Two catabolite activator protein molecules bind to the galactose promoter region of Escherichia coli in the presence of RNA polymerase

The catabolite activator protein (CAP) ofEscherichia coli , complexed with cAMP, is required for efficient initiation of transcription from the galactose P1 promoter (start site at +1) but not from the overlapping P2 promoter (start site at -5) [Musso, R. E., DiLauro, R., Adhya, S. & deCrombrugghe, B. (1977)Cell 12, 847-854]. We investigated the interactions between CAP/cAMP and thegal promoter region in the presence of RNA polymerase. DNase I protection experiments ofgal promoter restriction fragments revealed that CAP/cAMP protects the DNA from digestion between positions -50 and -25 and that RNA polymerase protects it from -35 to +10; however,gal DNA in the presence of both CAP/cAMP and RNA polymerase is protected from DNase I digestion between positions -68 and +15. Results of exonuclease III protection experiments show that RNA polymerase alone protects thegal DNA from -30 to +15; when both CAP/cAMP and RNA polymerase are present in the reaction, protection is afforded from -65 to +20. We directly quantified the amount of cAMP and CAP bound togal promoter DNA in the presence of RNA polymerase by selectively pelleting the ternary complexes (CAP/cAMP-RNA polymerase-gal promoter DNA) in a Beckman Airfuge. We found two CAP molecules specifically bound to thegal promoter, although only one cAMP molecule was found in the complex at low cAMP concentrations (but sufficient to support P1 transcription). Thus, both the DNA protection experiments and the centrifugation results indicate that RNA polymerase induces the binding of a second CAP molecule to thegal promoter in forming stable initiation complexes. It appears that the second CAP molecule is needed to stimulate initiation from the P1 promoter; this may be involved in regulating the relative rates at which transcription begins from the twogal start sites.