Correlation of the CRP-Operon Binding Affinity With the Activation of Transcription by CRP

Published

October 12, 2021

Author(s)

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

Abstract

The dependence of transcriptional activation by 3 -5 cyclic adenosine monophosphate (cAMP) receptor protein (CRP) on the CRP binding affinity of the operon was investigated by comparing the cyclic nucleotide (cNMP)-ligated CRP/mutants binding affinities of three synthetic operons with the in vitro transcription yields of these operons in the presence of the cNMP-ligated CRP/mutant. The three operons are the lacUV5 operon, the lacUV5 operon where the lac CRP binding site sequence was replaced by a CRP consensus binding site sequence , and replaced by the CRP binding sequence of the gal operon. The cNMP-ligated CRP/mutants consisted of combinations of cNMP, cAMP, 3 -5 cyclic guanosine monophosphate (cGMP), and 3 -5 cyclic inosine monophosphate (cIMP) with CRP, a T127L mutant which activates transcription in vivo in the presence of cGMP, a S128A mutant which does not activate transcription in vivo, and a T127L/S128A mutant which activates transcription in vivo in the absence of cAMP. By monitoring the yield of a short transcript, radio labeled AAUU, and leaving out the next ribonucleotide, guanosine triphosphate in the in vitro assay, any effect of CRP on the elongation and termination of the processes in the transcription was minimized. Transcription enhancement factors, determined from the ratio of the product yield in the presence of the cNMP-ligated CRP/mutant to the yield in the absence of CRP/mutant for each operon, ranged from 1 to 13 for the highest CRP binding site affinity (consensus sequence to cAMP-ligated CRP). The enhancement factors exhibited a linear correlation with the cNMP-ligated CRP/mutant binding affinities, indicating that the different levels of transcriptional activation by cNMP-ligated CRP/mutants are a linear function of the CRP binding affinity.

Citation

Journal of Molecular Biology

Pub Type

Journals

Keywords

binding constant, cyclic adenosine receptro protein, DNA, operon, RNA transcription

Citation

Wang, S. , Shi, Y. , Gorshkova, I. and Schwarz, F. (2021), Correlation of the CRP-Operon Binding Affinity With the Activation of Transcription by CRP, Journal of Molecular Biology (Accessed April 19, 2024)