S-Space College of Natural Sciences (자연과학대학) Dept. of Biological Sciences (생명과학부) Theses (Ph.D. / Sc.D._생명과학부)
Regulation of zinc import and export genes by zinc-responsive Zur in Streptomyces coelicolor
방선균 Streptomyces coelicolor에서 아연의 획득과 방출을 조절하는 Zur의 전사 조절기작
- 자연과학대학 생명과학부
- Issue Date
- 서울대학교 대학원
- Streptomyces coelicolor; Fur family; Zur; zinc homeostasis; znuA; zincexporter; zitB; activation mechanism; mechanism structure model
- 학위논문 (박사)-- 서울대학교 대학원 자연과학대학 생명과학부, 2017. 8. 노정혜.
- In various bacteria Zur (Zinc-uptake-regulator), a zinc-specific regulator of Fur family, regulates genes for zinc uptake to maintain zinc homeostasis. It has also been suggested to control zinc mobilization by regulating some ribosomal proteins. The antibiotics-producing soil bacterium Streptomyces coelicolor contains four genes for Fur family regulators, and one (named as zur) is located downstream of the znuACB operon encoding a putative zinc uptake transporter. Zinc specifically represses the level of znuA transcript by Zur. Zn-Zur also represses genes for ribosomal proteins L31 (rpmE) and L33 (rpmG) for zinc mobilization. Previous ChIP-chip analysis and expression analysis in delta-zur mutant revealed a unique target of Zur, SCO6751, which is predicted to be a zinc-exporter and the expression of which is activated by Zur. The activation mechanism of SCO6751, which later was named zitB, was investigated. The zitB over-expressing cells showed white phenotype, being defective in sporulation, and decreased intracellular zinc content. Zinc-dependent gene activation by Zur occurred in two phases: at sub-femtomolar zinc concentrations (phase I) with concomitant repression of zinc-uptake genes, and at over micromolar zinc conditions (phase II). DNase I footprinting on zitB DNA was performed with fixed amount of Zur (2.7 µM) and varying ZnSO4 from 2.5 to 10 µM. The results demonstrated that the Zur-footprint extended further upstream as zinc increased, up to -138 nt from TSS. This expansion in Zur-binding region is likely to lie behind the induction of zitB by zinc in phase II. It can be postulated that high concentrations of zinc could have caused some changes in Zur and its DNA-binding behavior. Whether high zinc induces Zur oligomerization in the absence of DNA was examined. Zur appears not to form oligomers beyond dimer by itself under high zinc condition. The physiological role of zitB upstream sequence in vivo was examined. The effectiveness of the zitB upstream region in zinc-dependent gene activation was examined in vivo by using a heterologous reporter gene (GUS). Recombinant pzitB-GUS fusion plasmids that contain the zitB regulatory region up to Zur binding motif (from +50 ~ -60 nt) or including expanded Zur-binding region (up to -228 nt) were constructed on pSET152-based integration vector. The S1 mapping of GUS transcripts demonstrated that the zitB regulatory region with the Zur-box motif only allowed only marginal gene activation, whereas the longer zitB upstream sequence enabled full activation of the reporter gene expression. A molecular model of the Zur2-DNA complex in low zinc condition was presented. As the level of zinc increases to micromolar range, oligomeric Zur binding was observed in vitro. At micromolar zinc, formation of hexameric or octameric Zur bindings were captured by EMSA with limited amount of Zur. From EMSA, the formation of super-retarded complex with higher concentration of Zur indicated the possibility of multimerization of Zur, with or without DNA conformational change, to underlie the activation mechanism of phase II. Taken together these findings reveal a novel mode of zinc-dependent gene activation and an ingenious strategy to use a single metallo-regulator to control both the uptake and export genes over a wide range of zinc concentrations.