RNA IN DNA VIRUS ASSEMBLY

噬菌体phi 29编码的174个碱基的前头RNA（pRNA）是 在19个内切酶对DNA基因产物的包装中必不可少 3复合物（DNA-gp 3）进入病毒前体衣壳或前头。 这一发现可能对病毒的组装具有普遍意义 在这个结构复杂的水平上。 最终目标是 确定pRNA构成phi 29 DNA的机制 包装机，并与蛋白质责成催化DNA-gp 3 易位 当前项目的目标是确定 pRNA的高级结构，因为它构成活性中心 DNA包装机的几何形状，并确定其相对 衣壳门蛋白gp 10和ATP结合蛋白gp 16。 phi 29 pRNA基因将通过定点和随机诱变 诱变和pRNA突变体鉴定和测序。 将分离特定突变体的抑制子并进行测序， 识别伪结和长程相互作用。 突变的RNA 将测试与DNA-gp 3包装中的prohead的相互作用 以及与gp 16复合时的ATP酶活性。光活化 叠氮基-ATP将用于鉴定 DNA-gp 3包装的蛋白质和/或pRNA成分 机 单独的pRNA的折叠和高级结构， 与prohead和/或gp 16复合的pRNA将通过以下方法进行研究： 用Fe（II）-EDTA和Rh（phen）2 phi 3+和衍生的碱裂解 与DTPA-FE（II）结合将在pRNA上的选定位点处掺入， 靶向裂解。 将在pRNA内寻找UV交联， pRNA-蛋白质复合物在催化条件下作为 三维结构。 当三级约束为 得到，将使用符号和数值计算方法 用于pRNA的三维建模。 将试图 使pRNA结晶并使pRNA和gp 16共结晶以用于X射线 衍射研究 最后，pRNA将被定位在前头部， 它离开新生病毒颗粒的步骤 研究了 pRNA-gp的质量、化学计量和形态 16和pRNA-连接体复合物的测定。

The 174-base bacteriophage phi29-encoded prohead RNA (pRNA) is essential in the packaging of the 19 kilobase pair DNA-gene product 3 complex (DNA-gp3) into the viral precursor capsid or prohead. This finding may have general significance for assembly of viruses at this level of structural complexity. The ultimate goal is to determine the mechanism by which pRNA constitutes the phi29 DNA packaging machine and enjoins with proteins to catalyze DNA-gp3 translocation. The goal of the current project is to determine the higher order structure of pRNA as it constitutes the active center of the DNA packaging machine and to determine its geometry relative to the capsid portal protein gpl0 and the ATP-binding protein gpl6. The phi29 pRNA gene will be mutagenized by site-directed and random mutagenesis and the pRNA mutants identified and sequenced. Suppressors of specific mutants will be isolated and sequenced to identify pseudoknots and long range interactions. The mutant RNAs will be tested for interactions with proheads in DNA-gp3 packaging and for ATPase activity when complexed to gpl6. Photoactivatable azido-ATP will be used to identify the ATPase active center(s) of the protein and/or pRNA constituents of the DNA-gp3 packaging machine. The folding and higher order structure of pRNA alone and pRNA complexed to the prohead and/or gp 16 will be investigated by cleavage with Fe(II)-EDTA and Rh(phen)2phi3+ and bases derivatized with DTPA-FE(II) will be incorporated at selected sites on pRNA to target cleavage. UV cross-links will be sought within pRNA and pRNA-protein complexes under conditions of catalysis as a probe of three-dimensional structure. When tertiary constraints are obtained, a symbolic and numerical computation method will be used for three-dimensional modeling of pRNA. Attempts will be made to crystallize pRNA and to cocrystallize pRNA and gpl6 for x-ray diffraction studies. Finally, pRNA will be localized on proheads, and the step at which it leaves the nascent viral particle will be investigated. The mass, stoichiometry and morphology of pRNA-gp 16 and pRNA-connector complexes will be determined.