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

• Published in: The Journal of biological chemistry 2000-10, Vol.275 (43), p.33457-33463
• Main Authors: Wang, Shenglun, Shi, Ying, Gorshkova, Inna, Schwarz, Frederick P.
• Format: Article
• Language: English
• Subjects: Base Sequence; Binding Sites; Carrier Proteins; Cyclic AMP - metabolism; Cyclic AMP Receptor Protein - metabolism; DNA-Directed RNA Polymerases - metabolism; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Transcription, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M004877200

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 lacUV5promoter (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 × 107m−1for cAMP-ligated CRP* to 1.8 ± 0.3 × 107m−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 synlacpromoter 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 thesyncon promoter to the RNA polymerase is exothermic, with a binding constant (Kb) = 2.1 ± 0.2 × 107m−1.