01.09 Nucleic Acid Metabolism
- Broad sense of metabolism
- Breakdown (salvage pathway)
- Synthesis (bases, nucleotides, strands)
- Nucleotide & strand structures
- How replication and transcription work
- Broad sense of metabolism
- Nucleic acid structure
- Purines: adenosine & guanosine
- Pyrimidines: cytosine, uracil & thymine
- 5-carbon sugar (pentose)
- Carbon naming system uses “primes”
- DNA is 2′ deoxy (i.e. no O at this position)
- Mono-, di-,tri
- Phosphate names: alpha, beta & gamma
- ATP is an example; NTP is any of the 4 bases
- The sugar-phosphate backbone
- Basepairings: A w/ T (or U); C w/ G
- Anti-parallel means one strand (reading left to right) is 5′ to 3′ while the other (reading left to right) is 3′ to 5′
- The double helix structure is crucial for understanding how DNA & RNA are synthesized
- Replication is DNA synthesis
- Unwinding of the chromatin
- DNA polymerase is the enzyme that catalyzes replication
- Leading strand vs. lagging strand synthesis
- Both occur 5′ to 3′ at the same time at the same place
- Lagging strand synthesis requires it to loop back on itself
- Transcription is RNA synthesis
- Unwinding of the chromatin
- RNA polymerase is the enzyme that catalyzes transcription
- Strand synthesis
- An RNA copy of a gene is made
- The RNA basepairs with the “antisense” strand and is an exact copy of the “sense” strand, except that U’s replace all T’s
- Occurs 5′ to 3′ and creates a single strand of RNA
- Polymerization reaction–what 5′ to 3′ means
- The 3’OH at the end of a strand attacks alpha phosphate of an NTP
- Pyrophosphate (PP) leaves thus creating a bond between the strand and a new nucleotide
- Nucleotides are composed of a purine or pyrimidine base attached to a ribose and the ribose can then have 1-3 phosphates attached to it; and strands are composed of an alternating sugar-ribose backbone; bases of a strand can interact in an antiparallel manner
- Replication is DNA synthesis; is catalyzed by DNA polymerases; occurs via simultaneous leading and lagging strand synthesis
- Transcription is RNA synthesis; is catalyzed by RNA polymerases; creates a single strand of RNA that has the same sequence as the sense strand of a gene.
- The polymerization reaction involves the 3’OH at the end of a strand attacking a nucleotide such that the nucleotide gets attached to the strand, releasing pyrophosphate (PP)
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Hey there. What we’re going to talk about today is nucleic acid metabolism. And we’re going to focus on structures, replication, and transcription. When we say nucleic acid metabolism, some people are going to think that really what we’re talking about is how nucleic acids that we eat get broken down and how they get synthesized. And this is definitely interesting. Breakdown can happen through what’s called a salvage pathway and synthesis can happen. Little small molecules can be used to create the, A, the C, the G, the T, and U et cetera…fascinating biochemistry here. But that’s not what I think is probably most useful for you at this point. What I’m going to focus on are nucleic acid structures, and the synthesis I’m going to talk about is replication…that’s going to be DNA synthesis and I’m going to talk about transcription, which is RNA synthesis.
Let’s start by looking at a nucleic acid structure. These things are the bases and the bases are when we talk about an A or a G or a C or a U or a T. These are those structures and they fall into two categories. Notice these guys, these are the purines and they have two rings. And the pyrimidines have just a single ring. One of the ways I try to memorize these is that the longer word is the smaller molecule and the shorter word is the bigger molecule. So it’s kind of like a reverse-memorization sort of thing. So these are the things that are the bases and they go right here on the ribose. This in yellow is the ribose and the bond that connects the ribose to the base is a special bond called the glycosidic bond. Any of these bases can go on there.
One of the things I want you to note here with uracil look how it’s almost exactly the same as thymine, except that thymine’s got a CH3 at this position. U is found in RNA and T is found in DNA. That there is one difference between RNA and DNA. But also over here, notice that this position…if we’ve got an O here, then that’s for RNA, the oxy. And then if there’s no O, then it’s for DNA, which is then deoxyribonucleic acid. Ribonucleic acid is RNA. Deoxyribonucleic acid is DNA. You will only find a T in the DNA and you’ll only find the Us in the RNA. Okay. Actually the truth is, you do sometimes find Us in DNA, but it’s an indication that something’s gone wrong if there’s a U in the DNA.
