Qin Yinping, Su Shengchang, Farrand Stephen K
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
J Biol Chem. 2007 Jul 6;282(27):19979-91. doi: 10.1074/jbc.M703332200. Epub 2007 May 1.
Conjugative transfer of Agrobacterium Ti plasmids is regulated by TraR, a quorum-sensing activator. Quorum dependence requires TraM, which binds to and inactivates TraR. In this study, we showed that TraR and TraM form a 151-kDa stable complex composed of two TraR and two TraM dimers both in vitro and in vivo. When interacted with TraR bound to tra box DNA, wild-type TraM formed a nucleoprotein complex of 77 kDa composed of one dimer of each protein and DNA. The complex converted to the 151-kDa species with concomitant release of DNA with a half-life of 1.6 h. TraR in the complex still retained tightly bound autoinducer. From these results, we conclude that TraM interacts in a two-step process with DNA-TraR to form a large, stable antiactivation complex. Mutagenesis identified residues of TraR important for interacting with TraM. These residues form two patches, possibly defining the binding interfaces. Consistent with this interpretation, comparison of the trypsin-digested polypeptides of TraR and of TraM with that of the TraR-TraM complex revealed that a tryptic site at position 177 of TraR around these patches is accessible on free TraR but is blocked by TraM in the complex. From these genetic and structural considerations, we constructed three-dimensional models of the complex that shed light on the mechanism of TraM-mediated inhibition of TraR and on TraM-mediated destabilization of the TraR-DNA complex.
根癌土壤杆菌Ti质粒的接合转移由群体感应激活因子TraR调控。群体依赖性需要TraM，它能结合并使TraR失活。在本研究中，我们发现TraR和TraM在体外和体内均形成由两个TraR和两个TraM二聚体组成的151 kDa稳定复合物。当野生型TraM与结合在tra盒DNA上的TraR相互作用时，形成了一个77 kDa的核蛋白复合物，该复合物由每种蛋白质的一个二聚体和DNA组成。该复合物转变为151 kDa的物种，同时DNA释放，半衰期为1.6小时。复合物中的TraR仍紧密结合着自身诱导剂。根据这些结果，我们得出结论，TraM与DNA-TraR通过两步过程相互作用，形成一个大的、稳定的抗激活复合物。诱变鉴定出TraR中对与TraM相互作用重要的残基。这些残基形成两个区域，可能定义了结合界面。与此解释一致的是，将TraR、TraM和TraR-TraM复合物经胰蛋白酶消化后的多肽进行比较，结果显示，在这些区域周围TraR第177位的一个胰蛋白酶作用位点在游离的TraR上是可及的，但在复合物中被TraM阻断。基于这些遗传学和结构方面的考虑，我们构建了该复合物的三维模型，这有助于阐明TraM介导的对TraR的抑制机制以及TraM介导的TraR-DNA复合物的去稳定化机制。
