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首页> 外文期刊>Journal of Molecular Biology >PURIFICATION OF KORA PROTEIN FROM BROAD HOST RANGE PLASMID RK2 - DEFINITION OF A HIERARCHY OF KORA OPERATORS
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PURIFICATION OF KORA PROTEIN FROM BROAD HOST RANGE PLASMID RK2 - DEFINITION OF A HIERARCHY OF KORA OPERATORS

机译：从广泛寄主质粒RK2中纯化KORA蛋白-定义KORA算子的层次结构。

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The KorA protein of promiscuous plasmid RK2 is encoded in the central control operon, which coordinates expression of genes for replication, transfer and partitioning. KorA is known to repress transcription from seven promoters on the plasmid genome but at the trfA promoter, for the vegetative replication genes, it also causes derepression of trbAp, the first promoter for the operon whose genes are required primarily for mating pair formation prior to conjugative transfer. We have overproduced and purified KorA (101 amino acid residues). Crosslinking indicates that it exists largely as a dimer in solution. Western blotting with specific antibodies suggests that there are approximately 4000 monomers per cell in exponential phase and about 600 in stationary phase. Footprinting confirmed the expected location of the KorA operator, and indicated that KorA and RNA polymerase can bind simultaneously in promoter regions. Gel retardation experiments with DNA fragments carrying each of the seven KorA operators revealed that there is a hierarchy of binding affinities. Highest affinity (K-app = 12 to 20 nM) occurs with operators containing the 12 bp inverted repeat GTTTAGCTAAAC (klaAp, korAp and trfAp), while lower affinities (K-app = 136 to 272 nM) occur with less perfect repeats (klcA, kleA, kleC, kfrA). In addition, specific DNA sequences flanking the 12 bp are necessary for the characteristic type I KorA binding affinity. This hierarchy may be important in providing a graded response in expression of the operons controlled by KorA as its concentration varies, as for example on transition from exponential to stationary phase. (C) 1995 Academic Press Limited [References: 53]
机译：混杂质粒RK2的KorA蛋白在中央控制操纵子中编码，该操纵子协调基因的表达以进行复制，转移和分配。已知KorA抑制质粒基因组上七个启动子的转录，但在trfA启动子上，对于营养性复制基因，它也会引起trbAp的阻遏，trbAp是操纵子的第一个启动子，其基因主要是在结合之前形成配对对所必需的。转让。我们已经过量生产和纯化了KorA（101个氨基酸残基）。交联表明它在溶液中主要以二聚体形式存在。用特异性抗体进行蛋白质印迹表明，每个细胞在指数期大约有4000个单体，在固定期大约有600个单体。足迹证实了KorA操纵子的预期位置，并表明KorA和RNA聚合酶可以在启动子区域同时结合。带有7个KorA操纵子中每个操纵子的DNA片段的凝胶阻滞实验表明，结合亲和力存在等级关系。含有12 bp反向重复序列GTTTAGCTAAAC（klaAp，korAp和trfAp）的算子具有最高亲和力（K-app = 12至20 nM），而亲和力较低（K-app = 136至272 nM）则重复序列较不完善（klcA ，kleA，kleC，kfrA）。另外，侧翼12 bp的特定DNA序列对于特征性I型KorA结合亲和力是必需的。该层次结构对于在KorA所控制的操纵子的浓度变化时（例如在从指数相过渡到固定相时）提供表达的分级响应中可能很重要。 （C）1995 Academic Press Limited [参考号：53]

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