Site for Cro Repression and CI Activation
Regulation of the early promoters is more complicated than has been indicated. At both pL and pR are three binding sites for both Cro and repressor, OL1, OL2, and OL3 on the left, and OR1, OR2, and OR3 on the right. Repressor bound at OL1 is sufficient to inactivate pL. Repressor
and Cro binding on the right is more interesting and complicated because there these proteins regulate the activities of both pR and pRM.
Table 14.2 Effects of Increasing Concentrations of Lambda Re-pressor, its Binding to Operators, and its Effects on the Promoters pRMand pR
stimulator or, at higher concentrations, as a repressor for pRM(Table 14.2). These different activities are accomplished by the follow-ing means. Repressor bound at OR1 inactivates pR, but repressor bound at OR1 and OR2 simultaneously represses pR and activates pRM. Repressor bound at OR3 inactivates pRM. Cro protein bound at OR3 represses pRM, and Cro bound to OR1 or OR2 represses pR (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 OR3 and shuts off the synthesis of CI repressor. Only later, after the level of Cro has risen still higher, does it bind to OR2 and OR1 and shut down the activity of pR. 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 OR1 and OR2 and shuts off pR and turns on pRM. At still higher concentrations, repressor binds to OR3 and shuts off pRM. 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 pRM and pR
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 pR and pRM. 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 plac-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 pR and pRM 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 pRM and then pR 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, pR was shut off and pRM was turned on. Then, at higher concentrations, pRM 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 OR3 is occupied, but as Cro levels are increased, OR2 and OR1 become occupied as well. The results with repressor are the reverse. OR1 has the highest apparent affinity for repressor, being 50% occupied at a concentration of 3 nM in vitro. At twice this concentration, OR2 is 50% occupied, but 25 times this concentration is required for OR3 to be 50% occupied.
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