Lecture 12

1) We have a messenger RNA that has been transcribed from a specific region of the chromosome starting from a promoter and going to a stop signal and that mRNA will include some particular sequence. Ribosome comes and starts scanning this message for the place to start. It finds the first AUG. There are messages that don’t start at AUG, and there are also messages that don’t start at the first AUG, because the ribosome is looking for something very special.

2) How does it accomplish this matching b/w codons and amino acids? When it reaches UAG, its a signal for stop, don’t put anymore amino acids. There are 3 stop signals. How does the right amino acid get put on the right tRNA? There’s a dedicated enzyme for that. Its aminoacetyl synthetase.

3) How does this happen physically? It happens in this vast machine called the ribosome. In the ribosome, there’s a cavity for each tRNA-codon-amino acid. When the right tRNA fits in the cavity, the ribosome catalyzes a peptide bond between the amino acids. Then it moves the sequence by one step, until stop. Is there a tRNA for stop? No, there’s some protein factor. This is the smallest two tape turing machine.

4) Variations on the theme: How does this central dogma vary among different kind of organisms? Eukaryotes, prokaryotes, viruses. Prokaryote – E.Coli. Prokaryote has no distinct nucleus. Do they all do the same?

5) DNA Replication: a) Eukaryotes – What’s the structure of our chromosomes? Its a long linear chromosome. We have 23 chromosomes and together they make up 3e9 nucleotides. A typical chromosome is 150e6 bases long, its a single connected molecule. One tricky bit of replicating DNA. In replicating the lagging DNA, since we go reverse, the chance that we will get a primer at the end is pretty low, and if we don’t get it right, it’ll be short a little. That’s nothing here, but it’ll again be short in the next cell division and so on. Its tricky to replicate linear chromosome on a lagging strand. A special little solution is used. The ends of the chromosomes are called telomeres. These have very specific structures. In the humans they repeat- TTAGGG again and again and again. There’s a special enzyme that’ll come along and add some extra telomere to the chromosome. What’s the enzyme that adds telomere, telomerase. What cells need to have telomerase – rapidly dividing cells. Cells that have stop dividing can shut off their telomerase. Cells that need to divide lots and lots of times, need to tidy up its telomerase. What cells care about having telomerase on? Cancer cells.

6) Prokaryotes differ because their genomes are not linear chromosomes. The typical prokaryote chromosome is a double stranded circular DNA. It doesn’t have this problem of telomeres, you just keep replicating around and you get to the end. You have much smaller genomes too – few million bases. Humans also have 1 circular chromosome, the mitochondria have their own chromosome, its a circle. The mitochondria arose as a symbiolic bacterium, that became a symbiot of eukaryotic cell. Because it lives in your cell, it has thrown out all its genes that it doesn’t need anymore, so the mitochondria genome is 16000 bases long.

7) What do viruses have? Some viruses have double stranded linear, some have double stranded circular. Some have single stranded circular DNA. They inject this into the cell. As soon as it gets into the cell however, its replicated to make a double stranded DNA which can be transcribed and so on. But it travels around as a single stranded DNA.

‘8) Some viruses actually consist not of DNA at all but of RNA, single stranded RNA. When it gets into the cell, what does it do? How does it convert to DNA. Reverse transcriptase. Where does it get this reverse transcriptase from? You yourself encode it on your RNA. You might decide to put in the genetic code for reverse transcriptase, and when that message gets into the cell, it will first act as an mRNA translate the reverse transcriptase enzyme, which will then reverse transcribe the the RNA into DNA. This is a +-strand virus, it encodes its own reverse transcriptase instructions on its RNA. There are minus strand viruses, which don’t have this, what they do is, in their own package bring in your own reverse transcriptase. RNA thus converted to DNA, then to double stranded DNA, which can then be slammed into and inserted into your own chromosomes. What virus does this? HIV. More generally, retro viruses are the viruses that can run this process of converting from RNA to DNA, and install DNA copies into your genome. How do you then get the DNA copy out of your genome? You don’t, it doesn’t come out. We have to make sure that the virus is shut down by other ways by inhibiting its products, etc. but you can’t get its DNA copy out of your genome.

9) So you want to inhibit AIDS virus, you need to make inhibitors of this aspect of replication. Inhibitors of reverse transcriptase.

10) Transcription Prokaryote genome has some kind of a promoter that tells RNA polymerase to come sit down here. RNA polymerase starts copying, eventually it hits a signal that says stop transcribing. Note this is not a stop codon, that is for translation. This mRNA then goes off. Nothing special about proks.

