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Multiple Regions on the Escherichia coliHeat Shock Transcription Factor ς32 Determine Core RNA Polymerase Binding Specificity

ABSTRACT We have analyzed the core RNA polymerase (RNAP) binding activity of the purified products of nine defective alleles of the rpoHgene, which encodes ς32 in Escherichia coli. All mutations studied here lie outside of the putative core RNAP binding regions 2.1 and 2.2. Based on the estimatedKss for the mutant sigma and core RNAP interaction determined by in vitro transcription and by glycerol gradient sedimentation, we have divided the mutants into three classes. The class III mutants showed greatly decreased affinity for core RNAP, whereas the class II mutants’ effect on core RNAP interaction was only clearly seen in the presence of ς70 competitor. The class I mutant behaved nearly identically to the wild type in core RNAP binding. Two point mutations in class III altered residues that were distant from one another. One was found in conserved region 4.2, and the other was in a region conserved only among heat shock sigma factors. These data suggest that there is more than one core RNAP binding region in ς32 and that differences in contact sites probably exist among sigma factors.