Miwa Y, Fujita Y
Department of Biochemistry, Hamamatsu University School of Medicine, Japan.
J Biol Chem. 1988 Sep 15;263(26):13252-7.
The GntR protein is a negative regulator involved in gluconate-inducible expression of the Bacillus subtilis gnt operon which is responsible for gluconate metabolism. The GntR protein has been purified to homogeneity from an overproducing Escherichia coli strain harboring a gntR gene-carrying plasmid. The total amino acid composition and the NH2-terminal amino acid sequence of the purified protein were essentially the same as those deduced from the nucleotide sequence of the gntR gene. Molecular weight determination by gel filtration revealed that the purified protein is in a highly polymerized form, but it likely exists as a dimer when highly diluted. The purified GntR protein was found to be specifically bound to DNA fragments carrying the promoter of the gnt operon in an electrophoretic mobility shift assay. This binding was specifically inhibited by the addition of gluconate or glucono-delta-lactone. The purified protein repressed in vitro transcription from the promoter of the gnt operon. This repression was suppressed by gluconate or glucono-delta-lactone. These results indicate that the GntR protein is a repressor for the gnt operon and that gluconate and glucono-delta-lactone are inducers for this operon.
GntR蛋白是一种负调控因子，参与枯草芽孢杆菌gnt操纵子的葡萄糖酸盐诱导表达，该操纵子负责葡萄糖酸盐代谢。已从携带gntR基因质粒的过量表达大肠杆菌菌株中纯化出均一的GntR蛋白。纯化蛋白的总氨基酸组成和NH2末端氨基酸序列与从gntR基因核苷酸序列推导的结果基本相同。凝胶过滤法测定分子量表明，纯化蛋白呈高度聚合形式，但在高度稀释时可能以二聚体形式存在。在电泳迁移率变动分析中，发现纯化的GntR蛋白与携带gnt操纵子启动子的DNA片段特异性结合。添加葡萄糖酸盐或葡萄糖酸-δ-内酯可特异性抑制这种结合。纯化蛋白抑制gnt操纵子启动子的体外转录。葡萄糖酸盐或葡萄糖酸-δ-内酯可抑制这种抑制作用。这些结果表明，GntR蛋白是gnt操纵子的阻遏物，葡萄糖酸盐和葡萄糖酸-δ-内酯是该操纵子的诱导物。
