Regulatory Region C of the E. coliHeat Shock Transcription Factor, σ 32, Constitutes a DnaK Binding Site and is Conserved Among Eubacteria

Abstract

The E. coliheat shock response is regulated at the transcriptional level through stress-dependent controls of the heat shock promoter-specific σ 32subunit of RNA polymerase. A key aspect of this regulation, the sensing of stress and transmission of this information to σ 32, involves the chaperone system formed by the DnaK, DnaJ and GrpE heat shock proteins. This system mediates stress-dependent controls of levels and activity of σ 32which rely, at least in part, on direct association of DnaK and DnaJ with σ 32. We identified DnaK binding sites within the σ 32sequence by probing a cellulose-bound peptide library scanning σ 32. Two sites with high affinity for DnaK, containing the motifs RKLFFNLR and LRNWRIVK, were located centrally and peripherally, respectively, to the region C of σ 32, previously implicated genetically in chaperone-dependent control of σ 32levels. Cloning and sequencing of rpoHhomologs from five Gram-negative proteobacteria revealed that region C, including the DnaK binding motif central to it, is highly conserved among σ 32homologs but missing in other σ factors. We propose that binding of DnaK to region C is central to a conserved regulatory mechanism allowing the sensing of stress by the heat shock gene transcription machinery.