MECHANISM OF POLYA TAIL FORMATION BY VACCINIA VIRUS

Poly(A)尾巴是一种几乎普遍存在的基因特征，然而它的作用(S)和它的合成机制(S)在任何生物体中都没有得到很好的描述。在各种尾部合成酶(PAPs的聚(A)聚合酶)中，痘苗病毒酶为研究PAP机制的重要方面提供了强有力的工具。因此，尽管它与蜂窝PAP具有相同的特征。病毒酶参与了一个相对简单的多聚腺苷酸化过程，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，也不与其他细胞过程耦合，如内切、mRNA剪接或蛋白质磷酸化。我建议利用病毒酶的直接作用来研究任何PAP作用的核心机制，即催化、易位和过程性。PAP是已知的最简单的RNA合成酶之一，关于PAP的机械论见解应该有助于更广泛地理解RNA合成和大分子机制。异源二聚体痘苗PAP的VP55(催化)亚基在产生约25-30个核苷酸长的尾巴后突然停止进行的引物延伸，而VP39亚基是VP55进一步延伸的过程因子。我们将使用各种互补技术(如定点光交联法、交联法-SELEX、标记转移光交联法和锚定的‘化学蛋白酶’法)来研究异二聚体和异二聚体-RNA三元复合体，以确定拓扑限制条件。例如，一个目标是确定VP39‘S多腺苷酸化特异的RNA接触位点(S)是否像蛋白质表面一样’完全‘位于明显的二聚界面的两部分之间。改善VP55过度表达的初步尝试应该有助于阐明其3D结构。将使用离散寡核苷酸和一种新的选择方案来研究VP55易位的RNA决定因素。VP55易位、引物锚定和进行性尾巴合成之间的关系将通过共价光交联型PAP-引物复合体建立，3‘OH-远端和近端引物-PAP接触位点的相对定位灵活性将通过设计用于’挤压‘和’拉伸‘它们的相对位置的寡核苷酸来表征。最后，为了研究金属离子在VP55的S催化中心的作用，我们将确定VP55中两个明显的二价阳离子结合位点中的哪一个是Mn2增强的；使用共价光交联型VP55-引物共轭化合物来确定二价金属在NTP结合和催化中的作用；通过电子顺磁共振定量VP55-金属离子的亲和力；以及使用不寻常的硫取代寡核苷酸来解决金属离子的配位问题。

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.