Region C of the Escherichia coli heat shock sigma factor RpoH (sigma 32) contains a turnover element for proteolysis by the FtsH protease.
Transcription of most heat shock genes in Escherichia coli is initiated by the alternative sigma factor sigma(32) (RpoH). At physiological temperatures, RpoH is rapidly degraded by chaperone-mediated FtsH-dependent proteolysis. Several RpoH residues critical for degradation are located in the highly conserved region 2.1. However, additional residues were predicted to be involved in this process. We introduced mutations in region C of RpoH and found that a double mutation (A131E, K134V) significantly stabilized RpoH against degradation by the FtsH protease. Single-point mutations at these positions only showed a slight effect on RpoH stability. Both double and single amino acid substitutions did not impair sigma factor activity as demonstrated by a groE-lacZ reporter gene fusion, Western blot analysis of heat shock gene expression and increased heat tolerance in the presence of these proteins. Combined mutations in regions 2.1 and C further stabilized RpoH. We also demonstrate that an RpoH fragment composed of residues 37-147 (including regions 2.1 and C) is degraded in an FtsH-dependent manner. We conclude that in addition to the previously described turnover element in region 2.1, a previously postulated second region important for proteolysis of RpoH by FtsH lies in region C of the sigma factor.
Obrist
Markus
M
Institute of Microbiology, ETH Zürich, Switzerland.
Langklotz
Sina
S
Milek
Sonja
S
Führer
Frank
F
Narberhaus
Franz
F
D049069:ATP-Dependent Proteases / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D055550:Protein Stability; D016415:Sequence Alignment; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D012808:Sigma Factor; C069774:heat-shock sigma factor 32; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli