Mechanism of Poly(a) Tail Formation by Vaccinia Virus

描述（由申请人提供）：尽管多聚(A)尾部是mRNA中几乎无处不在的特征，但添加多聚(A)尾部的酶的结构和动力学在很大程度上是一个黑盒子。痘苗病毒多聚(A)聚合酶（PAP）在非模板核酸聚合酶中似乎是独特的，它具有(A)先天的易位能力，(b)一个可以帮助聚合酶延长的加工因子。此外，牛痘酶在缺少诸如RNA切割信号识别、RNA内核分裂、mRNA剪接和蛋白质磷酸化等偶联过程的情况下添加尾巴是不寻常的。因此，痘苗病毒PAP为研究单链酶内聚合酶分子动力学的重要方面提供了一个强有力的工具。虽然分离的痘苗PAP异二聚体的催化亚基（VP55）可以持续延长引物，但在添加长度约为25 - 30nt的尾部后，聚腺苷酸化停止。VP39亚基是VP55对尾巴伸长的促进因子。在初始资助阶段对VP39的结构和性能进行了研究。第二个资助期继续研究VP55亚基的易位特性，以及异源二聚体和异源二聚体- rna三元复合物的形貌。最近，研究VP55的一个长期障碍，即其高水平表达被克服，为需要大量VP55蛋白的结构和功能研究打开了大门。目的1 esta竞争继续解决结构生物学和结构-功能关系的聚合酶-加工因子（VP55-VP39）异二聚体。目的2和3涉及聚合酶分子动力学。提出了在p.l.使用的技术。实验室（分子生物学方法，光交联，质谱）将通过实验室间合作，在停止流动荧光和原子力显微镜的应用上得到加强。预计所提出的方法将提供一个整体的结构和动力学的易位，非模板聚合酶在聚(a)尾部的形成过程中。

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.