INTERACTIONS BETWEEN RNA-POLYMERASE AND THE POSITIVE AND NEGATIVE REGULATORS OF TRANSCRIPTION AT THE ESCHERICHIA-COLI GAL OPERON

The simultaneous binding of Gal repressor(GalR), catabolite activator protein (CAP or CRP), and RNA polymerase (RNAP) to the promoter region of the Escherichia coil gal operon has been analyzed thermodynamicall y, by quantitative DNase I ''footprint'' titration analysis, and struc turally, by the use of hydroxyl radical ((OH)-O-.) and 5-phenylphenant hroline (5OPP) ''footprinting''. In the absence of regulatory proteins , the preference of RNAP for one (P1) of the two gal operon overlappin g promoters (P1 and P2) is -0.4 +/- 0.2 kcal/mol, indicating only a sm all energetic preference for P1. The simultaneous binding of CAP and R NAP occurs with 10-fold cooperativity, with greater than 99% of the CA P-RNAP complex present at the P1 promoter. This cooperativity is inhib ited by the binding of GalR to the upstream operator, O-E, but does no t result in the repartitioning of RNAP between the P1 and the P2 promo ters. These results suggest that the CAP-RNAP cooperativity and promot er partitioning are not linked and are consistent with a mechanism by which GalR binding to O-E represses transcription by inhibiting the CA P-RNAP cooperativity. It is suggested that the CAP-RNAP cooperativity is dependent upon contacts made by the complex with the upstream DNA a nd that GalR binding to O-E prevents these contacts from occurring. Ch anges in nuclease reactivity at the internal operator O-I (centered at +53.5) take place upon RNAP binding. These changes are dependent on t ine DNA sequence present at Or 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 an d the internal operator sequences without direct contact between repre ssor molecules bound at their cognate sites.