MECHANISM OF POLYA TAIL FORMATION BY VACCINIA VIRUS

poly（A）尾是一种几乎普遍存在的mRNA特征，但它的作用和合成机制在任何生物中都没有得到很好的表征。在各种尾部合成酶（PAP的聚（A）聚合酶）中，牛痘病毒酶提供了一种强有力的工具，用于研究PAP机制的重要方面。因此，尽管它与蜂窝PAP共享特性。病毒酶参与相对简单的多腺苷酸化过程，该过程既不与具有多个RNA信号的相互作用或辅助蛋白因子偶联，也不与其它细胞过程偶联，例如核酸内切裂解、mRNA剪接或蛋白磷酸化。我建议利用病毒酶的直接行动，调查机制中央任何PAP的行动，即催化，易位和持续合成能力。PAP是已知的最简单的RNA合成酶之一，对其机制的深入研究有助于对RNA合成和大分子机制有更全面的了解。VP55异源二聚体牛痘PAP的（催化）亚基在产生长度约25-30 nt的尾后突然停止进行性引物延伸，并且VP 39亚基是VP 55进一步延伸的持续合成因子，异源二聚体和异源二聚体-RNA三元复合物将使用各种互补技术进行研究（例如位点特异性光交联、交联-SELEX、具有标记转移的光交联和锚定的“化学蛋白酶”方法）以鉴定拓扑约束。例如，一个目的是确定VP 39的多聚腺苷酸化特异性RNA接触位点是否像在明显的二分二聚化界面的两个部分之间的蛋白质表面上的“完全”内。改善VP 55过表达的初步尝试应有助于阐明其3D结构。RNA决定因素的VP 55易位将使用离散的寡核苷酸和一种新的选择方案进行研究。将使用共价光交联的PAP-引物复合物来建立VP 55易位、引物锚定和进行性尾合成之间的关系，并且将使用设计成“挤压”和“拉伸”其相对位置的寡核苷酸来表征3'OH-远端和近端引物-PAP接触位点的相对定位的灵活性。最后，为了研究金属离子在VP 55催化中心的作用，我们将确定VP 55. ATP复合物中两个明显的二价阳离子结合位点中的哪一个是Mn 2+增强的;使用共-光交联的VP 55-引物缀合物鉴定二价金属在NTP结合和催化中的作用;通过EPR定量VP 55-金属离子亲和力;并使用不常见的硫取代的寡核苷酸解决金属离子配位。

The poly(A) tail is a virtually ubiquitous mRNA feature, yet neither its role(s) nor its mechanism(s) of synthesis are well characterized in any organism. Of the various tail-synthesizing enzymes (poly(A) polymerases of PAPs), the vaccinia virus enzyme provides a powerful tool with which to study important aspects of PAP mechanism. Thus, although it shares characteristics with the cellular PAPs. the viral enzyme participates in a relatively simple polyadenylylation process that is coupled to neither the interactions or auxiliary protein factors with multiple RNA signals, nor other cellular processes such as endonucleolytic cleavage, mRNA splicing or protein phosphorylation. I propose to capitalize upon the viral enzyme's directness of action to investigate mechanisms central the action of any PAP, namely catalysis, translocation and processivity. Mechanistic insights regarding PAP, of one of the simplest known RNA- synthetic enzymes, should contribute to a more general understanding of RNA synthesis and macromolecular machinery. The VP55 (catalytic) subunit of the heterodimeric vaccinia PAP abruptly ceases processive primer elongation after producing tails approximately 25-30 nt in length, and the VP39 subunit is a processivity factor for further elongation by VP55, the heterodimer and the heterodimer-RNA ternary complex will be investigated using various complementary techniques (such as site- specific photocrosslinking, crosslinking-SELEX, photocrosslinking with label-transfer and an anchored 'chemical protease' approach) to identify topological constraints. For example, one aim is to establish whether VP39's polyadenylylation-specific RNA contact site(s) like within a 'fully' on the protein surface between two parts of an apparently bipartite dimerization interface. Initial attempts to improve VP55 over- expression should facilitate elucidation of its 3D structure. RNA determinants of VP55 translocation will be investigated using both discrete oligonucleotides and a novel selection scheme. The relationship between VP55 translocation, primer anchoring and processive tail synthesis will be established using covalently-photocross-linked PAP- primer complexes and flexibility in the relative positioning of the 3' OH-distal and proximal primer-PAP contact sites will be characterized using oligonucleotides designed to 'squeeze' and 'stretch' their relative positions. Finally, to investigate roles for metal ions at VP55's catalytic center, we will determine which of the two apparent divalent cation binding sites in the VP55.ATP complex is Mn2+-enhanced; identify roles for divalent metals in NTP binding and catalysis using co-valently photocrosslinked VP55-primer conjugates; quantify VP55-metal ion affinities by EPR; and address metal ion coordination using unusual, sulfur-substituted oligonucleotides.