Conserved Region 2.1 of Escherichia coli Heat Shock Transcription Factor σ³² Is Required for Modulating both Metabolic Stability and Transcriptional Activity

ABSTRACT

Escherichia coli heat shock transcription factor σ³² is rapidly degraded in vivo, with a half-life of about 1 min. A set of proteins that includes the DnaK chaperone team (DnaK, DnaJ, GrpE) and ATP-dependent proteases (FtsH, HslUV, etc.) are involved in degradation of σ³². To gain further insight into the regulation of σ³² stability, we isolated σ³² mutants that were markedly stabilized. Many of the mutants had amino acid substitutions in the N-terminal half (residues 47 to 55) of region 2.1, a region highly conserved among bacterial σ factors. The half-lives ranged from about 2-fold to more than 10-fold longer than that of the wild-type protein. Besides greater stability, the levels of heat shock proteins, such as DnaK and GroEL, increased in cells producing stable σ³². Detailed analysis showed that some stable σ³² mutants have higher transcriptional activity than the wild type. These results indicate that the N-terminal half of region 2.1 is required for modulating both metabolic stability and the activity of σ³². The evidence suggests that σ³² stabilization does not result from an elevated affinity for core RNA polymerase. Region 2.1 may, therefore, be involved in interactions with the proteolytic machinery, including molecular chaperones.