title: Role of region C in regulation of the heat shock gene-specific sigma factor of Escherichia coli, sigma32.
abstract: Expression of heat shock genes is controlled in Escherichia coli by the antagonistic action of the sigma32 subunit of RNA polymerase and the DnaK chaperone system, which inactivates sigma32 by stress-dependent association and mediates sigma32 degradation by the FtsH protease. A stretch of 23 residues (R122 to Q144) conserved among sigma32 homologs, termed region C, was proposed to play a role in sigma32 degradation, and peptide analysis identified two potential DnaK binding sites central and peripheral to region C. Region C is thus a prime candidate for mediating stress control of sigma32, a hypothesis that we tested in the present study. A peptide comprising the central DnaK binding site was an excellent substrate for FtsH, while a peptide comprising the peripheral DnaK binding site was a poor substrate. Replacement of a single hydrophobic residue in each DnaK binding site by negatively charged residues (I123D and F137E) strongly decreased the binding of the peptides to DnaK and the degradation by FtsH. However, introduction of these and additional region C alterations into the sigma32 protein did not affect sigma32 degradation in vivo and in vitro or DnaK binding in vitro. These findings do not support a role for region C in sigma32 control by DnaK and FtsH. Instead, the sigma32 mutants had reduced affinities for RNA polymerase and decreased transcriptional activities in vitro and in vivo. Furthermore, cysteines inserted into region C allowed cysteine-specific cross-linking of sigma32 to RNA polymerase. Region C thus confers on sigma32 a competitive advantage over other sigma factors to bind RNA polymerase and thereby contributes to the rapidity of the heat shock response.
authors.affiliation: Institut für Biochemie und Molekularbiologie, Universität Freiburg, D-79104 Freiburg, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Arsène
authors.forename: T
authors.initials: T
authors.lastname: Tomoyasu
authors.forename: A
authors.initials: A
authors.lastname: Mogk
authors.forename: C
authors.initials: C
authors.lastname: Schirra
authors.forename: A
authors.initials: A
authors.lastname: Schulze-Specking
authors.forename: B
authors.initials: B
authors.lastname: Bukau
mesh_terms: D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001667:Binding, Competitive; D017124:Conserved Sequence; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006207:Half-Life; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012808:Sigma Factor; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D012808:Sigma Factor; D014157:Transcription Factors; C069774:heat-shock sigma factor 32; D012321:DNA-Directed RNA Polymerases; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli; D003545:Cysteine