Something else to note about this ribose…notice the numbering. We call this one here the 2′ position, 3′, 4′, 5′. All of the numbers–these carbons–they are indicated with a number followed by prime. If we look at the bases, these things don’t have primes on them. It’s just one, two, three on around. So we use prime to indicate that we’re talking about the ribose. You’ve probably heard about 5′ and 3′. That’s really important in the synthesis of DNA and RNA, so keep track of these two positions on the ribose. Up to this point, this is what we call a nucleoside. I’m just going to do a little abbreviation there, …~side, if you’ve got just a base and a ribose. With this OH over here at the 5′, it would be called a nucleoside. If you start putting phosphates on it, you’ve got a nucleo-tide (~tide), and you could have one, two or three phosphates coming off of this 5′ position. These guys are labeled. This would be considered the alpha (phosphate)…this is the alpha position. This is the beta position. And then this is the gamma position for the phosphate. You can have phosphates coming off of a 5′ OH. You can similarly have phosphates coming off the 3′ OH. When we draw a strand…which would be a phosphate followed by a base and then a phosphate followed by a base…each of these phosphates…like this guy right here…that is between the two bases. That means you’d have one base here and then a phosphate coming off of this, and that would hook into the 5′ of the next nucleotide down, and with its base. It’s kind of complicated, but this phosphate is sitting in between…it’s connecting to the 3′ OH of one ribose to the five-prime OH of the next. I’ll try and draw you an example of that and a little bit.
Let’s look at what strands are. So a strand can be either DNA or it can be RNA. And in this case, I’ve got a double helix. It could be a DNA double helix…we can think of it that way. It’s got a sugar phosphate backbone. That’s what the orange and the purple ribbons are…a sugar phosphate backbone. That means that we’re alternating ribose, phosphate, ribose, phosphate, and then off of each of those riboses is going to be a base. That’s what each of these is made of.
Now notice we would have the 5′ end over here and then the 3′ end over here. We can just draw those in. Let’s call this one the 5′ end of the purple side and this is the 3′ end over here. To make a double helix, you have to have anti-parallel interaction…anti-parallel interaction…between the two strands. That means that the orange strand’s 5′ end is over here, and it’s 3′ end is here. That’s what’s meant by anti-parallel. In the center, this is where the base pairing happens. So As pair with Ts and they do it through two hydrogen bonds. So I’ll just do two over there. Cs pair with Gs through three hydrogen bonds. You’ll notice everywhere here, it’s a blue and a red, blue and a red, red and a blue, blue and a red. Or it’s going to be the green and the yellow, green and the yellow. We can tell that this is DNA because we don’t even have a code here for the U. So this is T that’s in it. And so that’s how we know that this is DNA. That’s how the base pairing happens in the center of it. Honestly, it’s such a beautiful structure. I mean, it’s just a really, really neat thing. And you know, pretty much every organism has some kind of DNA blue print to it. So it’s pretty neat.
Now let’s take a look at replication. Like I had said, this is DNA synthesis. We start with a chromosome. Chromosomes are–we have 46 of them–they are amazingly wound up. That’s how many we have: 46 chromosomes in a single cell. If you were to pick one of your cells and take all 46 of those chromosomes out and unravel them, they honestly are going to extend about a meter in length, which is amazing. That’s your wingspan…if you put your arms out. All of that DNA needs to get wound up and wound up the wound up and wound up and wound up in order to fit inside of a little tiny cell. It’s really thin, you know, like you can’t see it, but it’s really long, strangely. What has to happen is that the DNA needs to get unwound so that we can have exposure of those bases that are on the inside. That’s how a DNA polymerase enzyme can come along and read that sequence and make a new copy of DNA. The way that it does that…the enzyme comes in and binds, and it is able to bring in these nucleotides. We had talked about A, C, T and G, I’m going to give you a little abbreviation here. We’re going to call those NTPs. And that N could be either a C, a G, or an A, or a T. So that’s the N on there, but they all have the triphosphate on them. And they’re going to come in and match with whatever base is present right here at the end. So in this case, we can look off the code and see that there’s an adenine there. And so this is going to be thymine in orange, coming in and base pairing with that A. And then the polymerase is going to carry out a reaction that allows this growing strand right here to add one more nucleotide onto the end of it. I’ll try and show you the chemistry of this in a little bit. So that’s how that part happens.
