Purification, characterization and mode of PdhR, the transcriptional represser 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 (P_pdh) in the absence of pyruvate. Thepdh 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 P_pdh 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 P_pdh 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. [ABSTRACT FROM AUTHOR]