Purification and characterization of the repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli K12.
The glpR gene encoding the repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli was cloned downstream from the strong pL promoter of bacteriophage lambda. This allowed overproduction of the repressor upon thermal induction of a cryptic lambda lysogen harboring the cI857 gene. The repressor was purified 40-fold to homogeneity from an induced strain. The purification scheme utilized polyethyleneimine and ammonium sulfate fractionation, followed by phosphocellulose and DEAE-Sephadex chromatography. Purification was monitored by measuring the binding of radiolabeled inducer (sn-glycerol 3-phosphate) to the repressor. The purified repressor migrated as a single band exhibiting a subunit molecular weight of 30,000 assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the repressor under nondenaturing conditions was 100,000-130,000 suggesting the repressor is a tetramer under native conditions. Interaction of the repressor with sn-glycerol 3-phosphate was studied using flow dialysis. Scatchard analysis of the data indicated four binding sites/repressor tetramer and a dissociation constant of 31 microM. Interaction of the repressor with DNA was studied using band-shift electrophoresis. The repressor specifically bound DNA fragments containing the control regions for the glpD, glpK, and glpT-A genes. Binding of DNA by the repressor was diminished in the presence of sn-glycerol 3-phosphate.
Larson
T J
TJ
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061.
Ye
S Z
SZ
Weissenborn
D L
DL
Hoffmann
H J
HJ
Schweizer
H
H
D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D005798:Genes, Bacterial; D005809:Genes, Regulator; D005994:Glycerophosphates / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*
D005994:Glycerophosphates; D012097:Repressor Proteins; D014157:Transcription Factors; C029620:alpha-glycerophosphoric acid