RNA IN DNA VIRUS ASSEMBLY

174碱基噬菌体phi29编码前导RNA(PRNA)是 19kb碱基对DNA-基因产物的包装中必不可少的 3复合体(DNA-GP3)形成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 在这种复杂的结构水平上。最终目标是 确定PRNA组成phi29 DNA的机制 包装机和蛋白质对DNA-GP3的催化作用 易位。当前项目的目标是确定 PRNA的高阶结构，因为它构成活性中心 以确定DNA包装机的几何关系 衣壳蛋白gpl0和三磷酸腺苷结合蛋白gpl6。 Phi29prna基因将通过定点和随机诱变。 诱变及PRNA突变体的鉴定和测序。 特定突变体的抑制子将被分离和测序以 识别伪结点和远程交互。突变的RNA 将测试与DNA-GP3包装中的探针的相互作用 当与gpl6络合时，对ATPase活性有影响。可光激活的 叠氮-三磷酸腺苷将被用来鉴定三磷酸腺苷酶活性中心(S) DNA-GP3包装的蛋白质和/或PRNA成分 机器。PRNA单独的折叠和高阶结构 与Prohead和/或GP 16络合的PRNA将由 Fe(II)-EDTA和Rh(Phen)_2Phi~(3+)的裂解及碱衍生物 与DTPA-FE(II)将在PRNA上的选定位置结合，以 目标乳沟。将在PRNA和PRNA中寻找UV交叉连接 催化条件下的PRNA-蛋白质络合物作为探针 三维结构。当第三级约束为 得到，将使用符号和数值计算方法 用于PRNA的三维建模。我们将尝试 使PRNA结晶，并使PRNA和gpl6共结晶用于x射线 衍射研究。最后，PRNA将被定位在PROCES上， 它离开新生病毒颗粒的步骤将是 调查过了。PRNA-GP的质量、化学计量和形态 16和PRNA-连接器复合体将被确定。

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.