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

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Abstract
The catabolite activator protein (CAP) of E. 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). The interactions between CAP/cAMP and the gal promoter region in the presence of RNA polymerase were investigated. DNase I protection experiments of gal 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; gal DNA in the presence of 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 the gal DNA from -30 to +15; when CAP/cAMP and RNA polymerase are present in the reaction, protection is afforded from -65 to +20. One directly quantified the amount of cAMP and CAP bound to gal 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. Two CAP molecules specifically bound to the gal promoter were found, although only 1 cAMP molecule was found in the complex at low cAMP concentrations (but sufficient to support P1 transcription). The DNA protection experiments and the centrifugation results indicate that RNA polymerase induces the binding of a second CAP molecule to the gal promoter in forming stable initiation complexes. The second CAP molecule is apparently needed to stimulate initiation from the P1 promoter; this may be involved in regulating the relative rates at which transcription begins from the 2 gal start sites.