Mechanism of Poly(a) Tail Formation by Vaccinia Virus

描述（由申请人提供）：虽然poly（A）尾几乎是mRNA的普遍特征，但添加它的酶的结构和动力学在很大程度上是一个黑盒。牛痘病毒多聚腺苷酸聚合酶（PAP）在非模板核酸聚合酶中似乎是独特的，具有（a）先天易位能力，（B）可帮助聚合酶延伸的持续合成因子。此外，牛痘酶在不存在偶联过程如RNA切割信号识别、RNA内切核酸裂解、mRNA剪接和蛋白质磷酸化的情况下添加尾部是不寻常的。因此，牛痘病毒PAP提供了一个强有力的工具，研究聚合酶分子动力学的重要方面的单链酶。尽管牛痘PAP异二聚体的分离的催化（VP 55）亚基可促发性延长引物，但在添加了约25 - 30 nt长的尾部后，聚腺苷酸化停止。VP 39亚基是VP 55尾延伸的持续合成因子。VP 39的结构和性质在最初的资助期间进行了研究。第二个资助期继续进行VP 55亚基的易位特性的表征，以及异二聚体和异二聚体-RNA三元复合物的拓扑结构。最近，VP 55研究的一个长期障碍，即其高水平表达被克服，为需要更大量VP 55蛋白的结构和功能方法打开了大门。目的我的竞争继续地址的结构生物学和结构功能的关系内的聚合酶-合成因子（VP 55-VP 39）异二聚体。目的2和3解决聚合酶分子动力学。建议在P.I.的实验室（分子生物学方法，光交联，质谱法）将通过实验室间的合作来加强停流荧光和原子力显微镜的应用。预计所提出的方法将提供一个完整的视图的结构和动力学的易位，非模板聚合酶在聚（A）尾的形成。

DESCRIPTION (provided by applicant): Although the poly (A) tail is a nearly ubiquitous feature of mRNA, the structure and dynamics of the enzyme that adds it are largely a black box. The vaccinia virus poly (A) polymerase (PAP) appears to be unique among the non-templated nucleic acid polymerases in possessing (a) an innate ability to translocate, (b) a processivity factor that can assist the polymerase in elongation. Moreover, the vaccinia enzyme is unusual in adding a tail in the absence of coupled processes such as RNA cleavage signal recognition, RNA endonucleolytic scission, mRNA splicing and protein phosphorylation. The vaccinia virus PAP therefore provides a powerful tool with which to study important aspects of polymerase molecular dynamics within a single-chain enzyme. Although the isolated catalytic (VP55) subunit of the vaccinia PAP heterodimer can processively elongate a primer, polyadenylation halts after tails approximately 25 - 30 nt in length have been added. The VP39 subunit is a processivity factor for tail elongation by VP55. VP39's structure and properties were studied during the initial funding period. The second funding period continued with a characterization of the translocational properties of the VP55 subunit, and the topography of both the heterodimer and the heterodimer-RNA ternary complex. Very recently, a long-standing obstacle to the study of VP55, namely its high-level expression was overcome, opening the door to structural and functional approaches requiring larger amounts of VP55 protein. Aim I of this competing continuation addresses the structural biology and structure-function relationships within the polymerase-processivity factor (VP55-VP39) heterodimer. Aims 2 and 3 address polymerase molecular dynamics. It is proposed that techniques in use in the P.l.'s lab (molecular biological approaches, photo crosslinking, mass spectrometry) will be augmented by inter-lab collaboration for the application of stopped-flow fluorescence and atomic force microscopy. It is expected that the proposed approaches will provide an integrated view of the structure and dynamics of a translocating, non-templated polymerase during formation of the poly (A) tail.