Mechanism of initiation of transcription by Escherichia coli RNA polymerase on supercoiled template

Abstract

DNA supercoiling is known to influence the pattern of gene expression in prokaryotes. Thus the mechanism of transcription initiation and the topological state of the template are intimately related. Using in vitro reconstituted transcription assays, composed of purified RNA polymerase and promoters in their natural topological state, we have conducted a detailed study of transcription initiation from T7 early promoters including the following steps: the formation of ternary complexes, acquisition of rifampicin resistance, release of sigma factor and the capacity for RNA chain elongation in complexes. We determined the order of these events and the length of the transcripts when each step occurred during initiation of transcription on supercoiled templates. The length of the transcripts varied in a promoter-specific manner. Analysis of abortive products formed during the initiation showed that stronger promoters go to the elongation mode at transcript lengths shorter than that required for weaker promoters.