11) Eukaryotes are different. It starts the same, there’s a promoter. RNA polymerase sits there, it starts transcribing. It stops. Then this RNA gets processed in interesting ways. At the 5′ end a funny modification is put on. A methyl G triphosphate is put on backwards. Gppp is put on at the 5′ end. This is called a cap. This is a signal for the cell saying this is a messenger RNA, get the ribosome on it, and so on. At the other end of the message, a long string of A’s is added. Its called a poly(A) tail. If you want to purify mRNA’s you can do so by using poly(T), because the poly(A) binds to it. The function of this A’s is to regulate the stability of the messages. If you don’t have the poly A tail, the message will be degraded rather rapidly. This poly A tail also acts like a clock that tells how long that mRNA sticks along.

12) But these are small modifications – The major difference is – only a small portion of the gene matters in order to make the protein. The mRNA gets made, it includes the whole sequence, and then the cell comes along and splices this message together. It is processed by clipping the unnecessary portions. That’s a mature RNA. This splicing is a remarkable phenomenon. Its a complex phenomenon. There’s a big body of enzymes that help accomplish that. Hence it’s not splicase, its splicosome. How does the splicosome know where to do this? There is information encoded in these messages. The sequence just at the end is a GU, and the sequence at each start is AG, but that’s obviously not enough information. We don’t have the complete information.

13) These bits that stay in are called exons, the bits that go out are called introns. This is confusing terminology. The introns are so named because they are intervening sequences. For a typical human gene, the length of the gene itself might be 30 thousand bases, but the mature mRNA might be 15 hundred bases. The clotting factor VIII gene, the gene that is mutated in individuals with haemophilia, that gene is 200 thousand bases long, and it gets spliced down to a mere 10 thousand bases. That’s nothing compared to Duschan muscular distrophy, that gene makes an immature mRNA of 2 million bases, its at it for hours. This gene is spliced down to 16 thousand messages.

14) Splicing mutations could be a problem, some errors could occur from errors in splicing. That does happen. There could be mutations that change a splicing, or create a new splicing, and all of that could screw up the gene.

15) Why do this? Why waste this energy? I might be able to encode multiple proteins with the same gene. One type of cell might splice up the gene one way, a different one might splice it another way, to produce a different protein. Alternative splicing could create multiple proteins. A typical gene in the human, has atleast two different splicings on average. How does the cell know to splice it one way or the other? We don’t fully know.

16) This is a huge overhead to do that, is it justified. There are other reasons- an evolutionary reason. Suppose a random event happens and a chromosome breaks. And suppose it sticks one part of the chromosome, to some other random part. If it lies in the code, that’s trouble, but if it lies in the intron, it created a new gene, that could still work. By having a random break b/w two genes in their introns, and slamming them up, you could make a gene that had a bunch of exons from one gene, and a bunch of exons from another gene, and it would get spliced up. Evolution might like that. Because it would be a very easy way to build new genes that would have a portion of one protein, and a portion of another protein. This kind of mix and match domain could be very useful. And when we look at a lots and lots of genes, we see genes that have similar first half but a different second half.

17) Why don’t bacteria have this? The metabolic cost is too high, it has to replicate every 20 mins, and all these extra bases would be bad news. Bacteria are more sophisticated than us because they are under rigorous evolutionary selection than we are. You might imagine that early life all had introns, and bacteria, competing to be more efficient got rid of their introns.

18) Small eukaryotes like yeast have introns, but very small in number. Only 5% of the genes have an intron. The bigger the genome size, the more the introns. The more pressure you are under to replicate rapidly, the less you can tolerate this interesting and complicated innovation.

19) Viruses depending on whether they are eukaryotic of prokaryotic do or do not have splicing.

20) Translation: Eukaryotes are simple. You get a gene, you get an mRNA, the ribosome goes to the mRNA, and it starts turning out one protein as it chugs along.

21) Prokaryotes: differ in an interesting way. I get a promoter that is transcribed into my mRNA. The mRNA can encode multiple independent proteins. Protein 1, 2, 3 simultaneously, and you have a poly cistronic message. Why have a single mRNA that encodes multiple distinct proteins each starting with its own ribosome start site? Efficiency. How about, make them multiple steps in a biochemical pathway? Have them encoded on a single messenger, so you’d have to only worry about regulating that once, you will make all the enzymes for the pathway. That’s exactly what bacteria do, they put all enzymes for a single pathway on a single message, so when they say, lets digest hexose this morning, they have all the ingredients on a single RNA. That’s because they are small genomes, they are pressed for space. This single unit, that has multiple genes encoded in a single message, is called an operon.

22) Viruses: Viruses have very little room, their genomes are tiny. A typical virus might have a genome of 5000 bases to 10000 bases, to 200 thousand bases. It wants to pack a lot of protein coding information in, and so viruses have come up with the most extraordinary way of doing that. Some viruses have gone to the extreme of having RNA’s that get made from them, and they can use the same sequence in 3 different reading frames, and get different proteins. In a prokaryotic or eukaryotic, only one of the reading frames is used. This is an extraordinary packing of information density.

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