Journal of Biological Chemistry 2000-10-27

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

S Wang, Y Shi, I Gorshkova, F P Schwarz

Index: J. Biol. Chem. 275 , 33457-33463, (2000)

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Abstract

The enhancement of the transcription of three synthetic promoters by cNMP-ligated 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, cGMP, and cIMP ligated with CRP, T127L CRP, S128A CRP, and T127L/S128A CRP. The transcriptional activation of a 152-base pair lacUV5 promoter (synlac promoter) with a CRP consensus binding site sequence (syncon promoter) was enhanced by an average factor of 12.3 +/- 0.5 with the cAMP-ligated complexes of CRP/mutants and cGMP-ligated T127L, although their promoter binding site affinities varied by a factor of 5. However, in the presence of bound RNA 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, indicating that the CRP/mutant interacts with the bound RNA polymerase, which would account for the near constancy of the enhancement factors. The corresponding 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 syncon promoter to the RNA polymerase is exothermic, with a binding constant (K(b)) = 2.1 +/- 0. 2 x 10(7) m(-1).