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首页> 外文期刊>Journal of Molecular Biology >PARTNER SWITCHING MECHANISMS IN INACTIVATION AND REJUVENATION OF ESCHERICHIA COLI DNA GYRASE BY F PLASMID PROTEINS LETD (CCDB) AND LETA (CCDA)
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PARTNER SWITCHING MECHANISMS IN INACTIVATION AND REJUVENATION OF ESCHERICHIA COLI DNA GYRASE BY F PLASMID PROTEINS LETD (CCDB) AND LETA (CCDA)

机译：F质粒LETD（CCDB）和LETA（CCDA）活化和恢复大肠杆菌DNA脱氧核糖核酸的伙伴转换机制

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Escherichia coil DNA gyrase, as well as a free form of its A subunit (GyrA), exists in an inactivated form in cells that overproduce the F plasmid protein LetD (CcdB). We found that the inactivated DNA gyrase and GyrA protein can be rejuvenated in vitro by another F plasmid protein, LetA (CcdA). Using this rejuvenation as an assay, we purified the inactivated GyrA protein to near homogeneity and found it to be complexed with the LetD protein. The complex has a molecular mass of 230 kDa and was suggested to be a complex of two molecules each of GyrA and LetD proteins. The GyrA-LetD complex, in the presence of purified GyrB protein, does not cause DNA cleavage. Therefore, the LetD protein in the GyrA-LetD complex inhibits the gyrase action by a mechanism different from one that involves trapping a covalently linked gyrase-DNA complex. In as much as a free form of the LetD protein has been shown to induce DNA cleavage by gyrase, the LetD protein seems to have two distinct modes of action on DNA gyrase. Rejuvenation of the inactivated GyrA protein by the LetA protein was achieved in vitro, and mechanisms governing this process were examined using the purified proteins. The rejuvenated GyrA protein sediments through sucrose gradients as a single protein species of 190 kDa and is indistinguishable from a free form of GyrA protein. In the same sedimentation experiment, the LetD protein was seen to be complexed with the added LetA protein. Thus, the LetA protein apparently rejuvenates the GyrA protein by removing the bound LetD protein from the inactivated form, followed by formation of a LetA-LetD complex. (C) 1996 Academic Press Limited [References: 32]
机译：大肠杆菌线圈DNA促旋酶及其游离形式的A亚基（GyrA）以灭活的形式存在于过量产生F质粒蛋白LetD（CcdB）的细胞中。我们发现灭活的DNA促旋酶和GyrA蛋白可以通过另一个F质粒蛋白LetA（CcdA）在体外恢复活力。使用该复兴作为测定，我们将灭活的GyrA蛋白纯化至接近均一，并发现它与LetD蛋白复合。该复合物的分子量为230 kDa，被认为是GyrA和LetD蛋白两个分子的复合物。在纯化的GyrB蛋白存在下，GyrA-LetD复合物不会引起DNA裂解。因此，GyrA-LetD复合物中的LetD蛋白通过一种不同于捕获共价连接的旋转酶-DNA复合物的机制来抑制旋转酶作用。已经显示，LedD蛋白的游离形式可诱导其被DNA促旋酶切割，但它似乎对DNA促旋酶具有两种不同的作用方式。在体外实现了由LetA蛋白对失活的GyrA蛋白的修复，并使用纯化的蛋白检查了控制该过程的机制。复兴的GyrA蛋白通过蔗糖梯度以190 kDa的单个蛋白质形式沉淀，与自由形式的GyrA蛋白没有区别。在同一沉降实验中，LetD蛋白与添加的LetA蛋白复合。因此，通过从失活的形式去除结合的LetD蛋白，然后形成LetA-LetD复合物，LetA蛋白显然使GyrA蛋白再生。 （C）1996 Academic Press Limited [参考：32]

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