Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur
Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur
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DOI：
10.1073/pnas.1017744108
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发表时间：
2011-03-22
影响因子：
11.1
通讯作者：
Roe, Jung-Hye
中科院分区：
文献类型：
--
作者：
Shin, Jung-Ho;Jung, Hoi Jong;Roe, Jung-Hye
锌是细胞必需的过渡金属之一。锌的过量或缺乏都是有害的，细胞利用高度敏感的锌结合调节剂来实现体内平衡。在这篇文章中，我们提出了一个晶体结构的活性Zur从天蓝色链霉菌与三个锌结合位点（C-，M-和D-位）。三个位点的突变差异影响孢子形成和靶基因的转录，例如C-和M-位点突变抑制孢子形成和解阻遏所有检测的靶基因，而D-位点突变不影响孢子形成和解阻遏仅敏感基因。代表性的金属网站突变体的生化和光谱分析表明，C-网站服务的结构作用，而M-和D-网站调节DNA结合活性的开关和微调器，分别。与突变对靶基因的差异效应一致，TPEN的锌螯合在体内对某些基因（znuA，rpmF 2）的去抑制作用比其他基因（rpmG 2，SCO 7682）更敏感。在体外不同的启动子上形成的TPEN-DNA复合物观察到类似的TPEN敏感性模式。敏感的启动子与Zur的结合亲和力低于不太敏感的启动子。对于两个启动子组，EDTA处理的apo-Zur在不同浓度的添加锌下获得其DNA结合活性，对于较不敏感和敏感的启动子，分别对应于4.5 × 10（-16）M和7.9 × 10（-16）M的游离锌浓度。靶基因的分级表达是响应锌可用性而微妙地调节β-DNA结合亲和力的巧妙结果。它使细菌能够以更高的灵敏度检测金属消耗并优化基因表达模式。
Zinc is one of the essential transition metals in cells. Excess or lack of zinc is detrimental, and cells exploit highly sensitive zinc-binding regulators to achieve homeostasis. In this article, we present a crystal structure of active Zur from Streptomyces coelicolor with three zinc-binding sites (C-, M-, and D-sites). Mutations of the three sites differentially affected sporulation and transcription of target genes, such that C-and M-site mutations inhibited sporulation and derepressed all target genes examined, whereas D-site mutations did not affect sporulation and derepressed only a sensitive gene. Biochemical and spectroscopic analyses of representative metal site mutants revealed that the C-site serves a structural role, whereas the M-and D-sites regulate DNA-binding activity as an on-off switch and a fine-tuner, respectively. Consistent with differential effect of mutations on target genes, zinc chelation by TPEN derepressed some genes (znuA, rpmF2) more sensitively than others (rpmG2, SCO7682) in vivo. Similar pattern of TPEN-sensitivity was observed for Zur-DNA complexes formed on different promoters in vitro. The sensitive promoters bound Zur with lower affinity than the less sensitive ones. EDTA-treated apo-Zur gained its DNA binding activity at different concentrations of added zinc for the two promoter groups, corresponding to free zinc concentrations of 4.5 x 10(-16) M and 7.9 x 10(-16) M for the less sensitive and sensitive promoters, respectively. The graded expression of target genes is a clever outcome of subtly modulating Zur-DNA binding affinities in response to zinc availability. It enables bacteria to detect metal depletion with improved sensitivity and optimize gene-expression pattern.