Abstract
Transcription initiation by Escherichia coli RNA polymerase at most promoters is associated with a reiterative synthesis and release of short abortive RNA products. We have investigated the mechanism of abortive RNA synthesis by using holoenzymes containing mutant sigma70 subunits with changes in region 3 (S506F and P504L), which reduce the ratio of abortive to full-length products. Binary complexes formed by these mutant enzymes at a modified lambdaPR promoter contained a smaller fraction of open complexes than for normal polymerase, suggesting an involvement of region 3 in melting duplex DNA or in maintenance of the open complex. The half-lives of the majority of binary complexes formed by the mutant enzymes were less than 1 min, in contrast to 30 min for the wild-type complexes. The time courses of transcription and pulse-labeling assays showed that moribund complexes, which generate only abortive products (Kubori, T., and Shimamoto, N. (1996) J. Mol. Biol. 256, 449-457), were formed by the mutant enzymes. However, they accumulated to a lesser extent than for the wild-type enzyme, due both to faster dissociation and conversion into inactive complexes. This is the main cause of the low degree of abortive transcription display...Continue Reading
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