Catalytic Mechanism of Vaccinia Virus Protein VP39 in 2

痘苗病毒蛋白VP39的催化机制2

• 批准号: 9604188
• 负责人: Paul Gershon
• 金额: $ 27万
• 依托单位: Texas A&M AgriLife Research
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-15 至 2000-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604188&HistoricalAwards=false
• 关键词: Catalytic; Mechanism; Vaccinia; Virus; Protein

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.

9604188 Gershon第1部分-技术 RNA甲基转移酶，即牛痘病毒蛋白VP 39，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。这种酶的三维结构，最近由PI确定，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甲基化碱基而不是糖部分，该系统也提供了一个不寻常的机会来检查核酸核糖甲基化。 VP 39是一种双功能蛋白，其第二个功能是作为poly（A）聚合酶持续合成因子，与mRNA的两端相互作用。本研究调查是否VP 39拥有一个以上的RNA结合位点，以满足其双重功能，使用多聚腺苷酸化和甲基转移酶底物之间的竞争分析野生型VP 39和突变体与取代的表面残基在可能的RNA结合位点。还将进行功能和RNA结合试验。 由于单体VP 39对加帽RNA的亲和力大于未加帽RNA，因此还将研究加帽RNA结合对VP 39与其二聚化配偶体VP 55相互作用的影响。 进一步的实验将旨在研究VP 39与加帽RNA相互作用的双分子甲基化反应序列，例如底物电离和催化中的特定残基的参与。还将确定帽2 '-0-甲基化是否与甲基碳中心处的倒位相关。 第2部分非技术性牛痘病毒是一种良性痘病毒，其恶名源于其作为疫苗的广泛使用，导致第一次全球根除人类疾病，即天花。牛痘的独特之处在于它能够自主表达其基因，而不需要它感染的细胞中存在的大多数机制。它通过携带它自己的宿主细胞酶的类似物来做到这一点。这些类似物中的许多比宿主细胞等价物更容易研究，因为它们可以更容易地大量生产和纯化，但保持同样的信息，分子信息。这些酶中的一种被命名为“帽特异性2 '-O-甲基转移酶”，在所有基因的表达中起作用，并且被认为是这类酶的原型。RNA甲基转移酶（即牛痘病毒蛋白VP 39）的三维结构为研究RNA甲基化的催化机制提供了物理基础。VP 39与加帽RNA在结构和生物化学上的相互作用的阐明是分子生物化学朝着理解反应机制的方向取得进展的一个例子。