RNA IN VIRAL DNA PACKAGING

174个碱基的噬菌体phi 29前头RNA（pRNA）对于将19个内切酶对DNA-基因产物3复合物（DNA-gp 3）体外包装到病毒前体衣壳或前头中是必需的。pRNA通过相同分子的分子间碱基配对形成新的环状六聚体，并且该六聚体与前头部的头尾连接器结合。 假设pRNA在DNA-gp 3和前头部的对接中起作用，识别DNA-gp 3的左端以启动包装，并且作为DNA易位ATP酶的组分。 pRNA在颈部和尾部组装期间离开DNA填充的头部，并且它不是成熟病毒体的一部分。 这种RNA依赖的DNA包装机器的结构和功能的研究可能对其他病毒，包括哺乳动物病毒的组装具有普遍意义。 本研究的最终目的是确定pRNA构成phi 29 DNA包装机并与连接器和包装ATP酶连接以催化DNA-gp 3易位到prohead中的机制。 本研究的主要目的是：1）利用核糖核酸酶足迹法和体外筛选与gp 16结合的适体研究pRNA上的gp 16相互作用结构域，2）研究pRNA作为DNA包装方向的决定因素，3）利用透射和扫描透射电子显微镜研究pRNA六聚体和与连接体或gp 16复合的pRNA的结构，4）研究pRNA与连接体或gp 16的相互作用。4）使用Fe（II）-EDTA作为游离pRNA和参与DNA包装的pRNA的三级结构的探针; 5）改进pRNA的分子模型; 6）产生pRNA和与连接子或gp 16复合的pRNA的衍射质量晶体;和7）表征λ和T3的DNA包装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.