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: 7250421
• 负责人: JOEL F SCHILDBACH
• 金额: $ 3.39万
• 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 2007-09-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7250421
• 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）：结合是一种重要的手段， 细菌物种之间的基因转移，并已牵连在 编码抗生素抗性的基因的传播。本研究的目的 计划是了解机制和调节质粒切口和 在细菌接合中启动DNA转移。质粒切口，a 转移单个质粒DNA链的先决条件，通过 松弛酶，结合和切割单链DNA的核酸酶。TraI，The 接合质粒F因子的松弛酶，是F接合 转移TraI具有松弛酶活性和解旋酶活性， 后者解旋和分离质粒DNA链，因为切割的链是 转移给接受者。Tral如何能够对抗双链 体内DNA，以及TraI如何在其松弛酶和解旋酶活性之间转换 是未知的。提出了实验来回答关于Tral的四个关键问题 和接合起始：如何是TraI切口和转移起始 受oriT DNA序列和结合它们的蛋白质的影响？什么 蛋白质-蛋白质相互作用影响TraI切口和DNA起始 转移，以及破坏它们的影响是什么？一种蛋白质 识别单链DNA的精确序列特异性如何 TraI结构和领域组织影响其活动？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 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，构象或功能性 TraI与DNA结合后发生的事件，以及结构的作用 和域组织在确定TraI功能中的作用。