[MUSIC] So in this second part of the introduction, we're going to look at the enzymes and the proteins that are part of the arabinose system. Arabinose has to be taken up by cells. Lactose, or glucose, or any other sugar. In the case of lactose, remember that the lactose permease, the product of the Y gene, is, in fact, the transport. In the case of a rabinose, there are two systems. There is a system which is called a high affinity system, but low capacity, and there is a system that is high capacity, low affinity. This system will work under different arabinose concentration on the outside. But we don't have to worry about that. These are the two system. FGH is the high affinity, low capacity. And E is the high capacity, low affinity. We're not going to discuss F,G, H, we are only going to discuss E in this paper. Arabinose gets in the cell, An arabinose once in the cell, will activate this protein RC to do a number of functions, activating functions, as well as repressing function. The main activating function is the induction of expression, transcription of the araB, araA, and araDG, the so-called bad opera, BAD. Those are the three enzymes that are necessary to use arabinose as a sole carbon source. Arabinose is first converted by araA Into L-ribulose. This is an epimeric that changes the position of the aldehyde group into a keto group. L-ribulose is then phosphorylated by a kinase, the product of the araBG. And then, the last enzyme sorry the first enzyme is an isomerase not an epimerase I'm sorry, all right, it's an isomerate then, the kinase and then, the epimerase that will transform L-ribulose into G cellulose phosphate. And this G cellulose phosphate will go into the central metabolism. Those are the only three enzymes necessary to use arabinose. RE's necessary for uptake, as well as FGH, which we will not discuss. AraC is only regulating the transcription of this gene, nothing else. Okay, now Schleif has worked most of his life on the arabinose system. Hence NLRSC, hence this love-hate relationship with the AraC protein. Schleif did one major experiment. He did an experiment that convinced, a biochemical experiment, that convinced finally everybody that AraC was a positive regulator of transcription. That was in vitro synthesis of the AraB enzyme, in a system, which is semi-purified, and makes protein, RNA protein. So he's worked, practically all his life on RSC. And RSC is a protein, which like many, many proteins, has two domains. It has an N-terminal domain that binds arabinose. The Schleif lab also showed that arabinose is the sugar that binds to the pocket, and a C-terminal domain that binds DNA. And these two domains are linked by a linker, and there's an N-terminal arm which is floating around. This is this AraC protein. In the absence of arabinose, this protein behaves like a repressor. So there is negative control. But it behaves like a repressor, because it binds to two element, 02 and i1. By binding to 02 and i1, it prevents expression from the PBAD promoter, the araBAD promoter. And it prevents expression of the araC promoter. So it turns off its own gene. Like lambda bound to R3 was turning off its own synthesis. Negative control. Once you add arabinose, this protein changes and becomes capable of binding next to each other, to these two sides I2 and I1. Arabinose here is represented by this red dot, and the terminal arm is closing the pocket. And that allows RNA polymerase to bind and initiate transcription. In addition there is an additional protein called Cap which helps transcription. So it looks like it is a little bit complicated. But it's not that complicated. The key issue is that there is a sight which does not exist in the lax system. And the site is called the eye site for inducer or inducibility. And, in fact, Englesberg, went on to isolate mutants in the eyesight. Now, his interpretation of the mutants, was that these mutants were helping the transition between this extended RSC confirmation, to disclose RCS confirmation. That was his interpretation at the time. That was wrong. He isolated many IC mutants. And eight of them were recovered 15 years later, sequenced, and in fact all these are IC mutants are promoter mutant for the BAD operand. They're not I mutant, they're promoter mutants, they're up mutants. And he, of course, he couldn't realize that, but they're compatibility with i mutant but in fact they are promoter mutants. So, you see that when you purely rely on enzyme assays and very simple experiments, you can reduce things that are perfectly correct, but the mechanism, the real mechanism may be quite different than what you think. Now it's still not known how the polymerase, RNA polymerase, RNA polymerase is represented here. This is RNA polymerase, this thing. RNA polymerase Is helped by CAP and AraC. It's believed that this help is multiple. One of the help is that AraC bind to I2, keeps the polymerase from unattaching to a poor promoter. That's one. The second is that CAP is touching, or talking to this little ball that is coming out of the RNA polymerase. This domain of protein. And there is a third interaction between this second ball, which is here. And nobody really knows what. Very recent experiments suggest that it in fact, this ball touches here, and not here. But this is really going into tiny details of mechanist. Now these experiments where- Or this model, was very much helped by some experiments that I want to show you and in particular, by a series of experiment that could have been a classical paper, but I choose another one. And this is the use of deletions to understand basic phenomena. In the next paper, in the paper you are going to read, you are going to see other deletions. These deletions, in this case, all come from the left of the region. They come somewhere from here, and they end at a different position. Division one ends within RSC. Division two extends a little bit, and division three extends more. These are the three deletions. Now, what is the phenotype of these deletion mutants? Of course, RCS's absent, so there is no activation. So there is no expression with all three deletions. Now, you can provide RCS interants from a plasmid, or an episome, or a conjugant, you can provide RSC. What happens when you provide RSC with deletion one, deletion three and deletion two? Something very different. When you provide rc with deletion one, you get a little bit of expression. One unit. If you add arabinose, you get 200 units. So this is inducable. With deletion two, you get 200 units in the presence of arabinose, but 20 in the absence. So this is also inducible. Because you go from 20 to 200. You induce ten-fold. This is induced 200-fold. But they're both inducible. And with deletion three, which cuts within the promoter, you don't get anything. So far so good. But what's this difference between deletion one and deletion two? This one is strongly inducible. This one is weakly inducible, deletion two. Or, you could say deletion one and deletion two can be turned on to maximum level, 200. But dilation two is missing something, so that even in the absence of aribinose, it is a little bit constitutive. Or a little bit derepressed. And in fact, this is exactly what's going on. Deletion one will end somewhere here. This is deletion one. Deletion two removes, O2. So it cuts all of this. This is deletion 2. So now this structure cannot form, this loop of the DNA cannot form. AraC has no choice but to bind to I1 and sometimes to I2. So you get a little bit of expression. It's like a leaky system. It's not repressed. This is the evidence for repression. And of course deletion three, you remove the promoter so you cut all the way into here, and of course you cannot induce with, and you cannot express with and without arabinose. So this would be deviation three. So, in fact, negative control came in the picture after positive control.