In vitro asymmetric binding of the pleiotropic regulatory protein, FruR, to the ace operator controlling glyoxylate shunt enzyme synthesis.
The fruR gene of Escherichia coli, which encodes the regulatory protein FruR, was cloned in the pT7-5 expression vector so as to overproduce a protein tagged with 6 histidine residues. By using a one-step chromatographic procedure, FruR was purified to near-homogeneity. Analysis of the protein under both denaturing and nondenaturing conditions indicated that it is a tetramer with a molecular mass of about 150 kilodaltons. The positions of interference between FruR and the operator of the acetate operon were examined. The number and nature of the nucleotides essential for FruR binding were determined by several different techniques: base methylation with dimethyl sulfate, base removal by formic acid and hydrazine, uracil interference, and hydroxyl radical footprinting. It was observed that FruR asymmetrically binds to a 16-base pair DNA sequence located 170 base pairs upstream from the transcriptional start point of the ace operon.
Cortay
J C
JC
Institut de Biologie et Chimie des Protéines, CNRS, Passage du Vercors, Lyon, France.
Nègre
D
D
Scarabel
M
M
Ramseier
T M
TM
Vartak
N B
NB
Reizer
J
J
Saier
M H
MH
Cozzone
A J
AJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D004269:DNA, Bacterial / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D006038:Glyoxylates / Q000378:metabolism*; D006900:Hydroxylation; D008745:Methylation; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014498:Uracil / Q000378:metabolism; D056968:Lac Repressors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004798:Enzymes; D029968:Escherichia coli Proteins; D006038:Glyoxylates; D012097:Repressor Proteins; D014498:Uracil; C489416:FruR protein, E coli; C068837:FruR protein, Bacteria; D056968:Lac Repressors