Lecture 13

1) Variations in bacteria, viruses and eukaryotes – bacteria have circular chromosomes, eukaryotes have linear chromosomes. Today we’ll talk about variation but not between organisms, but within organism from time to time and place to place. How some gene activities are turned on on some occasions and turned off on other occasions. This is very important for a multicellular organism like you, since it makes sure the same basic code is doing different things in different cells. Its also important for a bacteria to make sure its doing different things at different times depending upon its environment.

2) Which are the steps in the central dogma where you would like to regulate the activity of a gene. Could you regulate the activity of a gene by changing the DNA encoded in the genome? It does happen, not very common though.

3) Levels of regulation – DNA rearrangement. Your immune system creates new genes by rearranging locally some pieces of DNA. Some bacteria decide whether a gene should be on or off, by actually flipping around a segmenet of DNA in their chromosome.

4) The most common form is at the level of transcriptional regulation, whether or not a transcript gets made or how its processed is different. a) Initiation of mRNA – RNA polymerase should sit down at this gene, on this occasion, is a regulatable step. You only turn on alpha globin and beta globin used to make haemoglobin in red blood cells. b) Splicing choices that you make wrt your message – skip an exon say. c) regulate at the level of mRNA stability, persistence of the message, degradation of the message. In some cells, the message is protected so that it hangs around longer.

5) Regulation at the level of translation – If I give you an mRNA, is it automatically going to be translated? May be the cell has a way to sequester (wrap it up) the mRNA in some way so that it doesn’t get to the ribosome in some conditions. There are an exciting new set of genes called microRNAs, that encode 21-22 base pair segments, that are able to pair with a mRNA and interfere in some ways partially with its translatability. By the no of microRNAs, an organism can control how actively a mRNA is translated.

6) Post translational control – once a protein is made, the protein is modified in some way – the protein is inactive unless you put a phosphate group on it, and some enzyme comes along and puts a phosphate group on it. Or its inactive until you take off the phosphate group.

7) Every step can regulate whether you have a biochemical activity of a certain amount at a certain time, and every one of them is used. What we think is the most important, is initiation of mRNA. The fundamental place where you are going to regulate whether you are going to have the product of a gene, is whether or not you bother to transcribe its RNA.

8′) The amount of protein that a cell might make varies wildly. Your red blood cells, 80% of the protein they make is alpha or beta globin. That’s not true about any other cell.

9) Simplest case – gene regulation in bacteria – Lac operon in EColi. EColi likes carbon, it likes sugar, and its favorite sugar is glucose – for glycolysis. If there is no glucose, it can work on other sugars. It has a variety of pathways, that will shunt other sugars to glucose, which will allow you to go through glycolysis. Suppose you give it lactose, its a disaccharide, where you have got a glucose and a galactose. EColi is able to break lactose into glucose and galactose. It does that by a particular enzyme called beta-galactocytase, which breaks down galactocides, and it will give you glucose + galactose. How much beta-galactocytase does a Ecoli cell have around? A lot, when there’s only lactose around. 10% of protein can be beta-gal,when you have lactose but no glucose. When you have glucose around, you have almost none.

10) Why have this complex mechanism? Why not always have 1%, like a insurance policy, so what if your digestion is slow when there’s lactose around, life’s long. Competition. If the other bacteria has a mutation that allows it to make 4 times beta-gal, it will suck all the lactose. So if Ecoli has 0% when glucose, and 10% when none, you can conclude that it has worked that out through pretty rigorous competition. It doesn’t waste energy when you don’t need it, and when you do need it, it can compete hard.

11) How does it get the lactose in the cell? It has another protein which is a lactose permease, that makes the cell permeable to lactose, then the beta-gal can break the lactose down. Both these proteins, beta-gal and lactose permease get regulated.

12) How does it work? Structure of Lac Operon: What’s an operon? In bacteria you often made a transcript, that had multiple proteins encoded on it. A single mRNA could be made, and multiple starts for translation could be present, and this would be a good thing, if you wanted to make multiple proteins that were part of the same biochemical pathway. Such an object, a regulated piece of DNA, that makes a transcript encoding multiple polypeptides, is called an operon, because they are operated together.

