MECHANISM OF POLYA TAIL FORMATION BY VACCINIA VIRUS

多聚(A)尾实际上是一种普遍存在的mRNA特征，但它的作用和合成机制在任何生物体中都没有很好地表征。在各种尾部合成酶（PAP的聚(A)聚合酶）中，牛痘病毒酶为研究PAP机制的重要方面提供了强有力的工具。因此，尽管它与细胞pap具有相同的特征。病毒酶参与一个相对简单的多腺苷化过程，该过程既不耦合于与多个RNA信号的相互作用或辅助蛋白因子，也不耦合于其他细胞过程，如核内溶裂解、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的核心作用机制，即催化、易位和加工性。PAP是一种已知最简单的RNA合成酶，它的机制见解有助于对RNA合成和大分子机制有更全面的了解。异二聚体痘苗PAP的VP55（催化）亚基在产生大约25- 30nt长度的尾部后突然停止了引物的加工延伸，VP39亚基是VP55进一步延伸的一个加工因子，异二聚体和异二聚体- rna三元复合物将使用各种互补技术(如位点特异性光交联，交联selex，光交联与标签转移和锚定的“化学蛋白酶”方法)，以确定拓扑约束。例如，一个目的是确定VP39的聚腺苷化特异性RNA接触位点是否像在一个“完全”的蛋白质表面上的两个部分之间的明显的两部分二聚化界面。改善VP55过表达的初步尝试应该有助于阐明其三维结构。VP55易位的RNA决定因素将使用离散寡核苷酸和一种新的选择方案进行研究。VP55易位、引物锚定和过程尾合成之间的关系将通过共价光交联PAP-引物复合物建立，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为“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。ATP复合物是Mn2+增强的；利用共价光交联vp55引物偶联物确定二价金属在NTP结合和催化中的作用；EPR定量vp55 -金属离子亲和度；并使用不寻常的硫取代寡核苷酸来处理金属离子配位。

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.