The kinetics of formation of complexes between Escherichia coli RNA polymerase and the rrnB P1 and P2 promoters of Bacillus subtilis. Effects of guanosine tetraphosphate on select steps of transcription initiation.
The kinetics of formation and dissociation of heparin-resistant transcription initiation complexes between Escherichia coli RNA polymerase and the rrnB P1 and P2 promoters from Bacillus subtilis were investigated using a gel retardation assay. The results suggest that the formation of polymerase-promoter complexes proceeds by a three-step reaction mechanism. The bimolecular collision between free RNA polymerase and the promoter creates a heparin-sensitive complex, which then isomerizes to an initial, and then a subsequent, heparin-resistant complex. We propose that a sequential mechanism best describes the bimolecular collision and that the forward rate constants predominate in the overall rate of heparin-resistant complex formation. At 35 degrees C, the association of polymerase with P1 and P2 was very rapid (ka = 1.5 - 2.1 x 10(8) M-1 s-1; kf > or = 0.56 s-1). Direct information on the formation of the closed and intermediate transcription complexes and indirect information on the formation of open complexes suggest that guanosine tetraphosphate did not differentially affect any step between growth rate-regulated and non-growth rate-regulated rRNA promoters by more than 2-fold.
Wellington
S R
SR
Department of Microbiology, University of British Columbia, Vancouver, Canada.
Spiegelman
G B
GB
D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006159:Guanosine Tetraphosphate / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D013696:Temperature; D014158:Transcription, Genetic; D012407:rRNA Operon
D004269:DNA, Bacterial; D006159:Guanosine Tetraphosphate; D006493:Heparin; D012321:DNA-Directed RNA Polymerases