Molecular Analysis of Tral IE. Coli Helicase 1) Function

Tral IE 的分子分析。

• 批准号: 7904439
• 负责人: JOEL F SCHILDBACH
• 金额: $ 23.97万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904439
• 关键词: ATPase Domain; Amino Acids; Bacteria; Binding; Biochemical; 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; Location; Membrane; Membrane Proteins; Microbial Biofilms; Modeling; Molecular; Molecular Analysis; Molecular Conformation; Mutagenesis; 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; protein complex; research study; response; single molecule; stoichiometry; tool; unfoldase

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.

细菌结合是一个过程，从而将结合质粒从供体转移到接受者。 圆质粒作为单链DNA转移，因此必须裂解一个质粒链 在捐赠者中，并在接收者中结扎。对于共轭质粒F，Trai是 切割和连接过程。 trai是一种松弛酶或镍酶，将单链质粒DNA切割 显着的序列特异性。 DNA昵称，导致TRAI之间的稳定联系形成 DNA链发生在Trai参与称为Leashosom体的多蛋白质络合物时。在 除了其松弛酶活性外，TRAI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同一蛋白质中，这些活动之间的切换可能是 F共轭转移的重要调节步骤。在一种用于F传输的模型中，Trai裂解质粒 DNA作为松弛体的一部分，在接收信号启动转移后解离，飞行器前一端 从供体和受体中的DNA中，使用其解旋酶活性沿传入的DNA跟踪， 并将质粒结在一起以结束转移。结合遗传，生化， 单分子荧光和结构技术，我们提出了旨在回答的实验 关于TRAI和共轭启动的几个关键问题：我们可以观察到一个单独的松弛体 实时单元？如果是这样，相对于共轭孔，宽松体位于供体细胞内的位置 通过哪个DNA运输？当捐助者和接收者时，松弛体的位置会发生变化吗 相互影响？形成松弛体需要什么特征？被转移到 转移过程中的接收者，如果是的话，我们可以在接收者中检测到Trai吗？如果Trai被转移，是否转移了 在折叠或变性状态下？如果是变性的，VirB4同源物TRAC是否起到了进化酶的作用？做 当Trai与其弛豫酶及其解旋酶DNA底物相互作用时，它的构象会变化？可以 这种构象变化或DNA与松弛酶结合的负相关性和 TRAI的解旋酶区域解释了Trai角色之间的转换？研究不仅会提供 深入了解共轭转移所需的分子机制，但它们也将有助于我们 对大型多功能蛋白的调节机制的一般理解。细菌结合促进细菌之间的遗传交换，有助于基因组多样化和 肠道病原体的进化。共轭质粒也可以通过 生物膜形成和其他机制，是促进基因替代物的潜在工具 基因疗法。提出的实验将更好地理解 共轭，最终允许旨在改善人类健康的操作。