Fondos parafiscales

Lambda repressor acts as both a negative and a positive regulator of transcription. Repressor dimers normally bind cooperatively to adjacent operator sites OrI and Or2, the dimer at OrI

helping a second to bind to Or2, and block transcription of the early lytic genes when bound at these sites (reviewed by Hochschild and Ptashne, 1988). Stimulation of repressor transcription from Pr m appears to involve contact between repressor bound at Or2 and RNA polymerase bound

B

Figure 6.Xgenes and regulatory elements

A.A portion of the X genome. The arrows indicate the directions' and start sites of transcription of various genes. O lP l and OrPr are the left and right operator and promoter regions, respectively.

Pr m is the cl promoter active in a lysogen.

B. An expanded diagram of the X o r region. 0 r1 , O r2 and O r3 are the 17 bp repressor and Cro binding sites. The start sites of transcription from Pr and Pr m, which are located outside the

domains connected by a string of 40 amino acids. The amino terminal domain contacts the DNA and RNA polymerase (Pabo and Lewis, 1982), and the carboxyl domain which facilitates dimer formation and mediates interaction between dimers (Sauer et ai, 1979). Each of the three sites in Or can bind one repressor dimer. The sole role of OrI (a high affinity binding site for repressor) is to help ensure the occupancy of Or2 (a weak repressor binding site), which means that at higher concentrations repressor can bind to Or2 and activate transcription in the absence of repressor bound at OrI (Meyer et ai, 1980).

Positive control of transcription by repressor is mediated by the action of repressor at Or2

helping RNA polymerase to bind and begin transcription at Pr m (which governs transcription of cl in

a lysogen). This increases the rate of transcription -tenfold. The repressor dimer bound to Or2 increases the affinity of Pr m for RNA polymerase since protein-protein contacts as well as RNA

polymerase/DNA contacts are required to position the polymerase. A repressor dimer bound at Or2 therefore represses Pr by excluding the binding of RNA polymerase to that promoter (Meyer

et ai, 1980). This in turn prevents transcription to the right but facilitates transcription to the left (a

difference which is attributed to Or2 being slightly closer to Pr than to Pr m)- A repressor dimer

bound at OrI would block binding of RNA polymerase to Pr but would be too far away to affect

polymerase at Pr m which functions only at a very low level since no repressor is bound at Or2. Binding of a single repressor dimer at Or3 would block binding of RNA polymerase to Pr m and have

no effect on transcription from Pr. In summary, when Xrepressor acts as a positive regulator of

transcription, repressor dimers are nearly always bound at OrI and Or2 with Or3 being generally free of repressor. This arrangement of repressor at Or turns off transcription from Pr but turns on

Pr m- In this way repressor, but not Cro, is synthesised (reviewed by Ptashne, 1986b).

Interaction of repressor dimers with the three operator sites is dependent on two factors. The first is the affinity of repressor for each isolated site in Or - this is known as the “intrinsic" affinity of a site and can be measured using Or DNA in which neighbouring sites are mutated such that they no

longer bind repressor. The second factor affecting the order of binding is the interaction of repressor dimers at adjacent binding sites (Ptashne etaL, 1980). Just as repressor at Or 2 assists

binding of RNA polymerase to promoter Pr m. sorepressors can interact with each other to facilitate

binding.

The cooperative binding of repressor to Orwas demonstrated in In vitro studies of the binding

of repressor at various concentrations to wild type and mutant Or templates (Ptashne etaL, 1980).

The intrinsic affinities of Or2 and Or3 for repressor are approximately equal and are more than an order of magnitude lower than that of OrI . However, over a broad concentration range (including

that found in a lysogen) OrI and Or2 are filled and Or3 tends to be vacant. This is due to a

favourable interaction between repressor dimers bound at OrI and Or2 which ensures that these

sites are filled coordinateiy. Yet another favourable interaction between dimers bound at Or2 and

Or3 is apparent if no repressor is bound to OrI . This cooperativity of repressor binding has been ascribed by Ptashne and coworkers (1980) to protein-protein interactions as opposed to conformational changes in the DNA transmitted through the double helix (isolated amino domain of repressor binds to all three sites in Or non-cooperatively). It is believed that the flexibility in the

region connecting the amino and carboxyl domains could orient the carboxyl portion of a repressor bound at Or2 such that it could contact one neighbouring dimer but not another. This contrasts the

situation found with Cro/operator interactions in which Cro binds non-cooperatively. As with the repressor amino domain, mutations of any site in Or affects the affinity of that site alone for Cro

(Johnson etaL, 1979).

In a lysogen, repressor bound at OrI and Or2 maintain the cro gene in the off position while

stimulating transcription of Its own gene, cl. As cells grow and divide, repressor is constantly being synthesised. However, if the repressor concentration increases, perhaps due to ceil division being temporarily Inhibited, then repressor would bind also to Or3, thus turning off the cl gene. Once ceil

division had been resumed, and repressor concentration dropped to the proper level, the cl gene would then function to provide more repressor. In this way a constant level of repressor is

maintained in the cell despite changes in growth rate (reviewed by Ptashne, 1986b).

The concentration of repressor in a cell is high enough to ensure that OrI and Or2 are likely to be filled at any one time. This means that in the absence of an inducing agent the lysogenic state is stable and can thus be maintained more-or-less indefinitely. If the switch from the lysogenic to the lytic state is induced then the action of Cro comes into play.