Williams D R, Motallebi-Veshareh M, Thomas C M
School of Biological Sciences, University of Birmingham, Edgbaston, UK.
Nucleic Acids Res. 1993 Mar 11;21(5):1141-8. doi: 10.1093/nar/21.5.1141.
The KorB protein of broad-host-range plasmid RK2 is a transcriptional repressor involved in the control of genes for plasmid replication, conjugative transfer and stable maintenance. We have purified this protein close to homogeneity from cells harbouring an overexpression vector with the korB gene under the control of the tac promoter. KorB binds to restriction fragments bearing its proposed operator sequence, OB. Its interaction with this palindromic site was confirmed by DNaseI or hydroxyl radical footprinting at two OB sequences from RK2. Comparisons showed that the OB context affects the nature of the footprint. Our evidence suggests that KorB is a tetramer. As such, it may be able to bind two sites simultaneously on the same or on different DNA molecules. Using the korABF promoter, which is subject to KorB repression, we demonstrate by footprinting and restriction protection that KorB and RNA polymerase can bind simultaneously. Permanganate footprinting showed that KorB represses this promoter by preventing isomerization of the RNA polymerase-promoter complex from the closed to open form.
广宿主范围质粒RK2的KorB蛋白是一种转录阻遏物，参与控制质粒复制、接合转移和稳定维持相关基因。我们已从携带在tac启动子控制下带有korB基因的过表达载体的细胞中，将这种蛋白纯化至接近均一状态。KorB与带有其假定操纵序列OB的限制性片段结合。通过对来自RK2的两个OB序列进行DNaseI或羟基自由基足迹分析，证实了它与这个回文位点的相互作用。比较结果表明，OB上下文会影响足迹的性质。我们的证据表明KorB是一种四聚体。因此，它可能能够同时结合同一或不同DNA分子上的两个位点。使用受KorB抑制的korABF启动子，我们通过足迹分析和限制性保护证明，KorB和RNA聚合酶可以同时结合。高锰酸盐足迹分析表明，KorB通过阻止RNA聚合酶 - 启动子复合物从封闭形式异构化为开放形式来抑制该启动子。
