Quail M A, Guest J R
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Mol Microbiol. 1995 Feb;15(3):519-29. doi: 10.1111/j.1365-2958.1995.tb02265.x.
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.
大肠杆菌pdhR - aceEF - lpd操纵子的阻遏蛋白PdhR被扩增至总细胞蛋白的23%，并通过肝素 - 琼脂糖和阳离子交换色谱法纯化至同质。纯化后的蛋白是一种单体（相对分子质量29,300），在没有丙酮酸的情况下，它能特异性结合含有pdh启动子（Ppdh）的DNA片段。通过DNase I足迹法确定pdh操纵基因是一个具有间断二元对称的区域，即+11AATTGGTaagACCAATT+27，位于转录起始位点的下游。PdhR对来自Ppdh的体外转录的抑制作用超过1000倍，并且这种抑制作用被其共效应物丙酮酸以浓度依赖的方式拮抗。对Ppdh处RNA聚合酶结合的研究表明，在没有PdhR的情况下，RNA聚合酶保护 - 44至+21区域，但在有PdhR的情况下，在+9上游未检测到RNA聚合酶结合或保护。结论是PdhR通过结合以+19为中心的操纵基因位点来抑制转录，从而阻止RNA聚合酶的有效结合。
