Zeng G, Ye S, Larson T J
Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308, USA.
J Bacteriol. 1996 Dec;178(24):7080-9. doi: 10.1128/jb.178.24.7080-7089.1996.
The nucleotide sequence of the glpEGR operon of Escherichia coli was determined. The translational reading frame at the beginning, middle, and end of each gene was verified. The glpE gene encodes an acidic, cytoplasmic protein of 108 amino acids with a molecular weight of 12,082. The glpG gene encodes a basic, cytoplasmic membrane-associated protein of 276 amino acids with a molecular weight of 31,278. The functions of GlpE and GlpG are unknown. The glpR gene encodes the repressor for the glycerol 3-phosphate regulon, a protein predicted to contain 252 amino acids with a calculated molecular weight of 28,048. The amino acid sequence of the glp repressor was similar to several repressors of carbohydrate catabolic systems, including those of the glucitol (GutR), fucose (FucR), and deoxyribonucleoside (DeoR) systems of E. coli, as well as those of the lactose (LacR) and inositol (IolR) systems of gram-positive bacteria and agrocinopine (AccR) system of Agrobacterium tumefaciens. These repressors constitute a family of related proteins, all of which contain approximately 250 amino acids, possess a helix-turn-helix DNA-binding motif near the amino terminus, and bind a sugar phosphate molecule as the inducing signal. The DNA recognition helix of the glp repressor and the nucleotide sequence of the glp operator were very similar to those of the deo system. The presumptive recognition helix of the glp repressor was changed by site-directed mutagenesis to match that of the deo repressor or, in a separate construct, to abolish DNA binding. Neither altered form of the glp repressor recognized the glp or deo operator, either in vivo or in vitro. However, both altered forms of the glp repressor were negatively dominant to the wild-type glp repressor, indicating that the inability to bind DNA with high affinity was due to alteration of the DNA-binding domain, not to an inability to oligomerize or instability of the altered repressors. For the first time, analysis of repressors with altered DNA-binding domains has verified the assignment of the helix-turn-helix motif of the transcriptional regulators in the deoR family.
测定了大肠杆菌glpEGR操纵子的核苷酸序列。验证了每个基因起始、中间和末端的翻译阅读框。glpE基因编码一种酸性胞质蛋白，含108个氨基酸，分子量为12,082。glpG基因编码一种碱性胞质膜相关蛋白，含276个氨基酸，分子量为31,278。GlpE和GlpG的功能未知。glpR基因编码甘油3 - 磷酸调节子的阻遏蛋白，该蛋白预计含252个氨基酸，计算分子量为28,048。glp阻遏蛋白的氨基酸序列与碳水化合物分解代谢系统的几种阻遏蛋白相似，包括大肠杆菌的葡糖醇（GutR）、岩藻糖（FucR）和脱氧核糖核苷（DeoR）系统，以及革兰氏阳性菌的乳糖（LacR）和肌醇（IolR）系统，还有根癌土壤杆菌的农杆碱（AccR）系统。这些阻遏蛋白构成一个相关蛋白家族，它们都含约250个氨基酸，在氨基末端附近有一个螺旋 - 转角 - 螺旋DNA结合基序，并结合一个磷酸糖分子作为诱导信号。glp阻遏蛋白的DNA识别螺旋和glp操纵基因的核苷酸序列与deo系统的非常相似。通过定点诱变改变glp阻遏蛋白的推定识别螺旋，使其与deo阻遏蛋白的匹配，或者在另一个构建体中使其丧失DNA结合能力。无论是体内还是体外，glp阻遏蛋白的这两种改变形式都不能识别glp或deo操纵基因。然而，glp阻遏蛋白的这两种改变形式对野生型glp阻遏蛋白均呈负显性，表明无法高亲和力结合DNA是由于DNA结合结构域的改变，而不是由于改变后的阻遏蛋白无法寡聚或不稳定。首次对具有改变的DNA结合结构域的阻遏蛋白进行分析，验证了deoR家族转录调节因子的螺旋 - 转角 - 螺旋基序的归属。
