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 10(7) m(-)(1) for cAMP-ligated CRP* to 1.8 +/- 0. 3 x 10(7) 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 10(7) m(-1).
Highlights
§ To whom correspondence should be addressed: Center for Advanced Research in Biotechnology/National Institute of Standards and Technology, 9600 Gudelsky Dr, Rockville, MD 20850
The enhancement factors determined for the cAMP-ligated cAMP receptor protein (CRP)/mutants in the in vitro transcription assay were compared with their binding affinities to a shorter 104-bp syncon promoter (Fig. 1) with and without bound RNA polymerase, determined from isothermal titration calorimetric (ITC) measurements
This is indicated for the cAMP-ligated CRP and T127L and S128A mutants by a nearly constant ratio (2.2 Ϯ 0.4) of the syncon promoter in vitro enhancement factors to the corresponding in vivo enhancement factors determined with plasmids carrying the CRP binding site construct
Summary
Vol 275, No 43, Issue of October 27, pp. 33457–33463, 2000 Printed in U.S.A. RNA Polymerase-cNMP-ligated cAMP Receptor Protein (CRP) Mutant Interactions in the Enhancement of Transcription by CRP Mutants*. 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, their promoter binding site affinities varied by a factor of 5. The enhancement factors determined for the cAMP-ligated CRP/mutants in the in vitro transcription assay were compared with their binding affinities to a shorter 104-bp syncon promoter (Fig. 1) with and without bound RNA polymerase, determined from ITC measurements. The rest of the promoter sequence, including the RNA polymerase binding site and the transcription P1 start site, is the same in all three promoters, and changes in the amount of transcription product from these three promoters in the in vitro assay would presumably result exclusively from differences in the CRP binding site properties
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