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. 项目概要/摘要 174 个碱基的噬菌体 ¿ 29 前头 RNA (pRNA) 是运动的重要组成部分， 将 19 KB 对基因组 DNA-gp3 复合物 (DNA-gp3) 包装到病毒前体衣壳中 （前头）。该电机是已知最强的分子电机之一，可产生约 110 pN 的力。 pRNA 通过相同分子的分子间碱基配对形成新型环状六聚体。 多聚体结合到前头的头尾连接器上，在此处它表现为五聚体环 CryoEM-3D 重建然后 ATPase gp16 的多聚体与 pRNA 寡聚体结合。 构成运动的 ATP 水解亚基的 pRNA 被重新捕获以在对接中发挥作用。 前头上的 DNA-gp3，识别 DNA-gp3 的左端以启动包装，并在 DNA 转位 ATP 酶的组装和酶功能离开充满 DNA 的头部。 颈部和尾部组装过程中，它不是成熟病毒粒子的结构和研究的一部分。 这种依赖于 RNA 的 DNA 包装马达的功能可能对揭示 研究的最终目标是确定抗病毒药物的结构和作用。 pRNA 在 DNA 易位机制中的功能作用 目前的目标是： 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分子间假结的结构和必要的 A 螺旋的 CCA 凸起；3) 将使用溶液和定点羟基自由基探测来绘制图谱 含有和不含 ATP 酶 gp16 的 prohead/pRNA 复合物中的 pRNA 核苷酸；4) 定点 随机分离 pRNA 突变体，以探究 pRNA 在 DNA 易位中的功能； 5）具有部分功能的 pRNA 突变体的单分子激光镊研究将探测 pRNA 在 ¿ 中的作用29 DNA易位。