that's the amount of negative or unwindedness that the DNA must achieve to be able to compact it down. So I know this is confusing too. But think of it I think the most practical way to think of it this is percent unwindedness. Instead of thinking about delta LK and planes and things going around. So how underwound negative means underwound a positive number would be overwound. So on your slides this morning I discovered a mistake and this is now the corrected picture. So here's a relaxed DNA molecule if it's underwound by two turns it would form this structure. So in your slides if you printed them last night or earlier these two would be swapped. So make sure to put in the right place. So you end up getting a left-handed pitch of supercoil when you underwind. So we know that the B form the actually strands are in a right-handed helix. So here we have that right-handed helix twisting about itself in a left-handed orientation if the DNA is underwound. If it's overwound it would have positive supercoils and then it would be a right-handed super helical twist. So you have to imagine these pictures. And so these two forms all of these structures have the exact same sequence of DNA but they're topoisomers they're not the same molecule. They have different amount of supercoiling right and so they're chemically the same but structurally they're very very different. And so we refer to these as topoisomers and the enzymes that help to cause this super helical twist to occur in DNA are called topoisomerases. So here's the topoisomerases there's two types. These types there's actually lots and lots of subtypes there's different types within prokaryotes and eukaryotes this is just a general picture. One thing that's always the same is that the type 1 topoisomerase is change this linking number in increments of 1 and that's because of the actual mechanism of the topoisomerase that we'll see in a moment. They can in the way they work is they cleave one strand and only one strand of duplex DNA allow the other strand to pass through so every time they cleave and let the strand pass through you decrease the linking number by 1. Now type 1 topoisomerases can relax both positive and negative supercoils and this is one of them is called same as an obvious name. Now the type 2 topoisomerases change this linking number in increments of 2 so this is easy to remember cool type 1 increments of 1 type 2 increments of 2 and the way they do this is they cut both strands of the DNA and let the duplex DNA to pass through so that's how they change them by 2 and so these also type 2 topoisomerases can relax both positive or negative supercoiling but in prokaryotes they can introduce negative supercoiling by an ATP dependent process so they can actually use the highly hexagonic hydrolysis of ATP to drive to add strain to the molecule and so DNA gyrase for example is the prokaryotic version of the type 2 topoisomerase that can do this introduction of negative supercoiling. Okay so it's 2 topoisomerases this is one way that you can visualize topoisomers of DNA so this is a DNA gel and if you have highly supercoiled DNA it's going to have a smaller cross sectional area it's compacted down to a smaller space so it's going to tend to pass through a viscous matrix of gel quicker and so here you have for example highly supercoiled DNA and here's some relaxed DNA but now what we're going to do is add a topoisomerase 1 to this test tube that has this mixture and we're going to see that you form a ladder so each rung or step in the ladder is a change in linking number of 1 so these shifts here you're changing the linking or as you're decreasing the linking number here it's able to pass through the matrix of the gel differently. Okay so you're relieving or introducing strain so here in this case we're taking this highly supercoiled DNA relieving that strain through the topoisomerase and until it fully or is progressing towards the relaxed state so here we're just relaxing we're not introducing supercoiling so here's the mechanisms you actually have for topoisomerase 1 you grab one strain make a covalent bond to the strain, break it, but don't let go if you let go that's not good so you hold on to the two pieces take the other strain and pass it through the break yes, hi if you're unwinding the DNA it has a larger cross-sectional area so that's why the peaks are moving up because they don't come through the gel as easier as easily yeah I was thinking about that while I was speaking but I didn't want to mention it but now I've navigated the complexity thank you you helped me think through it alright any other questions that was a good question okay so this is the mechanism you just simply pass one strand through these are topo 1 you're going to make a covalent bond between a tyrosine hydroxyl and the phosphate so you're going to maintain a covalent bond and hold on to this other strand non-covalently pass the second strand through re-anneal the nick reform the phosphodiester bond okay so something else has to wind the DNA up and this thing is actually relaxed remember topo 1 is just relaxing the DNA topo 2 is a different mechanism you grab a duplex of DNA you're actually grabbing both so here's one duplex here's another segment of duplex they're obviously attached to each other outside of here and then through ATP hydrolysis you can actually introduce negative supercoils or in the absence of ATP this can also be used to relax either positive or negative supercoils as you break the duplex DNA pass the other duplex through and then re-anneal the DNA and release so that's pretty so within bacterial cells you can just make a bunch of supercoils so this is called plectonemic supercoiling so this is mostly as drawn here just a bunch of DNA and so if you wind the DNA you have a bunch of DNA gyrase a bunch of negative supercoils here it compacts the DNA to a certain extent but really you're going to need some other proteins to help you to compact it even more you can only get so far by just this plectonemic supercoiling of