Molecular Analysis of Tral IE. Coli Helicase 1) Function

Tral IE 的分子分析。

• 批准号: 7372703
• 负责人: JOEL F SCHILDBACH
• 金额: $ 32.72万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7372703
• 关键词: ATPase Domain; Amino Acids; Bacteria; Binding; Biochemical Genetics; Biochemistry; Cells; Characteristics; Cleaved cell; Complex; Crystallization; Crystallography; DNA; DNA Binding; DNA Helicase I; DNA relaxase; Diffusion; Electron Microscopy; Endonuclease I; Engineering; Evolution; F Factor; Fluorescence; Genetic; Genetic Conjugation; Genome; Health; Homologous Gene; Human; Individual; Ligation; Link; Localized; Location; Membrane; Membrane Proteins; Microbial Biofilms; Modeling; Molecular; Molecular Analysis; Molecular Conformation; Mutagenesis; Numbers; Pathogenesis; Pilum; Plasmids; Play; Positioning Attribute; Process; Protein Region; Proteins; Recruitment Activity; Regulation; Relative (related person); Research; Role; Shapes; Signal Transduction; Single-Stranded DNA; Site; Specificity; Staging; Structure; Techniques; Testing; Time; Variant; Work; design; enteric pathogen; gene replacement; gene therapy; helicase; improved; in vivo; insight; mutant; pathogen; physical state; plasmid DNA; research study; response; single molecule; stoichiometry; tool; unfoldase

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.

描述（由申请人提供）：细菌接合是接合质粒从供体转移到受体的过程。环状质粒作为单链DNA转移;因此必须在供体中切割一条质粒链并在受体中连接。对于接合质粒F，TraI是切割和连接过程中的中心参与者。TraI是一种松弛酶或切口酶，以显著的序列特异性切割单链质粒DNA。当TraI参与称为松弛体的多蛋白复合物时，DNA切口会发生，这会导致TraI和DNA链之间形成稳定的连接。除松弛酶活性外，TraI还具有解旋酶活性。有效的转移需要松弛酶和解旋酶活性包含在相同的蛋白质中，并且这些活性之间的切换可能是F接合转移中的重要调节步骤。在F转移的一个模型中，TraI切割作为松弛体一部分的质粒DNA，在接收到启动转移的信号时解离，引导DNA的前端离开供体并进入受体，利用其解旋酶活性沿着进入的DNA追踪，并将质粒末端连接在一起以结束转移。使用遗传，生物化学，单分子荧光和结构技术相结合，我们提出的实验设计来回答几个关键问题TraI和共轭起始：我们可以观察到单个松弛体在一个细胞中的真实的时间？如果是这样，相对于DNA转运的接合孔，松弛体位于供体细胞内的哪个位置？当供体和受体相互作用时，松弛体的位置会改变吗？形成松弛体需要TraI的哪些特征？TraI是否在转移过程中转移到接受者，如果是，我们能否在接受者中检测TraI？如果TraI被转移，它是以折叠或变性状态转移的吗？如果变性，VirB 4同源物TraC是否充当解折叠酶？当TraI与松弛酶和解旋酶DNA底物相互作用时，TraI的构象是否发生变化？这种构象变化或DNA与TraI的松弛酶和解旋酶区域结合的负协同性能否解释TraI的作用之间的转换？这项研究不仅将深入了解共轭转移所需的分子机制，而且还将有助于我们对大型多功能蛋白质的调控机制的全面理解。细菌接合促进细菌之间的遗传交换，有助于肠道病原体的基因组多样化和进化。结合质粒还可以通过生物膜形成和其他机制促进细菌致病，并且是在基因治疗期间促进基因替换的潜在工具。拟议的实验将提供对接合机制的更好理解，最终允许旨在改善人类健康的操作。