Role of Region C in Regulation of the Heat Shock Gene-Specific Sigma Factor of Escherichia coli, ς32

Expression of heat shock genes is controlled in Escherichia coli by the antagonistic action of the ς32 subunit of RNA polymerase and the DnaK chaperone system, which inactivates ς32 by stress-dependent association and mediates ς32 degradation by the FtsH protease. A stretch of 23 residues (R122 to Q144) conserved among ς32 homologs, termed region C, was proposed to play a role in ς32 degradation, and peptide analysis identified two potential DnaK binding sites central and peripheral to region C. Region C is thus a prime candidate for mediating stress control of ς32, a hypothesis that we tested in the present study. A peptide comprising the central DnaK binding site was an excellent substrate for FtsH, while a peptide comprising the peripheral DnaK binding site was a poor substrate. Replacement of a single hydrophobic residue in each DnaK binding site by negatively charged residues (I123D and F137E) strongly decreased the binding of the peptides to DnaK and the degradation by FtsH. However, introduction of these and additional region C alterations into the ς32 protein did not affect ς32 degradation in vivo and in vitro or DnaK binding in vitro. These findings do not support a role for region C in ς32 control by DnaK and FtsH. Instead, the ς32 mutants had reduced affinities for RNA polymerase and decreased transcriptional activities in vitro and in vivo. Furthermore, cysteines inserted into region C allowed cysteine-specific cross-linking of ς32 to RNA polymerase. Region C thus confers on ς32 a competitive advantage over other ς factors to bind RNA polymerase and thereby contributes to the rapidity of the heat shock response.