MOLECULAR ANALYSIS OF TRAL (E. COLI HELICASE I) FUNCTION

TRAL（大肠杆菌解旋酶 I）功能的分子分析

• 批准号: 7088590
• 负责人: JOEL F SCHILDBACH
• 金额: $ 0.88万
• 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 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7088590
• 关键词: DNA binding protein; Escherichia coli; bacterial proteins; helicase; microorganism conjugation; nucleic acid sequence; plasmids; protein protein interaction; protein structure function

DESCRIPTION: (provided by applicant): Conjugation is an important means of genetic transfer between bacterial species and has been implicated in the spread of genes encoding antibiotic resistance. The goal of this research program is to understand the mechanism and regulation of plasmid nicking and of initiation of DNA transfer in bacterial conjugation. Plasmid nicking, a prerequisite of transfer of a single plasmid DNA strand, is performed by relaxases, nucleases that bind and cleave single-stranded DNA. TraI, the relaxase of conjugative plasmid F Factor, is essential to F conjugative transfer. TraI possesses both a relaxase activity and a helicase activity, the latter unwinding and separating the plasmid DNA strands as the cut strand is transferred to the recipient. How Tral is able to act against double-stranded DNA in vivo, and how TraI converts between its relaxase and helicase activities are unknown. Experiments are proposed to answer four key questions about Tral and conjugation initiation: How is TraI nicking and transfer initiation influenced by oriT DNA sequences and the proteins that bind them? What protein-protein interactions influence TraI nicking and the initiation of DNA transfer, and what is the effect of disrupting them? How does a protein recognize single-stranded DNA with exquisite sequence specificity? How does TraI structure and domain organization influence its activity? These questions will be answered through in vivo studies that will identify Tral proteins, protein interactions and plasmid DNA sequences that influence transfer, and will determine the effects of these proteins and DNA sequences on plasmid nicking, transfer initiation, and transfer termination. In vitro studies will focus on determining the DNA sequences and distortions that TraI can recognize, the energetics of the TraI-DNA interaction, the conformational or functional events that occur subsequent to TraI binding to DNA, and the role of structure and domain organization in determining TraI function.

描述：（由申请人提供）：共轭是 细菌物种之间的遗传转移，并与 编码抗生素耐药性的基因传播。这项研究的目标 程序是了解质粒划分的机制和调节 在细菌结合中的DNA转移的启动。质粒刻痕，a 单个质粒DNA链转移的先决条件，由 松弛酶，结合并裂解单链DNA的核酸酶。 Trai， 结合质粒F因子的松弛酶对F偶联性至关重要 转移。 TRAI既具有弛豫酶活性，又具有解旋酶活性， 后者将切割链的放松并分离质粒DNA链 转移到收件人。 Tral如何对抗双重链 DNA在体内以及TRAI如何在其松弛酶和解旋酶活性之间转换 是未知的。提议实验回答有关TRAL的四个关键问题 和共轭启动：Trai划分和转移启动如何 受Orit DNA序列和结合它们的蛋白质的影响？什么 蛋白质 - 蛋白质相互作用会影响TRAI昵称和DNA的启动 转移，破坏它们有什么影响？蛋白质如何 识别具有精美序列特异性的单链DNA？怎么样 TRAI结构和领域组织会影响其活动？这些问题 将通过体内研究来回答，该研究将识别tral蛋白， 蛋白质相互作用和影响转移的质粒DNA序列，并且 将确定这些蛋白质和DNA序列对质粒的影响 划分，转移启动和转移终止。体外研究将 专注于确定TRAI可以识别的DNA序列和扭曲 TRAI-DNA相互作用，构象或功能的能量学 TRAI与DNA结合后发生的事件以及结构的作用 和域组织确定TRAI功能。