DNA goes to RNA goes to protein.
And DNA goes to itself.
DNA is replicated, it makes RNA, and RNA is used to make protein.
So the first step of that is “How does DNA give rise to more DNA?”
Well… How do you find an enzyme? How do you do biochemistry?
You’ve got to grind up the cell. You’ve got to choose a cell in which you are likely to find an enzyme, grind it up, break it into different fractions, and test each fraction. That’s all biochemists do, right?
So what cell might have the enzyme we’re looking for? What cells might be able to copy DNA?
How about ALL CELLS
Lets use a simple cell. What’s a simple cell?
Lets use bacteria!
So lets take some bacteria. We’ll grind it up. Fractionate it into different fractions. We’ll see if one of those fractions has the ability to copy DNA.
If we’re going to run an assay, we have to give it a substrate. What substrate would you like to give it? What do you think it needs?
It better have some free nucleotides. Otherwise how could it make DNA?
What else? Are you going to ask it to make DNA all by itself?
We want something that can copy one of the strands of the double helix. So what should we give it?
Half a helix. A strand of DNA. The strand to be used as a template. So lets give it a template strand.
So we’ll take a template strand of DNA. Here’s my template strand of DNA:
It has a sequence in there. A’s, T’s, G’s, and C’s, each with a phosphate.
G – phosphate, A – phosphate, T – phosphate, A – phosphate, A – phosphate, A – phosphate… I won’t write them any longer.
Each one’s got a phosphate in there. That’s the way it goes.
Alright. That’s the template.
We need floating around in the solution: some trinucleotides. Okay we’ve got some nucleotides floating around.
And now will this enzyme work?
We have an enzyme. We’ll try different fractions and see if it’s able to just install the right letters in the right place.
No. It turns out it needed one more thing. And the person who discovered this, Arthur Kornberg, thought of it.
It needed a head start. It needed a primer.
So the primer goes phosphate – C, phosphate – T, phosphate – A, phosphate – T, phosphate – T, phosphate – T.
So this is the 5′ end of DNA.
Remember the phosphate is hanging of the 5′ carbon, right? What’s in the other end. Let’s see. It ends in the hydroxyl 3′ end of the ribose.
Since this is antiparallel. This strand is going 5′ phosphate to 3′ hydroxyl. You’re going to need to know 5′ and 3′.
So there you go. If you hand it a primer to give it a head start, and you hand it a template, and you hand it some nucleotides – you then assay different fractions, and see “is one of them capable of extending this strand by putting in an A, putting in a T, putting in a C, putting in a C, putting in a G, blur-dur-dup?”
And… Arthur Kornberg discovered an enzyme that could do this. And the biochemists went nuts. They thought, “Wow, this is so cool.” Kornberg was able to discover an enzyme that could accomplish this.
The enzyme polymerizes DNA. Coincidentally, what is the enzyme called?
DNA polymerase. Excellent.
Now, notice what it does. It takes this triphosphate:
Puts it in here:
And joins it into a sugar-phosphate chain.
Where does it get the energy for that synthesis?
Hydrolysis of the triphosphate, right?
It’s the hydrolysis of the triphosphate. That’s the energy.
What direction is the synthesis proceeding?
It starts here at the 5′ end
and it moves, adding to the 3′ end.
So it’s 5′ to 3′ direction. That’s the direction it moves. It adds to the 3′ end. Adds the free nucleotides to the 3′ end.
Why not do it the other way?
You see, suppose we were going the other way. Suppose the primer was this way: 3′ to 5′.
As we added each base, the triphosphate would be on this strand, right?
And we’d be adding to the 3′ end here:
That means the energy supplied by the triphosphate would be on the growing strand, rather than in the free nucleotides.
Why would it be a terrible idea to put your energy source on the growing strand?
You know those triphosphate bonds are pretty unstable. They hydrolyze by themselves at some frequency. If you’re a free nucleotide and the triphosphate hydrolyzes, big deal. That free nucleotide loses its triphosphate. But what if I’m the growing strand and I lose my triphosphate? Heh.. heh…. there goes my chain.
So you know, life’s not stupid. It doesn’t do it that way. It does it this way. No one has ever found a polymerase that goes that way. They find them all going this way for just that reason.
That was why life evolved it that way. Because you want your triphosphates – those hydrolyzable triphosphates – to be floating around freely rather than invest the energy.
Just think about that. It’s kind of a cool thing. It helps us remember which way it’s going and why it is, and how it is. And it’s kind of interesting.
Alright, so Kornberg wins the Nobel Prize for this.
Good stuff. Very deserved.
But you know, there’s some questions.
Where does the primer come from in life? Kornberg gave this test tube a primer. But suppose I’m replicating some DNA.
So lets suppose I have a double strand of DNA
Now just open it up here:
→5′ to 3′
← 5′ to 3′
I need to get, like, a primer here.
Then the primer can be extended by polymerase. Well, where does the primer come from?
It turns out there is an enzyme specially devoted to making those primers. Kornberg didn’t know it but there is an enzyme.
And by coincidence it is called, primase.
Exactly. Primase makes the primer.
You need a primer here, and the primer is made by primase. Once primase makes a primer, polymerase can chug along and do it just fine.
Let’s check out the other strand.
Primer here. Polymerase chugs along. But now as this double helix opens up, what happens over here?
The synthesis is going this way ←. So what do I have to do here?
Another primer. We need another primer.
Then as it opens up more, what do I need?
So the two strands are experiencing a very different kind of replication. In one case, one primer in the 5′ to 3′ direction is enough to keep going. In the other strand, as it keeps opening up, you’ve gotta keep making primers.