S-Space College of Natural Sciences (자연과학대학) Dept. of Biological Sciences (생명과학부) Theses (Ph.D. / Sc.D._생명과학부)
Regulation of gene expression for Fe-SOD and Ni-SOD by nickel-responsive nur in streptomyces coelicolor : Streptomyces coelicolor 에서 니켈에 반응하는 Nur에 의한 슈퍼옥사이드 디스뮤타제 유전자발현의 조절
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- 자연과학대학 생명과학부
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- 서울대학교 대학원
- Nur ; 니켈 ; 슈퍼옥사이드 디스뮤타제
- 학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2014. 2. 노정혜.
- Nur (Nickel uptake regulator) is a nickel-responsive transcription factor the regulates nickel homeostasis and anti-oxidative response in S. coelicolor. Nur is a unique nickel-specific Fur-family regulator. In S. coelicolor, expression of the sodF and sodN genes is inversely regulated by Nur. Superoxide dismutases (SODs) are widely distributed enzymes that convert superoxides to hydrogen peroxide and molecular oxygen, using various metals as cofactors. Many actinobacteria contain genes for both Ni-containing (sodN) and Fe-containing (sodF) SODs. With sufficient nickel, Nur directly represses sodF transcription, while inducing sodN indirectly. Bioinformatic search revealed that a conserved 19 nt stretch upstream of sodN matches perfectly with the sodF downstream sequence. So we estimated that Nur could activates sodN gene through sodF transcripts, double repression mechanism. First we checked the existence of transcripts containing anti-sodN region by S1 mapping. We found that the sodF gene produced a stable small-sized RNA species (s-SodF) that harbors the anti-sodN sequence complementary to sodN mRNA from the 5 end up to the ribosome binding site and s-SodF is approximately ~90 nt confirmed by northern blotting. We could not detect any Nur box in nearby 5 end of s-SodF and s-SodF is sensitive to 5-monophosphate-specific exonuclease. These data strongly indicated that the s-SodF RNA is a likely processed product of sodF mRNA. In order to check the s-SodF effect on sodN expression, we introduced to △sodF mutant an overexpression plasmid for s-SodF RNA whose expression was driven by a strong ermE* promoter. In △sodF mutant, half-life of sodN is delayed to about 16min compared to WT (3min). But, in s-SodF overexpression strain, delayed half-life of sodN is restored to 7min. This result indicated that the s-SodF RNA caused a significant decrease in the half-life of the sodN mRNA. Therefore, Nur activates sodN expression through inhibiting the synthesis of sodF mRNA, from which inhibitory s-SodF RNA is generated. This reveals a novel mechanism by which antagonistic regulation of one gene is achieved by small RNA processed from the 3UTR of another genes mRNA.
Recently, we reported crystal structure of Nur and Nur is homodimer and two DNA binding domains (DB-domain) are attached to the dimeric core constructed by two dimerization domains (D-domain). It contains a unique nickel-specific metal site (Ni-site) and nonspecific common metal site (M-site) per each monomer. Nur also has two Cys-X–X Cys motif but no zinc coordination is shown in crystal structure. Electrophoretic mobility shift assay (EMSA) using Nur overexpression cell extract by PET system in E. coli showed that Ni-site and M-site not Cys4 site are both important for DNA binding activity of Nur. This result coincide with Crystal structure of Nur. In order to confirm the critical residues of Nur in vivo, we introduced to △nur mutant an integration vector for pnur::nur variants expression and we checked the sodF repression activity of Nur by S1 mapping. We found that Ni-site was still important for Nur in vivo but mutations of M-site residues could not affect repression activity of Nur. Interestingly, Cys96S, Cys133S and Cys136 which consist of two Cys-X–X Cys motif were confirmed as critical residues of Nur. So we tried to do EMSA using S. coelicolor cell extract and result showed that Ni-site and two Cys-X–X Cys motif not M-site are both important for Nur in vivo. This result match with S1 mapping data. These experiment suggest the possibility that Nur coordinate zinc in two Cys-X–X Cys motif and this Cys4-Zn site will be critical structural metal binding site like BsuPerR in vivo.
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