Translocation of DNA across the Agrobacterium envelope

大分子在细胞间的转运是生物医学的一个重要领域。研究的重点 本实验室是根癌农杆菌的IV型分泌（T4 S）系统。这个系统是祖先 与细菌接合系统相关且功能相似，以及最近描述的蛋白质 细菌病原体在感染过程中使用的易位系统。T4 S系统介导 通过A.根瘤菌细胞被膜。底物包括核蛋白 与DNA单链共价连接的VirD 2松弛酶等颗粒，以及 作为VirE 2效应蛋白。T4 S系统将这些底物转移到各种遗传上- 通过依赖于直接细胞与细胞接触的过程，这 由VirD 4和11个VirB亚基组装而成的T4 S系统是一个特别有吸引力的系统，用于详细的 由于A.根瘤菌 大量的菌株、构建体和其他分子工具，以及丰富的可用信息， 以及与之密切相关的转移系统。本实验室工作的总体目标是描述 完整的分子术语所需的动态过程：i）VirB/D4分泌的生物发生 通道，ii）分泌底物的识别，和iii）底物跨革兰氏阴性通道的易位 信封.这些研究将结合使用最先进的生物化学和基于结构的方法 在当前的资助周期内，该实验室开发了强大的体内技术， 模型DMA和蛋白质底物被募集到并通过分泌通道。第一，贡献 两种ATP酶，VirD 4底物受体和VirB 11，早期和晚期底物转移反应 将被确定。第二，一种新发现的DNA底物和ATP介导的 激活通道组装或门控所需的VirBIO亚基的构象开关将是 完全特征化。最后，功能上相互作用的内膜亚基对， VirB 6和VirB 8以及外膜相关亚基VirB 2和VirB 9向底物易位 将被表征。拟议的研究将开发一种机制， 对第四型分泌的了解前所未有的详细。

Translocation of macromolecules between cells is a major area of biomedical interest. The focus of study in this laboratory is a type IV secretion (T4S) system of Agrobacterium tumefaciens. This system is ancestrally related and functionally similar to bacterial conjugation systems, as well as recently described protein translocation systems used by bacterial pathogens during the course of infection. This T4S system mediates transfer of diverse substrates across the A. tumefaciens cell envelope. The substrates include nucleoprotein particle such as the VirD2 relaxase covalently linked to a single strand of DMA,and protein monomers such as the VirE2 effector protein. The T4S system translocates these substrates to a variety of phylogenetically- diverse bacterial and eukaryotic cell targets through a process dependent on direct cell-to-cell contact. This T4S system, assembled from VirD4 and 11 VirB subunits, is an especially attractive system for detailed mechanistic studies of macromolecular transport because of the ease of manipulation of A. tumefaciens, the large numbers strains, constructs, and other molecular tools at hand, and the wealth of information available about it and closely related transfer systems. The overall goal of work in this laboratory is to describe in complete molecular terms the dynamic processes required for: i) biogenesis of the VirB/D4 secretion channel, ii) recognition of secretion substrates, and iii) translocation of substrates across the Gram-negative envelope. The studies will use state-of-the-art biochemical and structure-based approaches in combination with powerful in vivo technologies developed in this laboratory during the current grant cycle to define how model DMAand protein substrates are recruited to and through the secretion channel. First, contributions of two ATPases, the VirD4 substrate receptor and VirB11, to early- and late-stage substrate transfer reactions will be determined. Second, a newly-discovered phenomenon of DNA substrate- and ATP-mediated activation of a conformational switch of the VirBIO subunit required for channel assembly or gating will be fully characterized. Finally, the contributions of functionally interacting pairs of inner membrane subunits, VirB6 and VirB8, and outer membrane-associated subunits, VirB2 and VirB9, to substrate translocation across the cell envelope will be characterized. The proposed studies will develop a mechanistic understanding of type IV secretion in unprecedented detail.