Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for oxidation and transcriptional activation.
OxyR is a redox-sensitive transcriptional regulator of the LysR family which activates the expression of genes important for the defense against hydrogen peroxide in Escherichia coli and Samonella typhimurium. OxyR is sensitive to oxidation and reduction, and only oxidized OxyR is able to activate transcription of its target genes. Using site-directed mutagenesis, we found that one cysteine residue (C-199) is critical for the redox sensitivity of OxyR, and a C-199-->S mutation appears to lock the OxyR protein in the reduced form. We also used a random mutagenesis approach to isolate eight constitutively active mutants. All of the mutations are located in the C-terminal half of the protein, and four of the mutations map near the critical C-199 residue. In vivo as well as in vitro transcription experiments showed that the constitutive mutant proteins were able to activate transcription under both oxidizing and reducing conditions, and DNase I footprints showed that this activation is due to the ability of the mutant proteins to induce cooperative binding of RNA polymerase. Unexpectedly, RNA polymerase was also found to reciprocally affect OxyR binding.
Kullik
I
I
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Toledano
M B
MB
Tartaglia
L A
LA
Storz
G
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics*; D011485:Protein Binding; D012097:Repressor Proteins; D012641:Selection, Genetic; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C499299:oxyR protein, E coli; C073470:LysR protein, Bacteria; D006861:Hydrogen Peroxide; D012321:DNA-Directed RNA Polymerases; D003545:Cysteine