MECHANISM OF POLY(A) TAIL FORMATION BY VACCINIA VIRUS

Poly(A)尾巴是真核生物和病毒的特征 信使核糖核酸，在信使核糖核酸的翻译和稳定性中起作用。然而， 其与信使核糖核酸的作用机制尚不完全清楚。这个 本文所述工作的目标是开发一种模型，在该模型中 详细定义了聚(A)尾巴的合成机理。牛痘 病毒提供了进行此类研究的强大工具，因为 它的Poly(A)尾巴伸长策略类似于 细胞聚腺苷酸化系统，而病毒系统相对 简单，因为没有耦合的过程，如RNA切割和 信号识别，以及缺乏涉及 备用加工地点选择。此外，一个详细的机制， 已知最简单的RNA合成之一的聚(A)聚合酶的分析 酶，应该说明一些更一般的问题 RNA聚合酶中的RNA-蛋白质交易。迄今为止，私家侦探已经 鉴定异源二聚体痘苗病毒Poly(A)编码基因 聚合酶，并对VP55和VP39亚基进行了鉴定。 高度纯净的形式。VP55和VP39被发现具有催化和 分别是刺激性亚基，其中VP55连续增加一个30- 35个核苷酸的聚(A)末端到信使核糖核酸的3‘末端，然后突然转变为 非过程性扩展，VP39使VP55保持过程性 尾巴伸长时超过30-35个新台币。私家侦探还显示 VP55通过尿苷酸残基与RNA特异性地相互作用，并且 在腺苷加成过程中相对于RNA的移位。突然性 因此，VP55在处理能力上的转变似乎是由于其 沿着新生的Poly(A)尾巴移位，它缺乏尿苷 稳定的互动。VP39被证明具有额外的功能，在2‘- M RNA 5‘端的O-甲基化。在这项提议中，一个更具竞争力的模式 将开发聚(A)尾部形成的新技术。最初，研究将是 为解决体内聚(A)尾巴形成的问题而进行的研究：它将 确定VP55基因是否是必需的；是否可以制造病毒 这对Poly(A)尾巴的延长是有条件的致命的； 体内尾巴长度受体内水平以外的其他因素调节 爸爸。第二，大分子的特性和专一性 将表征相互作用以确定RNA结合位置 特征，哪个亚基将PAP引导到3‘-OH， 5‘端帽子结构对VP39-RNA相互作用的贡献 一个亚基仅通过另一个亚基与RNA相互作用。第三，中国的区域 RNA和核苷酸结合以及二聚化所需的蛋白质将 将尝试使这两个亚基结晶。 最后，将进行功能实验，以确定是否 无论VP39是否相互作用，VP39都是一个滑动钳位型加工系数 以一种功能的方式与ATP结合，并调查 VP39的5‘和3’端修饰功能。上述研究是 期望提供对聚(A)的机制的详细了解 尾巴合成。

The poly(A) tail is a characteristic feature of eukaryotic and viral mRNAs,with roles in mRNA translation and stability. However, the mechanisms involved in its addition to mRNA are not fully understood. The goal of the work described herein is to develop a model in which the mechanism of synthesis of a poly(A) tail are defined in detail. Vaccinia virus provides a powerful tool with which to conduct such a study, since its strategy for poly(A) tail elongation is analogous to that employed by cellular polyadenylation systems, yet the viral system is relatively simple due to the absence of coupled processes such as RNA cleavage and signal recognition, and the absence of regulatory decisions involving alternate processing site choice. Furthermore, a detailed mechanistic analysis of poly(A) polymerase, of one of the simplest known RNA-synthetic enzymes, should illuminate a number of more general questions regarding RNA-protein transactions in RNA polymerases. To date, the P. I. has identified the genes encoding the heterodimeric vaccinia virus poly(A) polymerase, and characterized the individual subunits, VP55 and VP39, in highly purified form. VP55 and VP39 were found to be catalytic and stimulatory subunits, respectively, in which VP55 processively adds a 30 - 35 nt poly(A) tail to an mRNA 3' end followed by an abrupt transition to non-processive extension, and VP39 enables VP55 to retain processivity during elongation of the tail beyond 30 - 35 nt. The P. I. has also shown that VP55 interacts with RNA specifically via uridylate residues, and translocates with respect to the RNA during adenylate addition. The abrupt transition in processivity by VP55 thus appears to result from its translocation along the nascent poly(A) tail, which lacks uridylates for stable interaction. vP39 was shown to have an additional function, in 2'- O-methylation of the mRNA 5' cap. In this proposal, a more compete model of poly(A) tail formation will be developed. Initially, studies will be performed to address aspects of poly(A) tail formation in vivo: It will be determined whether the VP55 gene is essential; whether viruses can be made that arc conditionally lethal for poly(A) tail elongation; and whether in vivo tail lengths are regulated by factors other than in vivo levels of PAP. Second, characteristics and specificities of macromolecular interactions will be characterized to determine RNA binding site characteristics, which subunit guides the PAP to the 3'-OH, the contribution of the 5' cap structure to VP39-RNA interaction, and whether one subunit interacts with RNA solely via the other. Third, regions of the proteins required for RNA and nucleotide binding and for dimerization will be identified, and attempts will be made to crystallize the two subunits. Finally, functional experiments will be conducted to determine whether VP39 is a sliding clamp-type processivity factor, whether VP39 interacts with ATP in a functional manner, and to investigate possible links between the 5' and 3' end modification functions of VP39. The above studies are expected to provide a detailed understanding of the mechanism of poly(A) tail synthesis.