Purification, characterization and mode of action of PdhR, the transcriptional repressor of the pdhR-aceEF-lpd operon of Escherichia coli.
The repressor of the pdhR-aceEF-lpd operon of Escherichia coli, PdhR, was amplified to 23% of total cell protein and purified to homogeneity by heparin-agarose and cation-exchange chromatography. The purified protein is a monomer (M(r) 29,300) which binds specifically to DNA fragments containing the pdh promoter (Ppdh) in the absence of pyruvate. The pdh operator was identified by DNase I footprinting as a region of hyphenated dyad symmetry, +11AATTGGTaagACCAATT+27, situated just downstream of the transcript start site. In vitro transcription from Ppdh was repressed > 1000-fold by PdhR and this repression was antagonized in a concentration-dependent manner by its co-effector, pyruvate. Studies on RNA polymerase binding at Ppdh showed that RNA polymerase protects the -44 to +21 region in the absence of PdhR, but no RNA polymerase binding or protection upstream of +9 could be detected in the presence of PdhR. It is concluded that PdhR represses transcription by binding to an operator site centred at +19 such that effective binding of RNA polymerase is prevented.
Quail
M A
MA
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Guest
J R
JR
D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D008058:Dihydrolipoamide Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007655:Ketoglutarate Dehydrogenase Complex / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D011768:Pyruvate Dehydrogenase Complex / Q000096:biosynthesis / Q000235:genetics*; D011773:Pyruvates; D019289:Pyruvic Acid; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification*; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C084955:PdhR protein, E coli; D011768:Pyruvate Dehydrogenase Complex; D011773:Pyruvates; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D019289:Pyruvic Acid; D007655:Ketoglutarate Dehydrogenase Complex; C088796:lpd protein, E coli; D008058:Dihydrolipoamide Dehydrogenase; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D012321:DNA-Directed RNA Polymerases