[MUSIC] Now, in the second introduction, we're going to go into a little bit of detail into how lambda is expressed and functions in these two different programs, lysogeny and lysis. When lambda enters the cell, the lambda DNA enters the cell. Two genes are expressed immediately. One is the N gene, which is here. And the other one is the cro gene, which is here. If N is made and is active, then the expression, the transcription of the DNA into the RNA, to the messenger RNA, continues on both sides. Like here, and like here. And you express the genes from the left operon, and from the right operon. And these are genes we will see again Cro, O and P, and exo. Then comes the decision. And this decision is taken by measuring, if you want, the activity of the cell. If the cell is healthy, in good shape and active, or if the cell is in a miserable state. If the cell is healthy and happy the phage will go and make lices and express all the structure proteins that are part of the virus, and you get lices and you get phage release. If the conditions are not good, if the cell is unhealthy, unhappy, not enough food, problems of one kind or another, then the phage, the signs that it's too risky to go to the phase, because the cell may stop functioning before you achieve production of phages. So the safety mode under this condition is to go into lysogeny, in which case we express C1 int and you integrate into the chromosome. And this is called a prophage. It's the precursor of the phage. With lambda, the prophage is integrated into the chromosome with other lysogenic phages. The DNA can stay outside the chromosome as a little plasmid. But in the case of lambda, it's integrated. So a lysogen is a bacterium that carries lambda DNA, And it expresses only one gene, C1. Which calls for the repressor. All the other genes are silent. Now, we're going to look at this region around c1. But first, I would like to show you what is the phenotype of the phage that is mutant in this gene C1. Armin Kaiser discovered mutant phages that have a different. Now this is not a single plaque, this is a drop phage, layered over sensitive bacteria. And this paper which came out in 57, showed that this is what the Y type does. The Y type lambda, makes what are called turbid plaques, that is you have phage but you have bacteria. Lysogenic bacteria that grow inside the growth. That's a typical lysogenic phage. Now he got clear mutants hence the name C, C for clear. This is the first gene you identify and it was called c1, this is cII, and this is cIII. With cI, you can see that all the bacteria have lysed. You do see a few microcolonies inside. Those are bacteria that do not adsorb the phage. They are called resistant but most of the bacteria are killed. cI is absolutely clear. cII and cIII are somewhat clear, but not absolutely clear. So in fact, cII and cIII help establish repression, but cI is absolutely essential. So this is a very small part of the genome of lambda, a very small part of the lambda genome between the c1 gene and the cro gene. And this is this region between the two, which is where the switch works. This region is called the operator or OR, and OR covers two promoters. The promoters M and the promoters R. One drives expression of cl, one drives expression of cro. And this promoter is made of three boxes of similar but slightly different sequences. Now we have two proteins that will recognize the same piece of DNA and compete for each other. C1 Is a relatively medium sized protein, 300 amino acid, 250 amino acid. That forms dimers, and these dimers bind the DNA. Cro is one of the smallest protein known, it's only 66 amino acid long. It is capable of forming dimer, and it is capable of binding DNA. But there is no c region that can touch each other. So two protein binds to the same region. What happens when repressor binds to these regions? Okay, now this is a cartoon, okay. Again you have the same region, and now you have the repressor bound at the OR1 site, the site at the right. When this is the case, there is no transcription of from this side for the CI gene, and no transcription of this side. A repressor bound here at OR1 will block both transcription. Now, the repressor never binds to OR2 alone, or to OR3 alone, but it's drawn here to explain what would happen if it were to bind by itself. At OR2, when the repressor is bound at OR2, this right transcription is off, but the left transcription is on. Now, if OR3 would be the only occupied site, transcription would work at OR1 but would be off on the left. So you can see that by having the protein at different places, you can change which gene is expressed. If you have no protein, only this gene is expressed. If you have the protein at this side, none is expressed. And you can't have the two genes expressed at the same time. Now, what makes the system beautiful is the fact that the repressor doesn't bind with the same affinity, affinity does not like these three sides in the same way. The most, the best site is the first site for the repressor. The second best is the second, and the third is the weakest site. Which means that when there's no repressor, when there is no repressor, you get transcription this way. When there's a little bit of repressor, you get no transcription. If you have this, you get no transcription on the left and you get transcription on the right. And if you have occupation of the three sides, you get nothing. So no expression, you get this. No repressor, you get this. And if the repressor is slightly enough, you express this promoter and more, you stop expressing it. So it's not only a switch, but it's like a regulator. Cro, on the other hand, will bind first to this side and then to the others. So the first thing that cro does is to prevent expression from the repressor gene and then prevent expression from PR. So two genes that are counteracting each other. What was extremely helpful for working with lambda was the discovery of phage related to lambda, cousins. And these cousins have different repressor and different immunity region. Drawn here is the lambda immunity region. With the GNC encrow. The 434, this is one of these phages, we'll come back to it. With its immunity region. And this immunity region can be introduced in lambda to make a phage, to make mosaic, if you want, that is partly lambda, partly 434. And this one has the immunity of 434. The properties of these phages are the following, lambda will plate on an E coli K12 that is not lysogenic, were not played on lysogenic host. And were played on a host that is lysogenic for 434. A clear mutant of lambda, like the one I showed you before from isolated. One of these clear mutants, they will form clear plaques. They were not grown lysogen, and they will form clear plaques on the lysogen for 434. This is the equivalent to a lack minus mutant with a lack system. The virilant phage, and they've been called virilant because they grow on a lysogen. And they form clear plaques on the lysogen. That's why they were called virile phages. These phages are insensitive to the presence of another lysogen. And among the phage that have been used, there's 434 and there's another one we'll see, which is 21. Phage 21 has about the same properties as 434 or lambda except it's a third member. Okay, now you have the basic notion about lambda regulation that will help you read the very short paper from the [FOREIGN] and the PNS paper that is the reading for next week.
