Mutational analysis of RsrA, a zinc-binding anti-sigma factor with a thiol-disulphide redox switch

Paget, Mark S. B., Bae, Jae-Bum, Hahn, Mi-Young, Li, Wei, Kleanthous, Colin, Roe, Jung-Hye and Buttner, Mark J. (2001) Mutational analysis of RsrA, a zinc-binding anti-sigma factor with a thiol-disulphide redox switch. Molecular Microbiology, 39 (4). pp. 1036-1047. ISSN 1365-2958

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Abstract

In the Gram-positive bacterium, Streptomyces coelicolor A3(2), expression of the thioredoxin system is modulated by a sigma factor called σR in response to changes in the cytoplasmic thiol–disulphide status, and the activity of σR 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 σR-dependent transcription, confirming that RsrA is a negative regulator of σR 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 σR.

Item Type:	Article
Additional Information:	Funding information: This work was funded by BBSRC grant 208/P11071 (to M.J.B. and M.S.B.P.), by a grant-in-aid to the John Innes Centre from the BBSRC and by a grant from the Korean Science and Engineering Foundation through the Research Centre for Molecular Microbiology at Seoul National University (to J.H.R.).
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	LivePure Connector
Date Deposited:	17 Apr 2019 16:30
Last Modified:	24 Sep 2024 10:24
URI:	https://ueaeprints.uea.ac.uk/id/eprint/70636
DOI:	10.1046/j.1365-2958.2001.02298.x

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