RNA in Viral DNA Packaging

病毒 DNA 包装中的 RNA

• 批准号: 7072309
• 负责人: SHELLEY GRIMES
• 金额: $ 31.98万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7072309
• 关键词: RNA binding protein; X ray crystallography; adenosinetriphosphatase; bacterial virus; bacteriophage lambda; cryoelectron microscopy; intermolecular interaction; nanotechnology; nuclear magnetic resonance spectroscopy; nucleic acid sequence; nucleic acid structure; protein structure function; ribonuclease H; virus DNA; virus RNA; virus assembly; virus genetics; virus protein

DESCRIPTION (provided by applicant): The 174-base bacteriophage phi29 prohead RNA (pRNA) is essential for in vitro packaging of the 19-kilobase pair DNA-gp 3 complex (DNA-g3) into the viral precursor capsid (prohead). pRNA is an integral part of the phi29 DNA packaging motor, one of the strongest molecular motors characterized, pRNA forms a novel cyclic hexamer by intermolecular base pairing of identical molecules. This multimer binds to the head-tail connector of the prohead, the core of the packaging motor, where it appears as a pentameric ring by cryoEM 3-D reconstruction. A multimer of the packaging ATPase gp16 then binds the pRNA to complete the motor. pRNA is hypothesized to function in docking of the DNA-gp3 and the prohead, in recognition of the left end of DNA-gp3 to initiate packaging, and as a component of the DNA translocating ATPase. pRNA exits the DNA-filled head during neck and tail assembly, and it is not a part of the mature virion. Study of the structure and function of this RNA-dependent DNA packaging motor may have general significance for assembly of other viruses, including mammalian viruses. The ultimate goal of the research is to determine the structures and the mechanisms by which pRNA constitutes the phi29 DNA packaging motor to catalyze DNA-gp3 translocation into the prohead. The aims of the current project are to: 1) study the DNA packaging motor comprised of the connector, pRNA and the ATPase gp16 by cryoEM-3D reconstruction and X-ray crystallography; 2) determine the structure of the pRNA intermolecular pseudoknot by NMR; 3) study the packaging motor step size and coordination of pRNA-gp16 motor subunits by the use of laser optical tweezers; 4) determine the role of the N-terminus of gp 10 (connector) in binding pRNA; and 5) identify the DNA packaging RNAs of phages SPP1 and lambda.

描述（由申请人提供）：174碱基噬菌体PHI29 prohead RNA（PRNA）对于将19-千里酶对DNA-GP 3复合物（DNA-G3）体外包装至病毒前体capsid（Prohead）至关重要。 PRNA是PHI29 DNA包装电机的组成部分，这是表征最强的分子电动机之一，PRNA通过相同分子的分子间碱基对形成了一种新型的环状六聚体。该多聚体结合了Prohead的头尾连接器，Prohead（包装电机的核心），在该连接器中，它通过Cryoem 3-D重建作为五聚式环。包装ATPase GP16的多聚体结合PRNA以完成电动机。假设PRNA在DNA-GP3和Prohead的对接中起作用，以识别DNA-GP3的左端以启动包装，并且是DNA转移ATPase的组成部分。 PRNA在颈部和尾部组装过程中退出了DNA填充的头部，它不是成熟病毒体的一部分。研究该RNA依赖性DNA包装电机的结构和功能可能对包括哺乳动物病毒在内的其他病毒的组装具有一般意义。该研究的最终目的是确定PRNA构成PHI29 DNA包装运动的结构和机制，以将DNA-GP3易位催化为Prohead。当前项目的目的是：1）研究由Cryoem-3D重建和X射线晶体学组成的连接器，PRNA和ATPase GP16组成的DNA包装电机； 2）确定NMR的PRNA间分子假诺的结构； 3）研究通过使用激光光镊的包装运动步长和PRNA-GP16运动亚基的配位； 4）确定GP 10（连接器）N端在结合PRNA中的作用； 5）确定噬菌体SPP1和Lambda的DNA包装RNA。