Lecture 11

1) Central Dogma: DNA is replicated(replication) to make copies of DNA. It is read out(transcription) into the intermediate RNA, and then it is translated into protein.

2) DNA Replication: How do you demonstrate DNA replication? Take a DNA w/o bacteria, and show that you can copy it. Crack open the cell and purify an enzyme that can copy the DNA. Which cell? E Coli, its a bacteria, therefore simple.

3) What do we do? Add materials to testtube containing DNA? What else should we add? Nucleotides, since we know it is made of nucleotides. We’ll add some dATP, we’ll add some dGTP, dCTP, dTTP, all together known as dNTPs.

4) What else? We want to copy DNA, so put in a DNA template strand. We add enzymes and we hope that that is going to copy the DNA. But that’s too optimistic. In order to copy the DNA, its helpful to give it a start. So we also add a short complementary primer strand, with the hope that he would be able to purify an enzyme, which may not be able to start the synthesis of DNA, but would be able to extend the synthesis of DNA.

5) The primer strand is 5′-TpApCpGpTpA. The template strand is 3′-ApTpGpCpApTpTpApGpGpC – 5′

6) What is the enzyme expected to do – catalyze the addition of a triphosphate to the growing end of the DNA chain, the 3′ end. Where is it going to get the energy from? From the dehydration synthesis and breaking of the triphosphate bond. This hypothetical enzyme that can polymerize DNA like that is called polymerase. Note that the replication always goes 5′ – 3′. No one has ever found a DNA polymerization system where it goes the other way.

7) In the other case, the triphosphate would have to be on the long growing chain. Why do you care if the triphosphate is on the long grown chain or the monomer? The high energy triphosphate bonds are unstable. What if they should just spontaneously hydrolize? Its no big deal if one of the monomers hydrolizes from a triphosphate to a monophosphate, you can always find another.

8) What happens in an organism? It has a long chromosome and DNA replication is going along on this chromosome. Btw, where does the primer come from? What enzyme makes it? Primase.

9) For one strand its fine. But for the other strand, since replication can occur only one way, how does it replicate? Since the DNA is opening in the wrong direction for it. What it does is it creates new primers as the DNA opens, and polymerizes them little till it reaches the next primase. But how does it covalently ligate (join) them together. Turns out this is done by an enzyme called Ligase. This model has been experimentally proven. The slower strand is called the lagging strand, and the other is called the leading strand. The lagging strand tries to catch up with the leading strand. The fragments are called Okuzaki fragments.

10) Take a long chromosome. Even assume its circular, like bacteria have, imagine trying to replicate this. We are going to have interlaced double helices. There’s no way w/o cutting to separate them. It cuts it. You’ll need to cut the DNA, pass it through the other side. They are topoisomers. What enzyme does that? Topoisomerase. Drugs that inhibit topoisomerase are therefore good anti-cancer drugs.

11) Fidelity of DNA replication: Why don’t we put any other base, why just the right one? Because there is an energetic difference, between the right base and any base. If I know delta G, I know equilibrium constant, so I know how often DNA polymerase makes a mistake. Keq = 1000. So DNA polymerase gets it right 99.9% of the time. This is terrible. A typical gene is more than a 1000 letters. So we are going to make a mistake on an average on every gene.

12) So there’s proof reading. Two kinds of DNA proof reading that go on. DNA polymerase itself has a proof reading activity. Whenever DNA polymerase adds a base, it also has an activity that’ll remove a base. It doesn’t just add bases forward, it has exonuclease activity that removes bases backward. It adds more than it subtracts, and if there is a mismatched base, its much more likely to be subtract then add. Presence of a mismatch induces the enzyme to do its removal more than if it was a match. Therefore one error in 10^6.

13) Then there are DNA mismatch and repair enzymes that come after polymerase has done its job. They feel the DNA, any mismatch creates funny structures, they chop out some sequence, and that is then replaced. Now you can get down to one error in 10^8 bases.

