The ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum is an iron-responsive transcriptional repressor in vitro
The ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum is an iron-responsive transcriptional repressor in vitro
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DOI：
10.1074/jbc.m404924200
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发表时间：
2004-07-30
影响因子：
4.8
通讯作者：
O'Brian, MR
中科院分区：
文献类型：
--
作者：
Friedman, YE;O'Brian, MR
毛皮蛋白抑制细菌中铁反应基因的转录。 Fur 是一种锌金属蛋白的发现以及在体外研究中使用 Fe2+ 的替代金属对 Fur 是否是直接铁传感器提出了质疑。在本研究中，我们发现，在金属存在下，来自日本慢生根瘤菌 (BjFur) 的毛皮 (BjFur) 与其靶标 DNA 的亲和力在金属存在下增加了 30 倍，K-d 值约为 2 nM。 DNase I 足迹实验表明，在 Fe2+ 存在的情况下，BjFur 可以保护其在 irr 基因启动子内的结合位点，但在金属不存在的情况下则不然，这表明 DNA 结合是 Fe2+ 依赖性的。在没有金属的情况下，使用纯化的组分，BjFur 不会抑制 irr 启动子的体外转录，但 BjFur 在 Fe2+ 存在的情况下会抑制转录。因此，BjFur 是体外铁反应性转录抑制因子。从最近铜绿假单胞菌 Fur 晶体结构推断出的调节性 Fe2+ 结合位点（位点 1）和结构性 Zn2+ 结合位点（位点 2）由许多 Fur 蛋白（包括 BjFur）中高度保守的氨基酸组成。在位点 1 (BjFurS1) 或位点 2 (BjFurS2) 中含有取代的 BjFur 突变体以高亲和力结合 DNA，并以 Fe2+ 依赖性方式在体外抑制转录。有趣的是，BjFurS2 只有一个二聚体占据了 irr 启动子，而野生型和 BjFurS1 显示出一个或两个二聚体占据。我们认为，从铜绿假单胞菌毛皮结构推论的金属结合位点的假定功能不能外推到整个细菌毛皮蛋白。
The Fur protein represses transcription of iron-responsive genes in bacteria. The discovery that Fur is a zinc metalloprotein and the use of surrogate metals for Fe2+ for in vitro studies question whether Fur is a direct iron sensor. In the present study, we show that the affinity of Fur from Bradyrhizobium japonicum (BjFur) for its target DNA increases 30-fold in the presence of metal, with a K-d value of about 2 nM. DNase I footprinting experiments showed that BjFur protected its binding site within the irr gene promoter in the presence of Fe2+ but not in the absence of metal, showing that DNA binding is Fe2+-dependent. BjFur did not inhibit in vitro transcription from the irr promoter using purified components in the absence of metal, but BjFur repressed transcription in the presence of Fe2+. Thus, BjFur is an iron-responsive transcriptional repressor in vitro. A regulatory Fe2+-binding site (site 1) and a structural Zn2+-binding site (site 2) inferred from the recent crystal structure of Fur from Pseudomonas aeruginosa are composed of amino acids highly conserved in many Fur proteins, including BjFur. BjFur mutants containing substitutions in site 1 (BjFurS1) or site 2 (BjFurS2) bound DNA with high affinity and repressed transcription in vitro in an Fe2+-dependent manner. Interestingly, only a single dimer of BjFurS2 occupied the irr promoter, whereas the wild type and BjFurS1 displayed one- or two-dimer occupancy. We suggest that the putative functions for metal-binding sites deduced from the structure of P. aeruginosa Fur cannot be extrapolated to bacterial Fur proteins as a whole.