Cysteine-to-alanine replacements in the Escherichia coli SoxR protein and the role of the [2Fe-2S] centers in transcriptional activation
Cysteine-to-alanine replacements in the Escherichia coli SoxR protein and the role of the [2Fe-2S] centers in transcriptional activation
复制标题
DOI：
10.1093/nar/25.8.1469
复制
发表时间：
1997-04-15
影响因子：
14.9
通讯作者：
Demple, B
中科院分区：
文献类型：
--
作者：
Bradley, TM;Hidalgo, E;Demple, B
大肠杆菌soxRS调节子在两个转录阶段激活氧化应激和抗生素抗性基因，SoxR蛋白在暴露于过量超氧化物或一氧化氮的细胞中被激活，然后刺激soxS基因的转录，其产物反过来激活大于或等于10个调节子启动子，纯化的SoxR蛋白是一个同源二聚体，含有一对soxS体外转录所必需的[2Fe-2S]中心，[2Fe-2S]中心被认为是由SoxR中的四个半胱氨酸残基的C-末端簇锚定的。在这里，我们分析了单个半胱氨酸被丙氨酸残基（Cys->Ala）取代的突变SoxR衍生物。无细胞提取物中的突变体蛋白以野生型亲和力结合soxS启动子，但纯化后缺乏Fe或可检测到的体外soxS转录活性。体内电子顺磁共振测量表明Cys->Ala蛋白缺乏野生型SoxR所见的[2Fe-2S]中心，Cys->Ala突变体蛋白在体内响应百草枯时未能激活soxS表达，然而，当表达至约5%的细胞蛋白质时，Cys->Ala衍生物使基础soxS转录增加2-4倍。Cys 119->Ala突变体蛋白的过表达强烈干扰野生型SoxR响应百草枯的soxS活化，这些研究证明了[2Fe-2S]中心在体内SoxR激活中的重要作用;这些数据也可能表明SoxR的转录激活的氧化剂非依赖性机制。
The Escherichia coli soxRS regulon activates oxidative stress and antibiotic resistance genes in two transcriptional stages, SoxR protein becomes activated in cells exposed to excess superoxide or nitric oxide and then stimulates transcription of the soxS gene, whose product in turn activates greater than or equal to 10 regulon promoters, Purified SoxR protein is a homodimer containing a pair of [2Fe-2S] centers essential for soxS transcription in vitro, The [2Fe-2S] centers are thought to be anchored by a C-terminal cluster of four cysteine residues in SoxR, Here we analyze mutant SoxR derivatives with individual cysteines replaced by alanine residues (Cys-->Ala). The mutant proteins in cell-free extracts bound the soxS promoter with wild-type affinity, but upon purification lacked Fe or detectable transcriptional activity for soxS in vitro, Electron paramagnetic resonance measurements in vivo indicated that the Cys-->Ala proteins lacked the [2Fe-2S] centers seen for wild-type SoxR, The Cys-->Ala mutant proteins failed to activate soxS expression in vivo in response to paraquat, a superoxide- generating agent, However, when expressed to similar to 5% of the cell protein, the Cys-->Ala derivatives increased basal soxS transcription 2-4-fold, Overexpression of the Cys119-->Ala mutant protein strongly interfered with soxS activation by wild-type SoxR in response to paraquat, These studies demonstrate the essential role of the [2Fe-2S] centers for SoxR activation in vivo; the data may also indicate oxidant-independent mechanisms of transcriptional activation by SoxR.