Abstract
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.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Acetyltransferases / biosynthesis
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Acetyltransferases / genetics*
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Bacterial Proteins / biosynthesis
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Bacterial Proteins / genetics*
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Bacterial Proteins / isolation & purification*
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Base Sequence
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DNA, Bacterial / genetics
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DNA, Bacterial / metabolism
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DNA-Directed RNA Polymerases / metabolism
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Dihydrolipoamide Dehydrogenase / biosynthesis
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Dihydrolipoamide Dehydrogenase / genetics*
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Dihydrolipoyllysine-Residue Acetyltransferase
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Escherichia coli / genetics*
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Escherichia coli / metabolism
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Escherichia coli Proteins*
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Gene Expression Regulation, Bacterial* / drug effects
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Ketoglutarate Dehydrogenase Complex / biosynthesis
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Ketoglutarate Dehydrogenase Complex / genetics*
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Molecular Sequence Data
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Operon / genetics
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Pyruvate Dehydrogenase Complex / biosynthesis
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Pyruvate Dehydrogenase Complex / genetics*
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Pyruvates
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Pyruvic Acid
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Recombinant Fusion Proteins / metabolism
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Repressor Proteins / genetics
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Repressor Proteins / isolation & purification*
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Transcription Factors / genetics
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Transcription Factors / isolation & purification*
Substances
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Bacterial Proteins
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DNA, Bacterial
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Escherichia coli Proteins
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PdhR protein, E coli
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Pyruvate Dehydrogenase Complex
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Pyruvates
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Recombinant Fusion Proteins
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Repressor Proteins
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Transcription Factors
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Pyruvic Acid
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Ketoglutarate Dehydrogenase Complex
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lpd protein, E coli
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Dihydrolipoamide Dehydrogenase
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Acetyltransferases
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Dihydrolipoyllysine-Residue Acetyltransferase
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DNA-Directed RNA Polymerases