The chromosomal arsR gene of Escherichia coli encodes a trans-acting metalloregulatory protein
The chromosomal arsR gene of Escherichia coli encodes a trans-acting metalloregulatory protein
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DOI：
10.1074/jbc.271.5.2427
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发表时间：
1996-02-02
影响因子：
4.8
通讯作者：
Rosen, BP
中科院分区：
文献类型：
--
作者：
Xu, C;Shi, WP;Rosen, BP
质粒编码的砷抗性（ars）操纵子赋予对砷和锑的高水平抗性，而大肠杆菌染色体编码的ars操纵子赋予低水平抗性。通过引物延伸定位染色体ars mRNA的转录起始位点，并鉴定了推定的-10和-35启动子识别位点。arsR基因，在这个操纵子的第一个基因，克隆使用聚合酶链反应。表达并纯化arsR基因产物，即ArsR阻遏物。凝胶迁移率变动分析的结果表明，阻遏物是一种DNA结合蛋白，它与含有染色体ars启动子的DNA片段结合。通过DNA酶I足迹分析确定的特异性结合位点跨越启动子区域中的33个核苷酸，包括推定的-35启动子元件。通过构建和表达截短的arsR基因与成熟形式β-内酰胺酶（blaM '）的编码区之间的一系列框内融合物，表明ArsR是调节染色体ars操纵子表达的反式作用阻遏物。此外，染色体编码的阻遏物可以调节质粒R773的ars操纵子的表达，并且R773阻遏物可以交叉调节来自染色体操纵子的表达。
Plasmid-encoded arsenical resistance (ars) operons confer high level resistance to arsenicals and antimonials, while the chromosomally encoded ars operon of Escherichia coli bestows low level resistance. The transcriptional start site of the chromosomal ars mRNA was mapped by primer extension, and putative -10 and -35 promoter recognition sites were identified. The arsR gene, the first gene in this operon, was cloned using polymerase chain reaction. The arsR gene product, the ArsR repressor, was expressed and purified. The results of gel mobility shift assays indicated that the repressor is a DNA binding protein that binds to a fragment of DNA containing the chromosomal ars promoter. The specific binding site, as determined by DNase I footprint analysis, spans 33 nucleotides in the promoter region, including the putative -35 promoter element. By construction and expression of a series of in-frame fusions between truncated arsR genes and the coding region for the mature form of beta-lactamase (blaM'), it was shown that ArsR is a trans-acting repressor that regulates expression of the chromosomal ars operon. In addition, the chromosomally-encoded repressor can regulate expression of the ars operon of plasmid R773, and the R773 repressor can cross-regulate expression from the chromosomal operon.