RNA IN VIRAL DNA PACKAGING

174碱基的噬菌体phi29前头RNA(PRNA)是将19kb碱基对的DNA-基因产物3复合体(DNA-GP3)体外包装成病毒前体衣壳或前头所必需的。PRNA通过相同分子间的碱基配对形成一种新型的环状六聚体，该六聚体与前头的头部-尾部连接。PRNA被认为在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转位ATPase的一个组成部分。PRNA在颈部和尾部组装过程中离开充满DNA的头部，它不是成熟病毒粒子的一部分。这种依赖RNA的DNA包装机的结构和功能的研究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。本研究的最终目的是确定PRNA组成phi29 DNA包装机，并与连接体和包装ATPase结合以催化DNA-GP3转位到前部的机制。本项目的目标是：1)通过核糖核酸酶足迹和体外筛选gp16结合适体来研究PRNA上的gp16相互作用结构域；2)研究PRNA作为DNA包装取向的决定因素；3)通过透射和扫描电子显微镜研究PRNA六聚体和PRNA与连接体或gp16络合的结构；4)使用Fe(II)-EDTA作为探针，研究游离PRNA和参与DNA包装的PRNA的三级结构；5)完善PRNA的分子模型；6)制备PRNA和PRNA与连接体或gp16络合的衍射性晶体；以及7)表征DNA包装中的RNA。

The 174-base bacteriophage phi29 prohead RNA (pRNA) is essential for in vitro packaging of the 19 kilobase pair DNA-gene product 3 complex (DNA-gp3) into the viral precursor capsid or prohead. pRNA forms a novel cyclic hexamer by intermolecular base pairing of identical molecules, and this hexamer binds to the head-tail connector of the prohead. 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 machine may have general significance for assembly of other viruses, including mammalian viruses. The ultimate goal of the research is to determine the mechanisms by which pRNA constitutes the phi29 DNA packaging machine and joins with the connector and packaging ATPase to catalyze DNA-gp3 translocation into the prohead. The aims of the current project are to: 1) study the gp16 interactive domain on pRNA by ribonuclease footprinting and in vitro selection of aptamers for gp16 binding; 2) investigate pRNA as the determinant of orientation of DNA packaging; 3) study the structure of pRNA hexamers and pRNA complexed to connectors or gp16 by transmission and scanning transmission electron microscopy; 4) use Fe(II)-EDTA as a probe of tertiary structure of free pRNA and pRNA engaged in DNA packaging; 5) refine the molecular models of pRNA; 6) produce diffraction quality crystals of pRNA and pRNA complexed to connectors or gp16; and 7) characterize the DNA packaging RNAs of phages lambda and T3.