13) The lac operon has a promoter, and we’ll call it p-lac. The first gene is given the name lacZ, it happens to encode beta-galactocytase enzyme. Remember, they did mutant hunt, and they didn’t know what each gene was for, so they gave them names as letters. Next is lacY, that encodes the permease. There is also a lacA, and that encodes transacetylase. There’s another gene, before the promoter that’s called lacI, and it too has a promoter, which we can call pI, and this lacI encodes a very interesting protein. So this is monocistronic, the p-lac region is polycistronic, cistrons is the name for these regions that were translated into distinct proteins.

14) lacI encodes a very interesting protein which is called the lac repressor. The lac repressor is not an enzyme like beta-galactase, its not a self-service channel for putting in lactose, it is a DNA binding protein. It binds to DNA. It doesn’t bind to any DNA, it has a sequence preference. It particularly likes to recognize a specific sequence of nucleotides, and binds there. What is that sequence where this likes to bind? It binds after the p-lac promoter, and this is called the operator sequence, or the operator site. It overlaps the p-lac promoter site. Who likes to bind at the promoter site? RNA polymerase. What’s going to happen if the lac repressor protein is sitting there? RNA polymerase cannot bind.

15) What will happen in terms of the transcription of the lac operon? No mRNA. Are we done? No, sometimes we want to make beta-galactocytase, so we want to get the repressor off from there. When do we want it to come out? When there’s lactose present. So somehow, we need to build a sensory mechanism, that is able to tell if lactose is present, and send a signal to the repressor protein, and it comes off. What signal?

16) How about using lactose itself? If lactose binds to the repressor, it will fall off, because its more interested in the lactose, than in the DNA. How does this interest come about? The liking for lactose, may change some energy, some shape, so that it bends around in ways so that it can no longer bind to the DNA. What happens is allo steric change, which means other shape, so it just changes its shape on binding of lactose, and falls off. So in the presence of lactose, lacI does not bind, and the lac operon is transcribed.

17) But how is lactose going to get in, because the lactose permease is made by the same operon? Instead of having this strong repressor, what if build some sloppy repressor that occasionally falls off, and occasionally allowes transcription of the lac operon, then we’ll have some trace quantities of permease around. And even if we have a little lactose around, it will shift the equilibrium so that the repressor is off more, that will make more permease and so on. So instead of having no mRNA when repressor binds, have very little mRNA. So we have lacON and lacMostlyOff.

18′) How do we know this? Mutants at lac operon: Remember this is before the times of DNA sequencing, they wanted to collect mutants, that affected this process. In order to collect mutants that screwed up the regulation, they knew that beta-galactocytase was produced in much higher quantity if lactose was around.

19) Wild type Ecoli, when you had no lactose would produce very little beta-galactocytase, say 1 unit, and in the presence of lactose, would produce a lot, say a 1000 units of ß-galactocytase. The problem with playing around with this is, lactose is playing two different roles. Lactose is both the inducer of the expression of the gene, by virtue of binding to the repressor, but it is also the substrate of the enzyme, as beta-galactocytase gets made, it breaks down the lactose, so there is less lactose. If you wanted to really study the regulatory controls, you have the problem that the thing that is inducing the gene by binding to the repressor, is the thing that’s getting destroyed, by the product of the gene. So its going to make the kinetics of studying such a process really messy. It would be very nice if you could make a form of lactose, that could induce ß-galactocytase by inducing to the repressor, but wasn’t itself digested. Chemically, you can do that. You can make a molecule called IPTG. This can induce ßgal, but its not a substrate, it won’t get digested. It is also convenient to use a molecule called XGAL, this is not an inducer, but it is a substrate, it will be broken down by the enzyme, and when its broken down it turns blue. These two chemicals turned out to be very handy in studying the expression of the lac operon.

20) Instead of adding lactose, if I add IPTG, I’m going to get ßgal, when I don’t have IPTG, I won’t get ßgal, but then I don’t have the problem of this getting used up. What kind of a mutant might I look for? I’d look for a mutant, that in the absence of the inducer, IPTG, still produces a lot of ßgal. I can also look for mutants that no matter what do not produce ßgal – what would they likely be? They would be structural mutations affecting the coding sequence  of ßgal. But that’s not as interesting as mutations that block repression – that cause ßgal to be produced all of the time. How would I find such a mutant? I want to find a mutant that makes a lot of ßgal, even when there’s no IPTG.

