Mechanism of Poly(a) Tail Formation by Vaccinia Virus

描述(申请人提供)：尽管聚(A)尾巴是信使核糖核酸几乎无处不在的特征，但添加它的酶的结构和动力学在很大程度上是一个黑匣子。痘苗病毒多(A)聚合酶(PAP)在非模板核酸聚合酶中似乎是独一无二的，它具有(A)天生的易位能力，(B)有助于聚合酶延长的加工性因子。此外，在缺乏RNA切割信号识别、RNA内切、mRNA剪接和蛋白质磷酸化等耦合过程的情况下，痘苗病毒酶不寻常地增加了尾巴。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大的工具。虽然分离的痘苗病毒PAP异源二聚体的催化(VP55)亚基可以连续地延长引物，但在添加大约25-30个核苷酸长的尾巴后，多聚腺苷基化停止。VP39亚基是VP55延长尾巴的加工因子。对VP39‘S的结构和性能进行了研究。第二个资助期继续描述了VP55亚单位的易位性质，以及异二聚体和异二聚体-RNA三元复合体的形貌。最近，VP55研究的一个长期障碍，即它的高水平表达被克服了，为需要更多VP55蛋白的结构和功能方法打开了大门。这一竞争延续的目的I解决了聚合酶加工因子(VP55-VP39)异源二聚体内的结构生物学和结构-功能关系。目标2和3涉及聚合酶分子动力学。建议通过实验室间的协作，加强S实验室使用的技术(分子生物学方法、光交联法、质谱仪)，以应用停流荧光和原子力显微镜。预计所提出的方法将提供关于在聚(A)尾巴形成过程中移位的、非模板的聚合酶的结构和动力学的综合视图。

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.