RNA in Viral DNA Packaging

病毒 DNA 包装中的 RNA

• 批准号: 8261108
• 负责人: SHELLEY GRIMES
• 金额: $ 35.97万
• 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 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8261108
• 关键词: ATP phosphohydrolase; Adenoviruses; Antiviral Agents; Bacteriophages; Base Pairing; Binding; Capsid; Complex; Cryoelectron Microscopy; DNA; DNA Packaging; Docking; Double Stranded DNA Virus; Enzymes; Genomics; Goals; Head; Herpesviridae; Hydroxyl Radical; Infection; Left; Maps; Modeling; Molecular Motors; Motor; Neck; Nucleotides; Pharmaceutical Preparations; Process; RNA; Research; Role; Site; Solutions; Structure; Tail; Viral; Virion; Virus Diseases; X-Ray Crystallography; base; design; laser tweezer; mutant; novel; reconstruction; single molecule; viral DNA

6. PROJECT SUMMARY/ABSTRACT The 174-base bacteriophage ¿29 prohead RNA (pRNA) is an essential component of the motor that packages the 19-kilobase pair genomic DNA-gp3 complex (DNA-gp3) into the viral precursor capsid (prohead). This motor is one of the strongest molecular motors known, generating ~110 pN of force. pRNA forms a novel cyclic hexamer by intermolecular base pairing of identical molecules. This multimer binds to the head-tail connector of the prohead, where it appears as a pentameric ring by cryoEM-3D reconstruction. A multimer of the ATPase gp16 then binds to the pRNA oligomer to constitute the ATP-hydrolyzing subunits of the motor. pRNA is hypothesized to function in docking of the DNA-gp3 on the prohead, in recognition of the left end of DNA-gp3 to initiate packaging, and in assembly and enzyme function 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 in uncovering targets for antiviral agents. The ultimate goal of the research is to determine the structural and functional roles of pRNA in the mechanism of DNA translocation. The current aims are: 1) X-ray crystallography will be used to determine atomic structures of monomeric and oligomeric pRNAs; 2) NMR will be used to determine the structure of the pRNA intermolecular pseudoknot and the essential CCA bulge of the A-helix; 3) Solution and site-directed hydroxyl radical probing will be used to map pRNA nucleotides in prohead/pRNA complexes with and without the ATPase gp16; 4) Site-directed and random pRNA mutants will be isolated to interrogate function of the pRNA in DNA translocation; and 5) Single-molecule laser tweezers studies of pRNA mutants having partial function will probe the roles of pRNA in ¿29 DNA translocation.

6。项目摘要/摘要 174碱基细菌噬菌体»29 prohead RNA（PRNA）是电动机的重要组成部分 包裹19-千里酶对基因组DNA-GP3复合物（DNA-GP3）纳入病毒前体capsid （prohead）。该电动机是已知的强分子电动机之一，产生了〜110 pn的力。 PRNA通过相同分子的分子间碱基对形成一种新型的环状六聚体。这 多聚机结合了Prohead的头尾连接器，在该连接器中，它以五聚戒指的形式结合 冷冻3D重建。然后，ATPase GP16的多聚体与PRNA低聚物结合到 构成电动机的ATP-hydrolysing亚基。假设PRNA在对接中起作用 Prohead上的DNA-GP3，以识别DNA-GP3的左端以启动包装，并在 DNA易位ATPase的组装和酶功能。 PRNA退出了充满DNA的头部 在颈部和尾部组件期间，它不是成熟病毒体的一部分。研究结构和 该RNA依赖性DNA包装电机的功能可能具有一般意义 抗病毒药的靶标。该研究的最终目标是确定结构和 PRNA在DNA易位机理中的功能作用。目前的目的是：1）X射线 晶体学将用于确定单体和寡聚PRNA的原子结构。 2） NMR将用于确定PRNA间分子伪not的结构和必需品 CCA凸起A螺旋； 3）溶液和位置定向的羟基自由基探测将用于绘制 PRNA核动肽在有或没有ATPase GP16的Prohead/PRNA复合物中； 4）定向 将分离随机PRNA突变体以询问PRNA在DNA易位中的功能。 5）对具有部分功能的PRNA突变体的单分子激光镊子研究将探测 PRNA在29个DNA易位中的作用。