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: 6625618
• 负责人: JOEL F SCHILDBACH
• 金额: $ 34.16万
• 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/6625618
• 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）：共轭是一种重要的手段 细菌物种之间的遗传转移，并与 编码抗生素耐药性的基因的传播。本研究的目标 计划的目的是了解质粒切口的机制和调节 细菌接合中 DNA 转移的起始。质粒切口， 单个质粒 DNA 链转移的先决条件是 松弛酶，结合和切割单链 DNA 的核酸酶。特拉尔， 接合质粒 F 因子的松弛酶，对于 F 接合至关重要 转移。 TraI 同时具有松弛酶活性和解旋酶活性， 后者解旋并分离质粒 DNA 链，因为切割链是 转移给收件人。 Tral 如何对抗双链 体内 DNA 以及 TraI 如何在其松弛酶和解旋酶活性之间进行转换 未知。建议通过实验来回答有关 Tral 的四个关键问题 和缀合起始：TraI 切口和转移起始如何 受 oriT DNA 序列和结合它们的蛋白质的影响吗？什么 蛋白质-蛋白质相互作用影响 TraI 切口和 DNA 的起始 转移，扰乱它们会产生什么影响？蛋白质是怎样产生的 识别具有精确序列特异性的单链 DNA？怎么样 TraI 结构和领域组织影响其活动吗？这些问题 将通过识别 Tral 蛋白的体内研究来回答， 影响转移的蛋白质相互作用和质粒 DNA 序列，以及 将确定这些蛋白质和 DNA 序列对质粒的影响 切口、转移起始和转移终止。体外研究将 专注于确定 TraI 可以识别的 DNA 序列和扭曲， TraI-DNA 相互作用的能量学、构象或功能 TraI 与 DNA 结合后发生的事件以及结构的作用 和域组织来确定 TraI 功能。