The simultaneous binding of Gal repressor (GalR), catabolite activator protein (CAP or CRP), and RNA polymerase (RNAP) to the promoter region of the Escherichia coli gal operon has been analyzed thermodynamically, by quantitative DNase I 'footprint' titration analysis, and structurally, by the use of hydroxyl radical ((·)OH) and 5-phenylphenanthroline (5OPP) 'footprinting'. In the absence of regulatory proteins, the preference of RNAP for one (P1) of the two gal operon overlapping promoters (P1 and P2) is -0.4 ± 0.2 kcal/mol, indicating only a small energetic preference for P1. The simultaneous binding of CAP and RNAP occurs with 10-fold cooperativity, with greater than 99% of the CAP-RNAP complex present at the P1 promoter. This cooperativity is inhibited by the binding of GalR to the upstream operator, O(E), but does not result in the repartitioning of RNAP between the P1 and the P2 promoters. These results suggest that the CAP-RNAP cooperativity and promoter partitioning are not linked and are consistent with a mechanism by which GalR binding to O(E) represses transcription by inhibiting the CAP- RNAP cooperativity. It is suggested that the CAP-RNAP cooperativity is dependent upon contacts made by the complex with the upstream DNA and that GalR binding to O(E) prevents these contacts from occurring. Changes in nuclease reactivity at the internal operator O(I) (centered at +53.5) take place upon RNAP binding. These changes are dependent on the DNA sequence present at O(I) and on the presence or absence of CAP. They are independent of the helical phasing between the promoters and O(I) and of the distance between them. These results suggest that RNAP can directly communicate with events occurring at both the external and the internal operator sequences without direct contact between repressor molecules bound at their cognate sites.
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