Identification of a Turnover Element in Region 2.1 of Escherichia coli σ32 by a Bacterial One-Hybrid Approach

Abstract

Induction of the heat shock response in Escherichia coli requires the alternative sigma factor σ32 (RpoH). The cellular concentration of σ32 is controlled by proteolysis involving FtsH, other proteases, and the DnaKJ chaperone system. To identify individual σ32 residues critical for degradation, we used a recently developed bacterial one-hybrid system and screened for stabilized versions of σ32. The five single point mutations that rendered the sigma factor more stable mapped to positions L47, A50, and I54 in region 2.1. Strains expressing the stabilized σ32 variants exhibited elevated transcriptional activity, as determined by a groE-lacZ fusion. Structure calculations predicted that the three mutated residues line up on the same face of an α-helix in region 2.1, suggesting that they are positioned to interact with proteins of the degradation machinery.