Changes in conserved region 3 of Escherichia coli sigma 70 reduce abortive transcription and enhance promoter escape

Abstract

Mutations within the Escherichia coli rpoD gene encoding amino acid substitutions in conserved region 3 of the sigma(70) subunit of E. coli RNA polymerase restore normal stress responsiveness to strains devoid of the stress alarmone, guanosine-3',5'-(bis)pyrophosphate (ppGpp). The presence of a mutant protein, either sigma(70)(P504L) or sigma(70)(S506F), suppresses the physiological defects in strains devoid of ppGpp. In vitro, when reconstituted into RNA polymerase holoenzyme, these sigma mutants confer unique transcriptional properties, namely they reduce the probabilities of forming abortive RNAs. Here we investigated the behavior of these mutant enzymes during transcription of the highly abortive cellular promoter, gal P2. No differences between mutant and wild-type enzymes were observed prior to and including open complex formation. Remarkably, the mutant enzymes produced drastically reduced levels of gal P2 abortive RNAs and increased production of full-length gal P2 RNAs relative to the wild-type enzyme, leading to greatly reduced ratios of abortive to productive RNAs. These results are attributed mainly to a decreased formation of unproductive initial transcribing complexes with the mutant polymerases and increased rates of promoter escape. Altered transcription properties of these mutant polymerases arise from an alternative structure of the sigma(70) region 3.2 segment that permits efficient positioning of the nascent RNA into the RNA exit channel displacing sigma and facilitating sigma release.