Now, this is what’s called the leading strand and it’s kind of straight forward. We’re going from 5′ to 3′, polymerase is moving in this direction, with the red arrow here. Simultaneous to this, we’re going to be reading the other strand and making a copy of it. Here, polymerase again is being shown. And this is also going from 5′ to 3′. And we’re going to bring in a red guy to pair with the orange guy; a green guy to pair with the blue guy, et cetera. Now, the weird thing is these are the same protein. So it’s one and the same protein…it’s just hard for people to draw it. But it basically means that this lagging strand here…lagging strand…needs to be looped back around on itself so that both of these are happening in the same direction, and so that the polymerase can move together in the same direction. Again, this one is going to be looped around to go the other way. I’ve put a video in the links section. It’s a really cool video that gives you a better idea of what that looping around must look like.
Let’s look at transcription now. It’s somewhat similar, but remember this is going to be now RNA synthesis. Same idea…you’ve got wound up chromosomes, they need to get unwound. And then we’ve got an RNA polymerase as the enzyme in this case, that’s going to be carrying out the reaction. Otherwise it’s not all that different. A CTP is going to come in and it’s going to base pair here with that G and the C is going to get put there. And then a G is going to get put here to pair with that C and then likewise, we’re gonna do a C and a C. So this enzyme is again, catalyzing from 5′ to 3′, and it’s adding these nucleotides onto the end based on what the sequence is here in the gene that you’re wanting to express.
So let’s take a look at what happens during the actual chemistry of the polymerization reaction. What I’ve got drawn here is a strand, and we’re going to add a nucleotide onto the end of it. The way I’ve drawn it…this is essentially DNA because there’s no O sitting here at that position. So get that idea of it kind of…it’s a long strand…and this would be the 5′ end way out here. What we’re looking at here is that 3′ end. Each ribose in the strand has a 5′ and 3′. Here’s a 5′, here’s the 3′, then here’s another 5′. But what it means is for each nucleotide, it runs from a 5 to a 3 from a 5 to a 3. So when we say 5′ to 3′ synthesis, really the business end is here at that 3’…that’s what we’re looking at here.
Let’s go ahead and make an enzyme active site around this OH…this is the polymerase. And what it’s going to do is it’s going to have some base (an amino acid side chain) inside of its active site that’s going to grab onto this H, and what that does is, it leaves these electrons here free to go and attack. There’s a slight positive charge here on these phosphates, each one of them. The enzyme active site is going to orient an NTP so…in this case, the nucleotide triphosphate is a G–we’re going to add that onto the C that we have here…so these electrons are going to be attracted over here to the alpha phosphate…remember, this is the alpha one…and that’s going to cause this bond here to break. So that’s going to release pyrophosphate. And in the process, we are now going to have a bond that goes from this oxygen over to this phosphate, which now is connected to this ribose, which is connected to this G. So we’re essentially taking an A and a C and now we’re adding a G onto it. And then we’ll add the next one on as the polymerase…as we bring another NTP into the active site, and then the polymerase does the same thing on this particular 3′ OH. So this is how the chain elongation happens.
Now, one thing I do want to point out there’s this pyrophosphate that goes on to get broken into two phosphates, and this is really the driving force of the reaction. So this is really what makes life happen, which is I think really cool to think about thermodynamically. It becomes very irreversible. You can’t send this back in the other direction. And so this reaction goes, it goes, and that’s really why life happens. It’s because there is this chemical reaction that is very thermodynamically favorable to occur, and it pushes us forward into growing, which I think is kind of neat.
Here we are with the summary: We talked about structures and namely, we talked about purines and pyrimidines…those are the bases. We talked about ribose and how those have the prime numbers on them. And then we talked about phosphates, how you could have a nucleotide triphosphate with those three phosphates coming off the 5′ end, but you could also have phosphates going between each, off of the 5′ and the 3′, and that’s how you would make an actual strand. Now, we also talked about DNA synthesis and how it is catalyzed by an enzyme called DNA polymerase. And we talked about RNA synthesis and how it’s called transcription, and it is catalyzed by an RNA polymerase. And we took a look at the actual reaction…we looked at how 5′ to 3′ synthesis occurs, that the main thing is that there’s that 3′ OH that attacks a nucleotide triphosphate. And the result is that a pyrophosphate gets released and hydrolyzed, and that ends up being the driving force of the reaction.
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