Molecular Analysis of Tral IE. Coli Helicase 1) Function

描述(由申请人提供)：细菌接合是将接合的质粒从供体转移到接受者的过程。循环质粒以单链DNA的形式转移，因此必须在供体中切割一条质粒链，在受体中连接。对于结合质粒F，Trai是切割和连接过程中的中心角色。TRAI是一种松弛酶或镍酶，具有显著的序列特异性，能裂解单链质粒DNA。DNA缺口是在Trai参与称为松弛小体的多蛋白质复合体时发生的，它导致Trai和DNA链之间形成稳定的连接。TRAI除了具有松弛酶活性外，还具有解旋酶活性。有效的转移要求松弛酶和解旋酶活性包含在同一蛋白质中，而在这两种活性之间的切换可能是F接合转移的重要调节步骤。在一种F转移模型中，Trai裂解作为松弛小体的一部分的质粒DNA，在接收到启动转移的信号时解离，引导DNA的前端从供体进入接受者，利用其解旋酶活性沿着传入的DNA跟踪，并将质粒末端连接在一起以完成转移。利用遗传学、生物化学、单分子荧光和结构技术的组合，我们提出了旨在回答有关Trai和接合启动的几个关键问题的实验：我们能实时观察细胞中的单个弛豫体吗？如果是这样的话，相对于DNA转运的接合孔，松弛小体位于供体细胞内的什么位置？当供者和受者相互作用时，松弛小体的位置会改变吗？松弛小体的形成需要TRAI的哪些特征？在传输过程中是否将Trai传输到接收者，如果是，我们能否在接收者中检测到Trai？如果Trai被转移，它是以折叠还是变性状态转移的？如果变性，VirB4同源Trac是否起到解折叠酶的作用？当Trai与它的松弛酶和解旋酶DNA底物相互作用时，它的构象会改变吗？这样的构象变化或者DNA与TRAI的松弛酶和解旋酶区域的负协同作用是否可以解释TRAI角色之间的转换？这项研究不仅将为我们深入了解连接转移所需的分子机制提供帮助，而且还将有助于我们对大型多功能蛋白质的调控机制的总体理解。细菌接合促进了细菌之间的基因交换，有助于肠道病原菌的基因组多样化和进化。接合质粒也可以通过生物膜形成和其他机制促进细菌的发病，并且是促进基因治疗中基因替换的潜在工具。拟议中的实验将提供对结合机制的更好理解，最终允许设计用于改善人类健康的操作。

DESCRIPTION (provided by applicant): Bacterial conjugation is a process whereby a conjugative plasmid is transferred from a donor to a recipient. The circular plasmid is transferred as single-stranded DNA; therefore one plasmid strand must be cleaved in the donor and ligated in the recipient. For conjugative plasmid F, TraI is the central player in the cleavage and ligation processes. TraI, a relaxase or nickase, cleaves single-stranded plasmid DNA with remarkable sequence specificity. DNA nicking, which causes formation of a stable linkage between TraI and the DNA strand, occurs while TraI participates in a multi-protein complex called the relaxosome. In addition to its relaxase activity, TraI possesses a helicase activity. Efficient transfer requires that relaxase and helicase activities be contained within the same protein, and a switch between these activities may be an important regulatory step in F conjugative transfer. In one model for F transfer, TraI cleaves plasmid DNA as part of the relaxosome, dissociates upon receiving a signal initiating transfer, pilots the leading end of the DNA out of the donor and into the recipient, tracks along the incoming DNA using its helicase activity, and ligates the plasmid ends together to conclude transfer. Using a combination of genetic, biochemical, single molecule fluorescence and structural techniques, we propose experiments designed to answer several key questions about TraI and conjugation initiation: Can we observe individual relaxosomes in a cell in real time? If so, where is the relaxosome located within the donor cell relative to the conjugative pore through which the DNA is transported? Does the relaxosome location change when donors and recipients interact? What characteristics of TraI are required to form the relaxosome? Is TraI transferred to the recipient during transfer, and if so can we detect TraI in the recipient? If TraI is transferred, is it transferred in a folded or denatured state? If denatured, does the VirB4 homologue TraC act as an unfoldase? Does the conformation of TraI change when it interacts with its relaxase and its helicase DNA substrates? Could such a conformational change or could negative cooperativity of binding of DNA to the relaxase and helicase regions of TraI explain the conversion between roles of TraI? The research will not only provide insight into the molecular mechanisms required for conjugative transfer, but they will also contribute to our general understanding of the regulatory mechanisms of large multifunctional proteins. Bacterial conjugation facilitates genetic exchange between bacteria, assisting genome diversification and evolution of enteric pathogens. Conjugative plasmids also can contribute to bacterial pathogenesis via biofilm formation and other mechanisms, and are potential tools for facilitating gene replacement during gene therapy. The proposed experiments will provide a better understanding of the mechanism of conjugation, eventually allowing manipulations designed to improve human health.