327회 The redox-dependent modulation of RsrA, an anti-sigma factor that…

327회

연사 : 노 정 혜 , 서울대학교 자연과학대학

제목:  The redox-dependent modulation of RsrA, an anti-sigma factor that regulates thioredoxin operon in Streptomyces coelicolor

Abstract

SigR (R) is a sigma factor responsible for inducing the thioredoxin system in response to oxidative stress in Streptomyces coelicolor. RsrA specifically binds to R and inhibits R-directed transcription under reducing conditions. Exposure to H2O2 or thiol-specific oxidant diamide dissociates R-RsrA complex. RsrA contains 7 cysteine residues in 105 total amino acid residues. Using single and multiple cysteine substitutuin mutagenesis into serines, we found out that 4 cysteines at 11, 41, 44, and 61st position are necessary for the activity of RsrA, as judged by Rbinding assay as well as inhibition of R-directed transcription in vitro. The mutation at each of these positions caused loss of R-binding ability, suggesting that the free thiol groups, not just the absence of disulfide bonds between them, are necessary for RsrA function. Since the essential thiols include those in the conserved HXXXCXXC motif, a putative metal binding site, we measured the content of metals in both wild type and triple-substitution mutant purified from E. coli containing S. coelicolor rsrA gene in pET15b, by atomic absorption and ICP-emission spectral analyses. Metals such as Ca, Mg, and Zn were detected in varying amounts depending on the batch of preparation. The content of Zn was systematically lowered under oxidized condition, suggesting that it depends on the thiol-disulfide status of the cysteines in RsrA and Zn may be involved in redox-dependent modulation of RsrA. However, the RsrA which was renatured in the absence of any metals from the guanidine-HCltreated sample resumed the ability to bind R, implying that metals are not required for the binding activity of RsrA. SPR analysis using BIACORE also demonstrated that oxidized RsrA resumed Rbinding activity in the absence of any metals. Currently more investigation is under way to reveal the role of redox-dependent metal binding in modulation of RsrA activity.