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.

细菌接合是将接合的质粒从供体转移到受体的过程。 环状质粒以单链DNA的形式转移，因此必须切割一条质粒链 在供者体内和在受者体内结扎。对于接合质粒F，Trai是 卵裂和结扎过程。TRAI，一种松弛酶或镍酶，用来裂解单链质粒DNA 显著的序列特异性。DNA缺口，导致Trai之间形成稳定的连锁 当Trai参与称为松弛小体的多蛋白质复合体时，DNA链就会发生。在……里面 TRAI除具有松弛酶活性外，还具有解旋酶活性。有效的转移需要松弛酶 和解旋酶活性被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结合转移中的一个重要调节步骤。在F转移的一种模型中，Trai裂解质粒 DNA作为松弛小体的一部分，在收到启动转移的信号后解离，引导前端 从捐赠者到接受者的DNA，使用解旋酶活性沿着传入的DNA进行跟踪， 并将质粒末端连接在一起以完成转移。利用基因，生化， 单分子荧光和结构技术，我们提出的实验旨在回答 关于TRAI和接合起始的几个关键问题：我们能否在 实时手机吗？如果是这样，松弛小体位于供体细胞内相对于结合孔的什么位置？ DNA是通过它运输的吗？当供体和受体发生变化时，松弛小体的位置会发生变化吗？ 互动？松弛小体的形成需要TRAI的哪些特征？Trai是否被转移到 如果是这样的话，我们能在收件人身上检测到Trai吗？如果列车被转移，它会被转移吗？ 处于折叠状态还是变性状态？如果变性，VirB4同源Trac是否起到解折叠酶的作用？会吗？ 当Trai与其松弛酶和解旋酶DNA底物相互作用时，其构象发生了变化？可 这种构象变化或可能否定DNA与松弛酶结合的协同性 TRAI的解旋酶区域解释了TRAI？这项研究不仅将提供 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的 对大型多功能蛋白质的调控机制有一般的了解。细菌接合促进了细菌之间的基因交换，有助于基因组多样化和 肠道病原体的进化。接合质粒也可通过以下途径促进细菌致病 生物膜的形成和其他机制，是促进基因替换的潜在工具 基因疗法。拟议的实验将使我们更好地了解 结合，最终允许旨在改善人类健康的操作。