Xu C, Shi W, Rosen B P
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
J Biol Chem. 1996 Feb 2;271(5):2427-32. doi: 10.1074/jbc.271.5.2427.
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.
质粒编码的砷抗性（ars）操纵子赋予对砷化物和锑化物的高水平抗性，而大肠杆菌染色体编码的ars操纵子则赋予低水平抗性。通过引物延伸法确定了染色体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。使用聚合酶链反应克隆了该操纵子中的第一个基因arsR基因。表达并纯化了arsR基因产物，即ArsR阻遏蛋白。凝胶迁移率变动分析结果表明，该阻遏蛋白是一种DNA结合蛋白，可与包含染色体ars启动子的DNA片段结合。通过DNase I足迹分析确定的特异性结合位点在启动子区域跨越33个核苷酸，包括推定的-35启动子元件。通过构建并表达一系列截短的arsR基因与β-内酰胺酶成熟形式（blaM'）编码区之间的读框内融合体，表明ArsR是一种反式作用阻遏蛋白，可调节染色体ars操纵子的表达。此外，染色体编码的阻遏蛋白可调节质粒R773的ars操纵子的表达，而R773阻遏蛋白可交叉调节染色体操纵子的表达。
