Region C of the Escherichia coli heat shock sigma factor RpoH (σ³²) contains a turnover element for proteolysis by the FtsH protease

• Published in: FEMS microbiology letters 2009-01, Vol.290 (2), p.199-208
• Main Authors: Obrist, Markus, Langklotz, Sina, Milek, Sonja, Führer, Frank, Narberhaus, Franz
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Amino acids; ATP-Dependent Proteases - genetics; ATP-Dependent Proteases - metabolism; Bacteria; Bacteriology; Biological and medical sciences; Degradation; E coli; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; FtsH protease; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Regulation, Bacterial; Gene fusion; Heat shock; Heat shock factors; heat stress; Heat tolerance; Heat-Shock Proteins - chemistry; Heat-Shock Proteins - genetics; Heat-Shock Proteins - isolation & purification; Heat-Shock Proteins - metabolism; Hydrolysis; Metabolism. Enzymes; Microbiology; Molecular Sequence Data; Mutation; Mutation, Missense; Physiological effects; Protease; Protein Stability; Proteolysis; Reporter gene; Residues; RpoH; Sequence Alignment; Sigma factor; Sigma Factor - chemistry; Sigma Factor - genetics; Sigma Factor - isolation & purification; Sigma Factor - metabolism; Transcription
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.2008.01423.x

Transcription of most heat shock genes in Escherichia coli is initiated by the alternative sigma factor σ³² (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.