RNA in Viral DNA Packaging

病毒 DNA 包装中的 RNA

• 批准号: 6819522
• 负责人: SHELLEY GRIMES
• 金额: $ 21.26万
• 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/6819522
• 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 个碱基的噬菌体 phi29 前头 RNA (pRNA) 对于将 19 千碱基对 DNA-gp 3 复合物 (DNA-g3) 体外包装到病毒前体衣壳（前头）中至关重要。 pRNA 是 phi29 DNA 包装马达的组成部分，是最强的分子马达之一，pRNA 通过相同分子的分子间碱基配对形成新型环状六聚体。这种多聚体与前头的头尾连接器结合，前头是包装电机的核心，通过冷冻电镜 3-D 重建，它显示为五聚环。然后包装 ATP 酶 gp16 的多聚体与 pRNA 结合以完成马达。 pRNA 被假设在 DNA-gp3 和前头的对接中发挥作用，识别 DNA-gp3 的左端以启动包装，并作为 DNA 易位 ATP 酶的组成部分。 pRNA 在颈部和尾部组装过程中离开充满 DNA 的头部，并且它不是成熟病毒体的一部分。研究这种依赖RNA的DNA包装马达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普遍意义。该研究的最终目标是确定pRNA构成phi29 DNA包装马达以催化DNA-gp3易位至prohead的结构和机制。当前项目的目标是：1）通过cryoEM-3D重建和X射线晶体学研究由连接器、pRNA和ATP酶gp16组成的DNA包装马达； 2)通过NMR确定pRNA分子间假结的结构； 3）利用激光光镊研究pRNA-gp16运动亚基的包装运动步长和协调性； 4）确定gp 10（连接器）的N端在结合pRNA中的作用； 5) 鉴定噬菌体SPP1和lambda的DNA包装RNA。