CAP INTERACTS WITH RNA-POLYMERASE IN SOLUTION IN THE ABSENCE OF PROMOTER DNA

PROTEIN-PROTEIN interactions between transcription activator proteins and RNA polymerase or basal transcription factors have been suggested to be important for transcription activation1-8. Interactions between catabolite gene activator protein (CAP)9,10 and RNA polymerase have been proposed based on face-of-helix-dependent transcription activation by CAP11-13 and based on face-of-helix-dependent cooperative binding of CAP and RNA polymerase to promoter DNA14,15. Mutants of CAP specifically defective in transcription activation have been isolated (mutants defective in transcription activation, but not defective in DNA binding and DNA bending16-19). All such mutants contain amino-acid substitutions within a surface loop consisting of amino acids 152 to 166 of CAP16-19. Here we use the thermodynamically rigorous technique of fluorescence polarization20-23 to show that CAP interacts with RNA polymerase in solution in the absence of promoter DNA (K(D,app) = 2.8 x 10(-7) M), whereas [Ala158]CAP, a mutant of CAP specifically defective in transcription activation, does not.