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J Biol Chem, Vol. 273, Issue 16, 9872-9877, April 17, 1998

Reduction in Abortive Transcription from the P_R Promoter by Mutations in Region 3 of the ⁷⁰ Subunit of Escherichia coli RNA Polymerase

Ranjan Sen, Hiroki Nagai, V. James Hernandez^¶, and Nobuo Shimamoto

From the  Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka-411, Japan and ^¶ Microbiology, SUNY/Buffalo Medical School, Buffalo, New York 14214

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 ⁷⁰ 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 P_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.

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