RNA Polymerase-cNMP-Ligated cAMP Receptor Protein (CRP) Mutant Interactions in the Enhancement of Transcription by CRP Mutants
S. Wang, Y. Shi, I Gorshkova, Frederick P. Schwarz
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 107 M-1 for cAMP-ligated CRP* to 1.8 0.3 x 107 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 (Kb) = 2.1 0.2 X 107 M-1.
, Shi, Y.
, Gorshkova, I.
and Schwarz, F.
RNA Polymerase-cNMP-Ligated cAMP Receptor Protein (CRP) Mutant Interactions in the Enhancement of Transcription by CRP Mutants, Journal of Biological Chemistry
(Accessed November 30, 2023)