RNA Polymerase-cNMP-Ligated cAMP Receptor Protein (CRP) Mutant Interactions in the Enhancement of Transcription by CRP Mutants

Published

October 27, 2000

Author(s)

S. Wang, Y. Shi, I Gorshkova, Frederick P. Schwarz

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 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 (K_b) = 2.1 0.2 X 10⁷ M^-1.

Citation

Journal of Biological Chemistry

Volume

275

Issue

43

Pub Type

Journals

Keywords

RNA Polymerase-cNMP

Citation

Wang, S. , Shi, Y. , Gorshkova, I. and Schwarz, F. (2000), RNA Polymerase-cNMP-Ligated cAMP Receptor Protein (CRP) Mutant Interactions in the Enhancement of Transcription by CRP Mutants, Journal of Biological Chemistry (Accessed April 19, 2024)