Xu C, Rosen B P
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
J Biol Chem. 1997 Jun 20;272(25):15734-8. doi: 10.1074/jbc.272.25.15734.
Arsenical resistance (ars) operons produce resistance to trivalent and pentavalent salts of the metalloids arsenic and antimony in cells of Escherichia coli. The first gene in the operon, arsR, was previously shown to encode a homodimeric trans-acting metalloregulatory repressor protein. Dimerization of ArsR was investigated using the yeast two-hybrid system in which the ArsR protein was fused to the Saccharomyces cerevisiae GAL4 DNA-binding domain and GAL4 activation domain to produce chimeric proteins. Transcriptional activation of lacZ reporter indicated that dimerization of the ArsR is stable in yeast. The results indicated that residues 1-8 and 90-117 are not required for ArsR dimerization. The genes for a series of truncated ArsR proteins containing six histidine tags were constructed and the proteins purified. The mass of each recombinant protein, as determined by size exclusion chromatography, was consistent with the results from two-hybrid analysis. The results of beta-galactosidase assays in vivo and gel mobility shift assays in vitro showed that dimers retained the ability to bind to the ars promoter and to respond to inducer, whereas monomeric ArsRs did neither. These results suggest that a core sequence of about 80 residues has all of the information necessary for dimerization, repression, and metal recognition.
抗砷操纵子可使大肠杆菌细胞对类金属砷和锑的三价及五价盐产生抗性。操纵子中的第一个基因arsR，先前已证明其编码一种同二聚体反式作用金属调节阻遏蛋白。利用酵母双杂交系统研究了ArsR的二聚化，其中将ArsR蛋白与酿酒酵母GAL4 DNA结合结构域和GAL4激活结构域融合，以产生嵌合蛋白。lacZ报告基因的转录激活表明，ArsR在酵母中的二聚化是稳定的。结果表明，残基1 - 8和90 - 117对于ArsR二聚化并非必需。构建了一系列含有六个组氨酸标签的截短ArsR蛋白的基因，并对这些蛋白进行了纯化。通过尺寸排阻色谱法测定的每种重组蛋白的质量，与双杂交分析的结果一致。体内β-半乳糖苷酶测定和体外凝胶迁移率变动分析的结果表明，二聚体保留了与ars启动子结合并对诱导剂作出反应的能力，而单体ArsR则不具备这些能力。这些结果表明，约80个残基的核心序列具备二聚化、阻遏和金属识别所需的所有信息。
