Identification of a Turnover Element in Region 2.1 of Escherichia coli σ ³² by a Bacterial One-Hybrid Approach

Abstract

Induction of the heat shock response in Escherichia coli requires the alternative sigma factor 32 (RpoH). The cellular concentration of 32 is controlled by proteolysis involving FtsH, other proteases, and the DnaKJ chaperone system. To identify individual 32 residues critical for degradation, we used a recently developed bacterial one-hybrid system and screened for stabilized versions of 32 . 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 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 ␣-helix in region 2.1, suggesting that they are positioned to interact with proteins of the degradation machinery.

Region 2.1 of the Escherichia coli heat-shock sigma factor RpoH (σ 32) is necessary but not sufficient for degradation by the FtsH protease

“…The predicted three-dimensional structure suggested that amino acids at positions 47, 50 and 54 are positioned on the same side of the putative ahelix 12a in region 2.1 of RpoH (Obrist & Narberhaus, 2005). The sequence of region 2.1 from 21 bacterial species shows that several RpoH proteins contain a proline residue at position 47 (Fig.…”

“…Additional point mutations in region 2.1 that render E. coli RpoH more active in vivo Point mutations in RpoH region 2.1 at positions 47, 50, 51, 54 or 55 were shown to turn the sigma factor into a stabilized protein, resulting in enhanced transcription of an RpoH-dependent promoter in vivo (Horikoshi et al, 2004;Obrist & Narberhaus, 2005). The predicted three-dimensional structure suggested that amino acids at positions 47, 50 and 54 are positioned on the same side of the putative ahelix 12a in region 2.1 of RpoH (Obrist & Narberhaus, 2005).…”

“…In vivo degradation and sample preparation of E. coli DrpoH cells carrying pEC5217 derivatives was performed as previously described (Obrist & Narberhaus, 2005). Equivalent amounts of protein (10 mg) were separated on 12 % SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Hybond-C; Amersham).…”

“…2007 Blaszczak et al, 1999;McCarty et al, 1996;Nagai et al, 1994;Narberhaus & Balsiger, 2003). Recently, an important turnover element was narrowed down to the highly conserved region 2.1 (Horikoshi et al, 2004;Obrist & Narberhaus, 2005). Several point mutations in this region markedly stabilized RpoH against FtsH-mediated proteolysis.…”

“…Horikoshi et al (2004) provided evidence that the enhanced activity and stability of RpoH proteins mutated in region 2.1 is not the result of an increased interaction with RNAP core enzyme. Since the critical residues line up on the same face of a putative a-helix, we speculated that these amino acids might be exposed for interaction(s) with components of the proteolytic machinery, either chaperones or the FtsH protease itself (Obrist & Narberhaus, 2005).…”