RNA IN VIRAL DNA PACKAGING

174碱基噬菌体phi29前体RNA （pRNA）是将19千碱基对dna -基因产物3复合物（DNA-gp3）体外包装到病毒前体衣壳或前体中所必需的。pRNA通过相同分子的分子间碱基配对形成一种新的环状六聚体，这种六聚体结合在前体的首尾接头上。据推测，pRNA的功能包括对接DNA-gp3和前体，识别DNA-gp3的左端以启动包装，以及作为DNA易位atp酶的组成部分。pRNA在颈部和尾部组装过程中退出充满dna的头部，它不是成熟病毒粒子的一部分。研究这种依赖rna的DNA包装机的结构和功能，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构成phi29 DNA包装机并与连接器和包装atp酶结合催化DNA-gp3易位到前体的机制。目前项目的目标是：1)通过核糖核酸酶足迹和gp16结合适体的体外筛选研究gp16在pRNA上的相互作用结构域；2)研究pRNA作为DNA包装取向的决定因素；3)通过透射电镜和扫描电镜研究pRNA六聚体和与连接器或gp16络合的pRNA的结构；4)利用Fe(II)-EDTA作为游离pRNA和参与DNA包装的pRNA三级结构探针；5)完善pRNA的分子模型；6)生成具有衍射质量的pRNA晶体和与连接器或gp16络合的pRNA晶体；7)表征噬菌体lambda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.