14) How does it know which strand is wrong and which is correct? You leave some mark on the old strand. Bacteria do that. Methylation enzymes mark the old strands, and it takes time for the methylation enzymes to mark the new strands, and that leaves some time between.

15) About 1 person in 400, is heterozygous for the mismatch repair enzyme genes i.e. genes that encode the mismatch repair enzymes. What if you lost one of your copy of the repair enzyme gene? No problem, other will do the work. What if you lost both? High mutation rate, and cancer. A colon cancer is caused by mutations in the gene encoding the mismatch repair enzyme. Speed of a DNA polymerase is 2000 nucleotides/sec.

16) Cornberg’s enzyme, is it actually the right enzyme? Is it the enzyme the cells use to copy the DNA? A biochemist would say yes. A genetist would say, take out the component, and demonstrate the cell can’t replicate. 

17) They took many mutant bacteria, one at a time, they grew them up, and they did Cornberg’s purification to purify DNA polymerase. This is unbelievably tedious. Take each one, purify it, get DNA polymerase, ok its there, next one, and so on. Suppose you found a mutant that couldn’t make Cornberg’s DNA polymerase, but still grew? That would prove Cornberg’s enzyme is not necessary.

18) Cornberg’s enzyme although it can replicate DNA is not the enzyme that cells use to replicate their DNA. It turns out to be a relatively minor enzyme used to fill in gaps. The actual enzyme is DNA polymerase III. So this duality b/w biochemistry and genetics is very important.

19) Transcription: Where do we start on the DNA? Somewhere on the DNA there is some information, we want to make a copy of that information. Where to start? There’s a sign that says start. Such a thing is called a promoter. The promoter says here is the place to start copying the DNA into RNA. It gets an enzyme that starts at the promoter. What’s the difference b/w DNA and RNA? deoxy. That deoxy is important, otherwise the hydroxyl would interfere with the making of the double helix in the DNA. RNA doesn’t make the double helix.

20) What’s the difference b/w T and U? T has a  methyl group.

21) DNA is used as a template to copy a strand of RNA. Some important names – the strand that is being transcribed is called the transcribed strand. The other is called the non-transcribed strand. The transcribed strand is also called the non-coding strand, because the RNA contains the code of the non-transcribed strand.

22) How does it know where to stop? Stop signal, that says stop of transcription.

23) Orientation of genes along the chromosome, which way you read, is not a fixed thing across the entire length of the chromosome. So when I say transcribed strand, that’s just a local definition, that says wrt that gene, this strand is coding and this strand is non-coding. How does RNA polymerase know when to turn on a gene? How does it turn on the right genes on the right tissues? That’s gene regulation.

24) Translation: We take our RNA, what’s the direction its been copied? 5′-3′. Single stranded molecule. How is this RNA interpreted? On an abstract sense, it is interpreted by a triplet code – 3 letter codons. Does it start anywhere? It always starts on the same codon. It always starts at AUG, this is the initiator codon, and it encodes a methionine. The interesting challenge in the world is how do you get from a sequence of nucleotides to a sequence of amino acids?

25) Transcription is easy due to matching. How are we going to get amino acids to match specific RNA sequences? Initial ideas were physical. The RNA message would fold into some kind of a funny shape, that would just happen to match a lysine – Amino acids would be directly read off the RNA message. But amino acids have so many different properties, it just didn’t make sense.

26) Francis Crick said when I want a certain amino acid into a growing protein chain, I’m going to build an adaptor molecule b/w amino acid and the codon. Turned out there was an adaptor molecule which was made itself out of RNA, called transfer RNA, and tRNA matched up by base pairing to each codon, and had amino acids attached to it. How do the amino acids get stuck onto the right transfer RNAs? There’s a bunch of enzymes that do precisely that job, they look at the tRNA, attach amino acid.


Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google+ photo

You are commenting using your Google+ account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )


Connecting to %s

%d bloggers like this: