Catalytic Mechanism of Vaccinia Virus Protein VP39 in 2

9604188格尔逊第1部分-技术核糖核酸甲基转移酶，即。痘苗病毒蛋白VP39甲基化mRNA帽结构的倒数第二个核苷酸的核糖2‘-O-，导致I型mRNA帽的形成。最近P.I.测定的该酶的三维结构为研究RNA甲基化的催化机制提供了结构基础。由于其他结构上定义的甲基转移酶是甲基碱基而不是糖部分，这个系统也提供了一个不同寻常的机会来检查核酸核糖甲基化。VP39是一种双功能蛋白，其第二功能是作为聚(A)聚合酶加工因子，与两端的mRNA相互作用。本研究利用野生型VP39的聚腺苷酸化底物和甲基转移酶底物之间的竞争分析，以及在可能的RNA结合位点上表面残基被取代的突变体之间的竞争，研究VP39是否具有一个以上的RNA结合位点来满足其双重功能。还将进行功能和RNA结合分析。由于单体VP39对封闭式RNA的亲和力大于未封闭式RNA，因此也将研究封闭式RNA结合对VP39与其二聚伙伴VP55相互作用的影响。进一步的实验将致力于研究VP39与CAP RNA相互作用的双分子甲基化反应序列，如底物电离的参与，以及特定残基在催化中的参与。2‘-0-甲基化是否与甲基碳中心的倒置相关也将被确定。第二部分非技术性痘苗病毒是一种良性的痘病毒，其恶名源于它作为疫苗的广泛使用，导致了第一次在全球根除一种人类疾病，即天花。牛痘病毒实际上是独一无二的，它能够自主表达自己的基因，而不需要它感染的细胞中存在的大多数机器。它是通过携带它自己的宿主细胞酶的类似物来做到这一点的，而这是它其他人所需要的。其中许多类似物比宿主细胞等效物更容易研究，因为它们可以更容易地大量生产和纯化，同时保持同样信息量的分子信息。其中一种酶，命名为“帽特异性2‘-O-甲基转移酶”，在所有基因的表达中发挥作用，被认为是这类酶的原型。本研究旨在了解核糖核酸甲基转移酶的精确作用机制。痘苗病毒蛋白VP39为研究RNA甲基化的催化机制提供了物理基础。VP39与Caped RNA在结构和生化上相互作用的阐明，是分子生物化学在理解反应机制方面取得进展的一个例子。

9604188 Gershon Part 1-Technical RNA methyltransferase, viz. vaccinia virus protein VP39, methylates the ribose 2'-O- of the penultimate nucleotide of the mRNA cap structure leading to formation of the type I mRNA cap. The three-dimensional structure of this enzyme, recently determined by the P.I., affords a structural basis on which to investigate the catalytic mechanism of RNA methylation. Since other structurally-defined methyltransferases methylate base rather than sugar moieties, this system provides also an unusual opportunity to examine nucleic acid ribose methylation. VP39 is a bifunctional protein whose second function is as a poly(A) polymerase processivity factor, interacting with both mRNA ends. This study investigates whether VP39 possesses more than a single RNA binding site to satisfy its dual function, using competition assays between polyadenylation and methyltransferase substrates for wild-type VP39 and mutants with substituted surface residues at possible RNA binding sites. Functional and RNA binding assays will also be done. Due to the greater affinity of monomeric VP39 for capped than uncapped RNA, the effect on capped RNA binding on VP39 interaction with its dimerization partner, VP55, will also be investigated. Further experiments will aim at the study of the bimolecular methylation reaction sequence for the interaction of VP39 with capped RNA, such as the involvement of substrate ionization, and of specific residues in catalysis. Whether cap 2'-0-methylation correlates with inversion at the methyl carbon center will also be determined. Part 2 Non -Technical Vaccinia virus is a benign poxvirus whose notoriety stems from its extensive use as the vaccine that led to the first global eradication of a human disease, namely smallpox. Vaccinia is virtually unique in its ability to express its genes autonomously, without needing most of the machinery present in the cells it infects. It does this by carrying its own analogs of host cell enzymes that it would other requi re. Many of these analogs are much easier to study than the host cell equivalent, because they can more easily be produced in large quantities and purified, yet maintain an equally informative, molecular information. One of these enzymes, named "cap specific 2'-O-methyltransferase", plays a role in the expression of all genes, and is considered a prototype for this class of enzymes. This study is aimed at understanding the precise mechanism by which the enzyme operates.The three-dimensional structure of RNA methyltransferase, viz. vaccinia virus protein VP39, affords a physical basis on which to investigate the catalytic mechanism of RNA methylation. The elucidation of the interaction of VP39 with capped RNA, structurally and biochemically, is one example of advances in Molecular Biochemistry towards the understanding of reaction mechanisms.