Structure and Function of Phi29 hexameric pRNA

Phi29 六聚体 pRNA 的结构和功能

• 批准号: 6871533
• 负责人: PEIXUAN GUO
• 金额: $ 27.56万
• 依托单位: PURDUE UNIVERSITY
• 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 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6871533
• 关键词: DNA virus; adenosinetriphosphatase; affinity labeling; bacterial virus; biophysics; chemical models; chemical structure function; crosslink; electron microscopy; enzyme activity; fluorescence resonance energy transfer; intermolecular interaction; intracellular transport; matrix assisted laser desorption ionization; molecular site; nucleocapsid; stoichiometry; virus DNA; virus RNA; virus assembly; virus infection mechanism

DESCRIPTION (provided by applicant): Migration or translocation of proteins or nucleic acids through barriers or cell membranes is a common process in biological systems. One of the most complex and intricate translocation processes is viral genomic DNA packaging. A strikingly common feature in the maturation of linear dsDNA viruses, including herpes viruses, adenoviruses, and the ds-DNA bacteriophages, is that the lengthy genome is translocated with remarkable velocity into a limited space within a preformed protein shell and packaged into near-crystalline density. A DNA-packaging motor accomplishes this energetically unfavorable motion reaction. The bacterial virus phi29 DNA packaging motor contains six pRNA molecules, which bind ATP and form a hexamer as a vital part of the motor. This pRNA contains two function domains, one domain is for binding to the protein component of the motor, and the other is the DNA-packaging domain, but the detailed structure and function of this domain is unknown. We hypothesize that the alternation between contraction and relaxation, driven by ATP hydrolysis, of each member of the hexameric RNA ring could rotate the DNA transportation machinery. Our long-term objective is to elucidate the biochemical and biophysical mechanism of this RNA-containing motor, thus providing fundamental information concerning antiviral therapy, bioenergy conversion, macromolecular interactions, nanomotors as parts for nanodevices, and the general mechanism(s) of biomotors. For instance, the motor components of cytomegalovirus have been used as targets for antiviral drug design. The short-term objective of this renewed grant is to continue the study on the structure and function of the hexamer pRNA, focusing on the structure and function of the DNA packaging domain. The stoichiometry of pRNA on the motor before, during and after DNA packaging will be confirmed. ATP-binding and possible ATPase activity of pRNA will be further investigated by crosslinking and ATPase assays under various conditions. Photoaffinity crosslinking will be used to determine the target, if any, the DNA-packaging domain binds or is adjacent to. The specific pRNA/protein interactions will be probed to refine our understanding of the 3D structure of the motor complex. Probing for such precise interactions involves the detection of both the specific nucleotides of pRNA that bind to the connector and the specific amino acids of the connector protein that contacts the pRNA. Fluorescent beads or nanotubes will be used to label the components to allow direct observation of the direction of motion.

描述（由申请人提供）：蛋白质或核酸通过障碍物或细胞膜的迁移或易位是生物系统中的常见过程。最复杂，最复杂的易位过程之一是病毒基因组DNA包装。线性dsDNA病毒的成熟中的一个非常共同的特征，包括疱疹病毒，腺病毒和DS-DNA噬菌体，是长时间的基因组以显着的速度转化为预先形成的蛋白质壳中的有限空间，并包装成近晶体的密度。 DNA包装电动机实现了这种能量不利的运动反应。 细菌病毒PHI29 DNA包装电机包含六个PRNA分子，它们结合ATP并形成六聚体作为电动机的重要部分。该PRNA包含两个函数域，一个域是用于与电动机的蛋白质成分结合，另一个域是DNA包装域，但是该域的详细结构和功能尚不清楚。我们假设，由ATP水解驱动的收缩与放松之间的交替，六聚体RNA环的每个成员都可以旋转DNA传输机械。我们的长期目标是阐明该含RNA的电动机的生化和生物物理机制，从而提供有关抗病毒疗法，生物能转化，大分子相互作用，纳米电视部分的纳米运动剂的基本信息，以及纳米模块的一部分以及生物动物的一般机制。例如，巨细胞病毒的运动成分已被用作抗病毒药物设计的靶标。这种新授予的赠款的短期目标是继续研究己酰胺PRNA的结构和功能，重点关注DNA包装域的结构和功能。在DNA包装之前，期间和之后，PRNA的化学计量学将得到确认。在各种条件下，通过交联和ATPase分析将进一步研究PRNA的ATP结合和可能的ATPase活性。光性交联将用于确定DNA包装域结合或与之相邻的目标（如果有）。将探测特定的PRNA/蛋白质相互作用，以完善我们对运动复合物3D结构的理解。对这种精确相互作用进行探测涉及检测与接触PRNA的连接器蛋白的pRNA的特异性核苷酸和连接器的特定氨基酸的检测。荧光珠或纳米管将用于标记组件以直接观察运动方向。