CAP interacts with RNA polymerase in solution in the absence of promoter DNA

Abstract

PROTEIN-PROTEIN interactions between transcription activator proteins and RNA polymerase or basal transcription factors have been suggested to be important for transcription activation^1–8. Interactions between catabolite gene activator protein (CAP)^9,10 and RNA polymerase have been proposed based on face-of-helix-dependent transcription activation by CAP^11–13 and based on face-of-helix-dependent cooperative binding of CAP and RNA polymerase to promoter DNA^14,15. Mutants of CAP specifically defective in transcription activation have been isolated (mutants defective in transcription activation, but not defective in DNA binding and DNA bending^16–19). All such mutants contain amino-acid substitutions within a surface loop consisting of amino acids 152 to 166 of CAP^16–19. Here we use the thermodynamically rigorous technique of fluorescence polarization^20–23 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 [Alal58]CAP, a mutant of CAP specifically defective in transcription activation, does not.