Topological unwinding of strong and weak promoters by RNA polymerase. A comparison between the lac wild-type and the UV5 sites of XXXX .

@entity268 @entity377 @entity1132 @entity5538 @entity8832 @entity56639 @entity526
Two @entity526 control regions have been compared in their ability to be unwound by RNA polymerase during formation of the transcriptionally active ("open") complex: the wild-type control region, consisting of two overlapping binding sites @entity8832 , both weakly transcribed, and an "up" P1 mutant, the strong lac L8UV5 promoter. The final complexes were characterized by their topological unwinding, by DNase I and @entity56639 footprints, as well as by methylation of unpaired @entity5538 residues. At the wild-type control region, the RNA polymerase footprint is weak, and single-strand formation is incomplete and slow. The same signals are strong, complete and quickly established at lac L8UV5; yet the final complexes were found to be equally unwound (by 1.7 turns) in the absence of @entity377 substrates as well as during an abortive initiation cycle. At the lac wild-type region, open complex formation occurs slowly enough to permit the measurement of the extent of a single-stranded region and of topological unwinding during the latency period. Not all the final species are active and unwinding appears to precede, in time, full open-complex formation. At the lac UV5 promoter the same conclusion was reached by a different method involving those changes in the various parameters that characterize open-complex formation monitored by an abortive initiation assay, conducted at increasing levels of template superhelicity. From both approaches we conclude that, at these promoters, the formation of the single-stranded region occurs at the expense of a negative change in linking number, initially stored in a closed intermediate, perhaps as negative writhing. Furthermore, abortive transcription assays indicate that the specific initiation efficiency of the species stored at both promoters, @entity8832 , on the wild-type template is increased as a whole with increasing superhelicity (conversion of inactive species to active ones, increased efficiency of active ones). We conclude that negative supercoiling is not an extra-regulatory element of the @entity1132 system, allowing modulation of expression of the wild-type promoter to the profit of P1. Instead, @entity8832 , in the absence of active catabolite receptor protein (CRP- @entity268 ), appear to be equally weak and to be equally affected by negative supercoiling in the range of superhelical densities examined. The physiological importance of the P1-P2 competition in the regulation of expression in this region is thus questioned. The major effect of CRP- @entity268 stimulation appears to be the direct activation at the P1 promoter.
