Reduction in abortive transcription from the λP_R promoter by mutations in Region 3 of the ζ⁷⁰ subunit of Escherichia coli RNA Polymerase

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 ζ⁷⁰ 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 λP_R 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 displayed by the mutant enzymes on this promoter.