Identification of a turnover element in region 2.1 of Escherichia coli σ32 by a bacterial one-hybrid approach

Induction of the heat shock response in Escherichia coli requires the alternative sigma factor sigma(32) (RpoH). The cellular concentration of sigma(32) is controlled by proteolysis involving FtsH, other proteases, and the DnaKj chaperone system. To identify individual sigma(32) residues critical for degradation, we used a recently developed bacterial one-hybrid system and screened for stabilized versions of sigma(32). The five single point mutations that rendered the sigma factor more stable mapped to positions L47, A50, and 154 in region 2.1. Strains expressing the stabilized sigma(32) 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 et-helix in region 2.1, suggesting that they are positioned to interact with proteins of the degradation machinery.