RNA polymerase-cNMP-ligated cAMP receptor protein (CRP) mutant interactions in the enhancement of transcription by CRP mutants

The enhancement of the transcription of three synthetic promoters by cNMP-l igated cAMP receptor protein (CRP)/mutant complexes was determined from the transcription yields of a short AAUU transcript in an abortive initiation in vitro transcription assay, The cNMP-ligated CRP and mutants were cAMP, c GMP, and cIMP ligated with CRP, T127L CRP, S128A CRP, and T127L/S128A CRP, The transcriptional activation of a 152-base pair lacUV5 promoter (synlac p romoter) with a CRP consensus binding site sequence (syncon promoter) was e nhanced by an average factor of 12.3 +/- 0.5 with the cAMP-ligated complexe s of CRP/mutants and cGMP-ligated T127L, although their promoter binding si te affinities varied by a factor of 5. However, in the presence of bound RN A polymerase, the binding affinities only ranged from 0.8 +/- 0.2 x 10(7) M -1 for cAMP-ligated CRP* to 1.8 +/- 0.3 x 10(7) M-1 for cAMP-ligated CRP, i ndicating that the CRP/mutant interacts with the bound RNA polymerase, whic h would account for the near constancy of the enhancement factors. The corr esponding enhancement factors for the synlac promoter and a promoter with a different CRP binding site sequence (syngal promoter) were also nearly the same, 7.2 +/- 0.7 and 6 +/- 1, respectively, The binding reaction of the s yncon promoter to the RNA polymerase is exothermic, with a binding constant (K-b) = 2.1 +/- 0.2 x 10(7) m(-1).