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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
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | 노정혜 | - |
dc.contributor.author | 김혜미 | - |
dc.date.accessioned | 2017-07-14T00:47:06Z | - |
dc.date.available | 2017-07-14T00:47:06Z | - |
dc.date.issued | 2014-02 | - |
dc.identifier.other | 000000017008 | - |
dc.identifier.uri | https://hdl.handle.net/10371/121379 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2014. 2. 노정혜. | - |
dc.description.abstract | 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. | - |
dc.description.tableofcontents | ABSTRACT i
CONTENTS iii LIST OF FIGURES vii LIST OF TABLES x ABBREVIATIONS xi CHAPTER I. INTRODUCTION 1 I.1. Biology of Streptomyces coelicolor 2 I.2. Superoxide dismutase (SOD) system 3 I.2.1. Regulation of SOD system in bacteria 3 I.3. Bacterial Metalloregulators 4 I.3.1. Fur family regulators 6 I.3.2. Structural features of Fur family members 7 I.4. Role and regulation of Nickel 13 I.5. Nur in S. coelicolor 13 I.6. Small regulatory RNAs in bacteria 14 I.6.1. In Hfq-containg bacteria 17 I.6.2. In Actinobacteria with no Hfq homologs 18 I.7. RNA processing 19 CHAPTER II. MATERIALS AND METHODS 21 II.1. Strains and growth conditions 22 II.1.1. Streptomyces coelicolor 22 II.1.2. Escherichia coli 22 II.2. DNA manipulations 23 II.3. Polymerase Chain Reaction (PCR) 23 II.4. Construction of sodF transcript overproducing strain 23 II.5. RNA analysis 25 II.5.1. S1 mapping and northern analyses 25 II.5.2. 5' RACE 26 II.5.3. Exonuclease digestion of RNA 27 II.6. Overproduction of S. coelicolor Nur variants from E. coli 27 II.7. Complementation and expression of S. coelicolor Nur variants in vivo 28 II.8. Electrophoretic mobility shift assay (EMSA) 30 II.9. Western blotting 30 CHAPTER III. RESULTS 32 III.1. Inverse Regulation of Fe-SOD and Ni-SOD by Nur 33 III.1.1. Presence of complementarity between the sense strands of sodF and sodN genes 33 III.1.2. Verification of transcripts existence encompassing the anti-sodN sequence 37 III.1.3. Sequence and structure information of s-SodF 40 III.1.3.1. The 5 and 3 boundaries of s-SodF RNA 40 III.1.3.2. Secondary structure of s-SodF 45 III.1.4. s-SodF producing mechanism 45 III.1.4.1. Confirmation of another promoter of s-SodF 45 III.1.4.2. The 5 phosphorylation status of s-SodF 46 III.1.4.3. Test for possible RNases related to processing s-SodF 49 III.1.5. Function of s-SodF in vivo 49 III.1.5.1. sodN mRNA half life in sodF and sodF2 mutants 49 III.1.5.2. s-SodF RNA decreases the stability of sodN mRNA 51 III.1.6. Mutations in the anti-sodN region of s-SodF inactivate its inhibitory function 55 III.1.7. Growth phase-dependent antagonistic expression of sodN and sodF 58 III.1.7.1. sod genes expression in various growth phase 58 III.1.7.2. half life of sodN mRNA in various growth phase 62 III.1.8. Nickel responsiveness of sod genes 67 III.1.8.1. Nickel sensitivity of sod mutants 67 III.1.8.2. Responsive level of sod transcription to nickel 67 III.2. Determination of critical residues for Nur activity 73 III.2.1. Prediction of metal coordination ligands of Nur 73 III.2.2. Crystal structure of Nur 79 III.2.3. Metal binding sites of Nur 80 III.2.3.1. Metal-site (M-site) 80 III.2.3.2. Ni-site 80 III.2.3.3. Possible Zinc binding site (two Cys-X-X-Cys motif) 82 III.2.4.Verification of the various metal binding residues in Nur in vitro 83 III.2.4.1. Various Nur protein expression in E. coli 83 III.2.4.2. The binding activity of Nur variants in vitro 83 III.2.5.Verification of the various metal binding residues in Nur in vivo 84 III.2.5.1. Various Nur protein expression in S. coelicolor 84 III.2.5.2. The complementation effects of various Nur variant proteins in vivo 88 III.2.5.3. The binding activity of Nur variants in vivo 88 III.3. Overall prospect about Nur 90 CHAPTER IV. DISCUSSION 97 IV.1. General signification of s-SodF regulation 98 IV.1.1. Small regulatory RNA produced from a functional mRNA inhibits the expression of an antagonistically regulated gene 98 IV.1.2. Predicted occurrence of similar regulation 98 IV.1.3. Inverse regulation of isoenzymes and antagonistic proteins 105 IV.2. Prospects for Future Studies 106 References 107 국문초록 117 감사의 글 120 | - |
dc.format | application/pdf | - |
dc.format.extent | 4956509 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Nur | - |
dc.subject | 니켈 | - |
dc.subject | 슈퍼옥사이드 디스뮤타제 | - |
dc.subject.ddc | 570 | - |
dc.title | Regulation of gene expression for Fe-SOD and Ni-SOD by nickel-responsive nur in streptomyces coelicolor | - |
dc.title.alternative | Streptomyces coelicolor 에서 니켈에 반응하는 Nur에 의한 슈퍼옥사이드 디스뮤타제 유전자발현의 조절 | - |
dc.type | Thesis | - |
dc.description.degree | Doctor | - |
dc.citation.pages | xi, 121 | - |
dc.contributor.affiliation | 자연과학대학 생명과학부 | - |
dc.date.awarded | 2014-02 | - |
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