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Mutational analysis of RsrA, a zinc‐binding anti‐sigma factor with a thiol–disulphide redox switch
Author(s) -
Paget Mark S. B.,
Bae JaeBum,
Hahn MiYoung,
Li Wei,
Kleanthous Colin,
Roe JungHye,
Buttner Mark J.
Publication year - 2001
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2001.02298.x
Subject(s) - sigma factor , biology , streptomyces coelicolor , thioredoxin reductase , thioredoxin , mutant , biochemistry , thiol , redox , cysteine , chemistry , rna polymerase , enzyme , escherichia coli , gene , organic chemistry
In the Gram‐positive bacterium, Streptomyces coelicolor A3(2), expression of the thioredoxin system is modulated by a sigma factor called σ R in response to changes in the cytoplasmic thiol–disulphide status, and the activity of σ R is controlled post‐translationally by an anti‐sigma factor, RsrA. In vitro , the anti‐sigma factor activity of RsrA, which contains seven cysteines, correlates with its thiol–disulphide redox status. Here, we investigate the function of RsrA in vivo . A constructed rsrA null mutant had very high constitutive levels of disulphide reductase activity and σ R ‐dependent transcription, confirming that RsrA is a negative regulator of σ R and a key sensor of thiol–disulphide status. Targeted mutagenesis revealed that three of the seven cysteines in RsrA (C11, C41 and C44) were essential for anti‐sigma factor activity and that a mutant RsrA protein containing only these three cysteines was active and still redox sensitive in vivo . We also show that RsrA is a metalloprotein, containing near‐stoichiometric amounts of zinc. On the basis of these data, we propose that a thiol–disulphide redox switch is formed between two of C11, C41 and C44, and that all three residues play an essential role in anti‐sigma factor activity in their reduced state, perhaps by acting as ligands for zinc. Unexpectedly, rsrA null mutants were blocked in sporulation, probably as a consequence of an increase in the level of free σ R .