Purification, characterization and mode of action of PdhR, the transcriptional repressor of the pdhR–aceEF–Ipd operon of Escherichia coli

1365-2958

Blackwell Publishing Journal Backfiles 1879-2005

Biology

Medicine

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 catlon-exchange chromatography. The purified protein is a monomer (Mr 29300) 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.

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