Yoshida K, Fujita Y, Sarai A
Department of Biotechnology, Faculty of Engineering, Fukuyama University, Japan.
J Mol Biol. 1993 May 20;231(2):167-74. doi: 10.1006/jmbi.1993.1270.
The Bacillus subtilis gnt operon is negatively regulated by the gnt repressor (GntR, 243 amino acids), which is antagonized by gluconate. The GntR protein belongs to a new family of bacterial regulatory proteins (GntR family). To locate the DNA-binding domain of the GntR protein, we obtained mutations of this protein, by hydroxylamine mutagenesis, which diminish its operator binding ability. Sequence analysis of these mutations indicated that the mutant GntR proteins (GntR43L, GntR66T, GntR74K and GntR75Q) had amino acid substitutions (Ser43 to Leu, Ala66 to Thr, Glu74 to Lys and Arg75 to Gln), respectively. They were all located within the N-terminal conserved region of the GntR family. In vivo and in vitro analysis of these GntR proteins indicated that their relative operator binding abilities became weaker in the order of GntR (wild type), GntR66T, GntR75Q, GntR74K and GntR43L. The equilibrium dissociation constants of GntR (wild type), GntR66T, GntR75Q and GntR74K as to operator binding were determined by gel retardation assays to be 0.43, 2.6, 4.2 and 8.8 M x 10(-10), respectively.
枯草芽孢杆菌gnt操纵子受gnt阻遏蛋白（GntR，243个氨基酸）负调控，葡萄糖酸盐可拮抗该阻遏蛋白。GntR蛋白属于一类新的细菌调控蛋白家族（GntR家族）。为了定位GntR蛋白的DNA结合结构域，我们通过羟胺诱变获得了该蛋白的突变体，这些突变体降低了其与操纵基因的结合能力。对这些突变的序列分析表明，突变的GntR蛋白（GntR43L、GntR66T、GntR74K和GntR75Q）分别发生了氨基酸替换（Ser43替换为Leu、Ala66替换为Thr、Glu74替换为Lys以及Arg75替换为Gln）。它们均位于GntR家族的N端保守区域内。对这些GntR蛋白进行体内和体外分析表明，它们与操纵基因的相对结合能力按GntR（野生型）、GntR66T、GntR75Q、GntR74K和GntR43L的顺序依次减弱。通过凝胶阻滞试验测定GntR（野生型）、GntR66T、GntR75Q和GntR74K与操纵基因结合的平衡解离常数分别为0.43、2.6、4.2和8.8×10⁻¹⁰M。
