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J. Biol. Chem., Vol. 275, Issue 43, 33457-33463, October 27, 2000

This Article Full Text Full Text (PDF) All Versions of this Article: 275/43/33457    most recent M004877200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, S. Articles by Schwarz, F. P. Search for Related Content PubMed PubMed Citation Articles by Wang, S. Articles by Schwarz, F. P. Social Bookmarking                      What's this?

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

Shenglun Wang, Ying Shi, Inna Gorshkova, and Frederick P. Schwarz^§

From the Center for Advanced Research in Biotechnology/National Institute of Standards and Technology, Rockville, Maryland 20850

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 × 10⁷ M¹ for cAMP-ligated CRP* to 1.8 ± 0.3 × 10⁷ M¹ 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 × 10⁷ M¹.

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