A mutant σ32 with a small deletion in conserved region 3 of σ has reduced affinity for core RNA polymerase

Abstract

σ70, encoded by rpoD, is the major σ factor in Escherichia coli. rpoD285 (rpoD800) is a small deletion mutation in rpoD that confers a temperature-sensitive growth phenotype because the mutant σ70 is rapidly degraded at high temperature. Extragenic mutations which reduce the rate of degradation of RpoD285 σ70 permit growth at high temperature. One class of such suppressors is located in rpoH, the gene encoding σ32, an alternative σ factor required for transcription of the heat shock genes. One of these, rpoH113, is incompatible with rpoD+. We determined the mechanism of incompatibility. Although RpoH113 σ32 continues to be made when wild-type σ70 is present, cells show reduced ability to express heat shock genes and to transcribe from heat shock promoters. Glycerol gradient fractionation of σ32 into the holoenzyme and free sigma suggests that RpoH113 σ32 has a lower binding affinity for core RNA polymerase than does wild-type σ32. The presence of wild-type σ70 exacerbates this defect. We suggest that the reduced ability of RpoH113 σ32 to compete with wild-type σ70 for core RNA polymerase explains the incompatibility between rpoH113 and rpoD+. The rpoH113 cells would have reduced amounts of σ32 holoenzyme and thus be unable to express sufficient amounts of the essential heat shock proteins to maintain viability.