A MUTANT SIGMA-32 WITH A SMALL DELETION IN CONSERVED REGION-3 OF SIGMA HAS REDUCED AFFINITY FOR CORE RNA-POLYMERASE

Sigma-70 encoded by rpoD, is the major cr factor in Escherichia coli. rpoD285 (rpoD800) is a small deletion mutation in rpoD that confers a temperature-sensitive growth phenotype because the mutant sigma-70 is rapidly degraded at high temperature. Extragenic mutations which reduce the rate of degradation of RpoD285 sigma-70 permit growth at high temperature. One class of such suppressors is located in rpoH, the gene encoding sigma-32, an alternative sigma-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 sigma-32 continues to be made when wild-type sigma-70 is present, cells show reduced ability to express heat shock genes and to transcribe from heat shock promoters. Glycerol gradient fractionation of sigma-32 into the holoenzyme and free sigma suggests that RpoH113 sigma-32 has a lower binding affinity for core RNA polymerase than does wild-type sigma-32 . The presence of wild-type sigma-70 exacerbates this defect. We suggest that the reduced ability of RpoH113 sigma-32 to compete with wild-type sigma-70 for core RNA polymerase explains the incompatibility between rpoH113 and rpoD+. The rpoH113 cells would have reduced amounts of sigma-32 holoenzyme and thus be unable to express sufficient amounts of the essential heat shock proteins to maintain viability.