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 sigma(32) subunit of RNA polymerase and the DnaK chaperone system, which inactivates sigma(32) by stress dependent association and mediates sigma(32) degradation by the FtsH protease. A stretch of 23 residues (R122 to Q144) conserved among sigma(32) homologs, termed region C, was proposed to play a role in sigma(32) 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 sigma(32), 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 sigma(32) protein did not affect sigma(32) degradation in vivo and in vitro or DnaK binding in vitro. These findings do not support a role for region C in sigma(32) control by DnaK and FtsH. Instead, the sigma(32) 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 sigma(32) to RNA polymerase. Region C thus confers on sigma(32) a competitive advantage over other sigma factors to bind RNA polymerase and thereby contributes to the rapidity of the heat shock response.