21) Put Ecoli on a plate, and don’t put IPTG. How do I tell if anybody is producing lot of ßgal? Put XGAL, and if anybody is producing a lot of ßgal, they turn blue. So mutants were found that were constitutive mutants – means they were expressing all the time, no longer regulated.

22) Characterising these constitutive mutants: They fell into two different classes. Mutant no 1 are operator constitutive – they have defective operator sequence. Mutations have occurred at the operator site. Mutant no. 2 have a defective repressor protein, the gene for the repressor protein.

23) How could I tell the difference? – some mutation in the operator site causes the repressor protein not to bind there anymore, defective repressor also doesn’t bind even if the operator site is good. One way to tell the difference is by crossing them to wild type, and asking whether they are dominant or recessive and things like that. The problem is EColi is not a diploid. So you cannot cross two EColi and make a diploid EColi, its a prokaryote, it has only one genome. But turns out you can make temporary diploids, you can make temporary diploids, partial diploids out of EColi because turns out you can make bacteria, which have a bacterilal chromosome, also engage in sex, and in the course of bacterial sex, plasmids can be transferred, for eg – an F-factor is able to be transferred from another bacteria, and through the wonders of partial mero diploidy, you can temporarily get Ecolis that are partially diploid.

24) Suppose the repressor is wild type, the operator is wild type, and the lacZ gene is wild type, and suppose I have no IPTG, then I’ll have 1 unit of ßgal. When I add inducer, I get 1000 units of ßgal. Suppose I had a operator constitutive mutation, then the operator site is defective, ßgal is going to be expressed all the time, even in the absence. Suppose I made the following diploid – I+O+Z+ over I+OconstitutiveZ+. What would be the phenotype? In the absence of IPTG, 1001 ßgal, and in the presence of IPTG, it would be 2000 ßgal. So the operator constitutive site looked like it was dominant.

25) Lets try I+O+Z+ over I+OconstitutiveZ-.  In the absence of IPTG, 1 unit, but in the presence, 1000 units. What if I reverse, I+O+Z- over I+O-Z+, then in the absence, 1000 units, in the presence 1000 units. From this experiment, you can tell, that the operator site is only operating the chromosome it is physically on, that it doesn’t make a protein that floats around, it works in cis (cis means on the same chromosome).

26) Lets look at the lack of the repressor mutants – I+O+Z+ over IconstitutiveO+Z+, in the absence, 2 units, in the presence, 2000 units. note this. If I give you I+O+Z- over IconstitutiveO+Z+, then in the absence, 1 unit, in the presence 1000 units. As long as I have a working copy of repressor, it will work on both chromosomes. It makes a product that diffuses around, and it is said to work in trans i.e. across.

27) Operator works in cis, mutation on the operator only affects the chromosome it lives on, whereas a functional copy of the lac repressor will float around because its a protein, that’s how they knew the difference. They proved their model by proving that these 2 kinds of mutations had very different properties. Operator chromosome only affected the physical chromosome on which they occurred, whereas a functional copy of the repressor could act on any chromosome.

28′) What about glucose? Glucose Control: When lactose its present, operator comes off, and RNAP sits, but wait! RNAP is not supposed to sit down if glucose is present – we need another sensor to tell if glucose is present or not, or if there is low glucose. There is another site before the promoter, on which a completely different protein binds, and this protein is the cyclic AMP regulatory protein. In the cell, when there is low amounts of glucose, we have high amounts of cAMP. Whereas lactose is used directly as the signal, cAMP is used as the signal here. cAMP helps RNAP, instead of being a repressor, its an activator, it makes it more attractive for RNAP to bind. Just like the repressor wasn’t perfect, the promoter isn’t perfect, and unless RNAP gets help from the cAMP, it doesn’t work. wow

29) We have 2 controls – a negative regulator responding to an environmental cue, a positive activator responding to an environmental cue, helping polymerase decide whether to transcribe or not, basically that’s how a human egg goes to an adult and lives its entire life, minus a few other details. That’s a sketch of how you turn genes on and off.

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