Site for Cro Repression and CI Activation

Site for Cro Repression and CI Activation

Regulation of the early promoters is more complicated than has been indicated. At both p_L and p_R are three binding sites for both Cro and repressor, O_L1, O_L2, and O_L3 on the left, and O_R1, O_R2, and O_R3 on the right. Repressor bound at O_L1 is sufficient to inactivate p_L. Repressor

and Cro binding on the right is more interesting and complicated because there these proteins regulate the activities of both p_R and p_RM.

Table 14.2 Effects of Increasing Concentrations of Lambda Re-pressor, its Binding to Operators, and its Effects on the Promoters p_RMand p_R

stimulator or, at higher concentrations, as a repressor for p_RM(Table 14.2). These different activities are accomplished by the follow-ing means. Repressor bound at O_R1 inactivates p_R, but repressor bound at O_R1 and O_R2 simultaneously represses p_R and activates p_RM. Repressor bound at O_R3 inactivates p_RM. Cro protein bound at O_R3 represses p_RM, and Cro bound to O_R1 or O_R2 represses p_R (Table 14.3).

Despite the fact that Cro and repressor bind to virtually the same sequences, at least as assayed by DNAse protection and the behavior of mutations lying in the three operator sites on the right, their binding is not the same. As the level of Cro begins to rise in cells, for example during a lytic infective cycle, Cro binds first to O_R3 and shuts off the synthesis of CI repressor. Only later, after the level of Cro has risen still higher, does it bind to O_R2 and O_R1 and shut down the activity of p_R. On the other hand, during a phage developmental cycle that will result in the production of a lysogen, as repressor begins to accumulate it first binds to O_R1 and O_R2 and shuts off p_R and turns on p_RM. At still higher concentrations, repressor binds to O_R3 and shuts off p_RM. The basis for the differential affinity of repressor and Cro for the three operators lies in the slight sequence differences among the sites and the structural differences in the proteins. Repressor and Cro “read” the sequences with different emphasis on different bases.

Table 14.3 Effects of Increasing Concentrations of Cro Protein, itsBinding to Operators, and its Effects on the Promoters p_RM and p_R

We owe the preceding picture to a series of clever in vivo and in vitro experiments by Ptashne and his collaborators. Two operations had to be performed to examine the in vivo effects of Cro and repressor on the activities of p_R and p_RM. The synthesis of the two proteins had to be decoupled and a means had to be found of varying the level of one protein while examining the activity of each of the promoters. Genetic engineering came to the rescue. In one case Cro protein synthesis was placed under control of the lac promoter via a p_lac-cro fusion (Fig. 14.11). Since the lac promoter was still regulated by lac repressor, the level of Cro in cells could be varied by varying the concentration of lac inducer, IPTG, added to the culture medium. Quantitating the activities of p_R and p_RM was facilitated by fusing either promoter to the β-galactosidase gene. Hence, although IPTG was added to cells and β-galactosidase was measured, the results elucidated behavior not of the lac operon, but of lambda phage.

As the intracellular concentration of Cro was increased, first p_RM and then p_R was repressed (Fig. 14.12). These promoters showed much

Figure 14.12 Response of thepRand pRM promoters to increasing concentrations of Cro protein.

different responses to repressor (Fig. 14.13). At one concentration, p_R was shut off and p_RM was turned on. Then, at higher concentrations, p_RM was shut off as well. In vitro transcription experiments using DNA fragments several hundred base pairs long yielded the same results.

Quantitation of the binding of Cro and repressor to the three opera-tors was done with DNAse footprinting. At the lowest Cro concentra-tions, only O_R3 is occupied, but as Cro levels are increased, O_R2 and O_R1 become occupied as well. The results with repressor are the reverse. O_R1 has the highest apparent affinity for repressor, being 50% occupied at a concentration of 3 nM in vitro. At twice this concentration, O_R2 is 50% occupied, but 25 times this concentration is required for O_R3 to be 50% occupied.