REPRESSOR FOR SN-GLYCEROL 3-PHOSPHATE REGULON OF ESCHERICHIA-COLI K-12 - PRIMARY STRUCTURE AND IDENTIFICATION OF THE DNA-BINDING DOMAIN

The nucleotide sequence of the glpEGR operon of Escherichia coli was d etermined. The translational reading frame at the beginning, middle, a nd end of each gene was verified. The glpE encodes an acidic, cytoplas mic 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 Gl pE and GlpG are unknown. The glpR gene encodes the repressor for the g lycerol 3-phosphate regulon, a protein predicted to contain 252 amino acids with a calculated molecular weight of 28,048. The amino acid seq uence of the glp repressor was similar to several repressors of carboh ydrate catabolic systems, including those of the glucitol (GutR), fuco se (FucR), and deoxyribonucleoside (DeoR) systems of E. coli, as well as those of the lactose (LacR) and inositol (IolR) systems of gram-pos itive bacteria and agrocinopine (AccR) system of Agrobacterium tumefac iens. These repressors constitute a family of related proteins, all of which contain approximately 250 amino acids, possess a helix-turn-hel ix DNA-binding motif near the amino terminus, and bind a sugar phospha te molecule as the inducing signal. The DNA recognition helix of the g lp 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 matc h that of the deo repressor or, in a separate construct, to abolish DN A binding. Neither altered form of the glp repressor recognized the gl p or deo operator, either in vivo or in vitro. However, both altered f orms of the glp repressor were negatively dominant to the wild-type gl p repressor, indicating that the inability to bind DNA with high affin ity was due to alteration of the DNA-binding domain, not to an inabili ty to oligomerize or instability of the altered repressors. For the fi rst time, analysis of repressors with altered DNA-binding domains has verified the assignment of the helix-turn-helix motif of the transcrip tional regulators in the deoR family.