the DNA alone and so there's here's the plectonemic supercoiling but in a more compacted form of supercoiling would be the solenoidal supercoiling so if we had some way some little solenoids that would squeeze things of very similar shape to the telephone cord of your and so the way that we do this is by binding histone proteins so we have negative supercoils in other words underwound DNA wrapped around and stabilized by binding to histones and so these histone particles come together to form a nucleosome and so these can be seen in an electron micrograph as beads on a string and so there's about 146 base pairs of nucleotide bound as you go in each nucleosome particle or nucleosome segment and so this is one way you can package DNA into nucleosomes there's a variety of protein constituents of nucleosomes H2 through H4 are actually in the core and H1 is at the at the entry point and it's regulating the assembly here so it's not actually drawn here this is just H2 through 4 and they're pretty small sequences but they also can have little arms so you can have little arms hanging out of here and you can have what's called a histone code where a large number of post translational modification of those arms which affects the ability of nucleosomes to be disassembled and also can affect association of nucleosomes with each other so in your textbook they have a picture of nucleosomes all stacked together with these arms going in between them and so here's the DNA it's negatively supercoiled or unwound around these histone proteins and that's not going to work nothing works okay so here's a picture so here's the histones the histones interact with the DNA and they unwind some of it so if they unwind the DNA say you start with completely relaxed DNA you add your histones you get under wound DNA around the histones then you're going to obviously going to have over wound DNA outside of the histone so the act of binding the DNA to the histone unwinds it and you're left with this positive supercoil on the outside and the role of the topoisomerase at least in eukaryotic cells where this can occur is to relieve this strain to stabilize the underwinding around the histones by removing the positive supercoils that form as a necessary result of underwinding around the histones okay and so you can so this line your textbook is really blown in in this section should go underneath here remember it's a left handed left handed right handed yeah left handed helix maybe it's right it might actually be drawn right it's hard to see I think it's wrong but whatever but you get the general idea if you wind something up there you have an opposite reaction and the topoisomerase relieves that so the net output so the linking number here is unchanged delta linking number is unchanged we're over or we're underwound here overwound here the net effect is to not change the linking number but as soon as the topoisomerase comes in it removes this overwinding and then we have net underwinding so that's why about because all this DNA is wrapped around the histone proteins okay alright and then we can take those nucleosomes and have those wrap around each other and now we step back into artistic renditions because we really you know up to the nucleosomes we had it we know sort of where the histones are how the thing is winding around there at this point there's a variety of different models of how we can get even more twist into this DNA so these are so called 30 nanometer particles and so we can have the nucleosomes remember each one you have DNA wrapping around histone proteins at each position but this the backbone here can itself wrap around itself and so here you see a left handed twist to this 30 nanometer particle I'm not sure that there's experimental evidence for a left handed twist but it's consistent with the super helical density that we tend to see in cells okay and so these particles once the nucleosomes have achieved this 30 nanometer particle they tend to be transcriptionally repressed when they're not in this 30 nanometer particle it's a lot easier to express the genes so to express the genes we're going to have to actively get the things out of this more condensed form okay and so we can take this even further we can bring it up to make here you have think of this as a line now and that line can now wrap around even more especially during replication of the DNA during cell division you can have formation of rosettes and so we have a variety of other proteins that can help to bend the 30 nanometer particle into a helix here so it can just step all the way back with the nucleosomes okay that's sad that that won't work alright yeah that's too bad I'll post those movies online okay so that's that, any questions? okay so we could go over the exam QI so I clearly have been teaching this class too long I have accurately accessed the difficulty of exams this exam is a lot harder and I apologize I try to make it consistent but this one was harder so you shouldn't be penalized for that and so what we've done is as I sent in the email we've actually adjusted this I looked at the high score and the high score was a 94% I said that's just not right the two questions were just impossible so we moved that up so we added 6 points to everywhere now the high score is 100% and here I'm showing you including that adjustment that everybody who took this exam received I'm showing the overlay of how people did last year and this year the averages when you make that adjustment turn out to be identical the justification was possible questions but as it turns out the averages are rock solid good but you and only you received the added bonus of having the sapling which defers some of the pressure that you need to do well on the exam so actually after the second exam you're doing better than they did last year and it doesn't feel like that I feel like I definitely challenged you not to do that again but you guys did well considering it was very difficult to exam Sarah can do regrades I don't know if she has something to say