Characterization of the interaction of the glp repressor of Escherichia coli K-12 with single and tandem glp operator variants

Abstract

The glp operons of Escherichia coli are negatively controlled by the glp repressor. Comparison of the repressor-binding affinities for consensus and altered consensus operators in vivo showed that all base substitutions at positions 3, 4, 5, and 8 from the center of the palindromic operator caused a striking decrease in repressor binding. Substitutions at other positions had a severe to no The proteins that catalyze the steps required for the utilization of glycerol, glycerol-3-phosphate (glycerol-P), and glycerophosphodiesters are encoded by the glp regulon of Escherichia coli (14). The glp regulon is composed of five operons located at three different regions on the linkage map of E. coli. Transcription of the glp operons is negatively regulated by the glp repressor, a tetrameric protein encoded by the glpR gene (13,17). Negative control is mediated by binding of the glp repressor to its operator sites within the glp operons. The affinity of the repressor for its operators is decreased in the presence of glycerol-P, the inducer for the regulon (14). Thirteen operators have been identified in the glp operons by using DNase I footprinting (12,22,23). The operators match more or less well the consensus operator 5'-WATGTTCGWT AWCGAACA TW-3' (W is A or T, and the dot indicates the center of symmetry) (22). Tandemly repeated repressor-binding sites are present in the control regions of the glpA CB, glpD, and glpFKX operons (12,20,22,23 Cloning of operator DNA. Plasmid vector pGEM3Z (Promega) was digested with BamHI and Pstl and purified following electrophoresis on low-gelling-temperature agarose. Equimolar amounts of the oligonucleotides to be cloned were mixed in 10 mM Tris-HCl (pH 8)-50 mM NaCl-1 mM EDTA and heated to 85°C. The annealing mixture was allowed to cool to room temperature for several hours. A 40-fold molar excess of annealed oligonucleotides was mixed and ligated (3 h) to 150 ng of cleaved vector DNA in 0.04 ml. An aliquot of the ligation mixture was used for transformation of strain DH5otF'.

Action at a distance for glp repressor control of glpTQ transcription in Escherichia coli K‐12

“…A common theme for repression of the four catabolic glp operons appears to be proximal tandemly arranged operators Weissenborn et al, 1992), to which GlpR binds co-operatively (Zhao et al, 1994). Remote operator sites located about 400 bp away from the proximal operators contribute additionally to repression of both glpT and glpD (Yang and Larson, 1996).…”

“…Therefore, these mutations do not change the amino acid sequences. The positions chosen for alteration of O T 1 or O T 3 were those shown to be crucial for binding of GlpR (Zhao et al, 1994). Positions mutated in IHF1 and IHF2 were chosen because they belong to the 13 bp IHF consensus sequence.…”

“…As previous studies on repression of glpT were performed using fusions with the glpT gene truncated upstream of the translational start point, the contributions of putative remote regulatory elements may have been overlooked. In order to obtain a more complete picture of glpT transcriptional regulation, the entire gene was searched for sequences resembling the glp operator consensus (half-site consensus is WAT-KYTCGWW, where W is A or T, K is G or T, and Y is C or T; Zhao et al, 1994). Three potential glp operators were found (O T 1 to O T 3; Fig.…”

Plasticity in Repressor-DNA Interactions Neutralizes Loss of Symmetry in Bipartite Operators

“…Sequence deviations have been seen in target sites that are recognized by the same transcription factor. Examples include glp repressors of Escherichia coli and NdgR from Streptomyces coelicolor, which recognize 13 and 19 different operator sequences, respectively (6,7). The sequence differences suggest the possibility of variations in the mode of recognition by a single transcription factor when recognizing target sequences.…”

Differencesin Genetic and Transcriptional Organization of theglpTQOperons betweenHaemophilus influenzaeType b andNontypeableStrains

“…Potential catabolic and transcriptional regulation sites with homology to consensus sequences upstream of glpT in E. coli were also found upstream of glpT in Eagan and 772. They include two catabolic activator protein binding sites (4), two operator sites with the potential of binding to the E. coli glp repressor protein (29), and a sequence homologous to the integration host factor (IHF) (9) (Fig. 2).…”