VACCINIA VIRUS BIOCHEMICAL GENETICS

近年来，越来越清楚的是，转录 伸长是真核生物调控的重要控制点 基因表达。我们最近的工作证明了复制后基因 转录延伸在痘苗病毒感染过程中受到调节。 对这一规则的研究对于理解这一规则具有重要意义 特别是牛痘病毒的基因表达，而该系统可能证明 是研究转录调控的重要模型 一般真核生物的伸长。 这个项目的目标是了解牛痘的规则。 病毒复制后(中晚期)基因转录 伸长率。该项目的中心是两个牛痘基因，G2R和A18R， 它们似乎调控复制后基因的转录延伸 与之配套的活动。基因实验表明， A18R基因产物是一种DNA解旋酶，它限制了 病毒RNA聚合酶，因此起负转录作用 延伸率，而G2R基因产物增强加工性 病毒RNA聚合酶的活性，因此可作为阳性 转录延伸因子。生物化学和遗传学实验 将其他几种病毒基因产物牵连到对 复制后转录延长，包括病毒晚期 转录起始因子(H5R)--病毒多聚合酶亚基 (J3R)和两个病毒RNA聚合酶亚基(J4R和A24R)。我们的 工作假说是这些基因产物可能一起工作。 作为转录延伸复合体，调节3‘端的形成 中晚期痘苗病毒mRNAs。我们打算测试一下， 通过1)体外转录来完善和扩展这一假设 延长试验，2)现有病毒突变体的表型分析 转录延伸缺陷，以及3)分离和 额外伸长缺陷突变体的特征。

In recent years it has become increasingly clear that transcription elongation is an important control point for regulation of eucaryotic gene expression. Our recent work demonstrates that postreplicative gene transcription elongation is regulated during vaccinia virus infection. A study of this regulation is important for understanding regulation of vaccina virus gene expression in particular, and the system may prove to be an important model for study of regulation of transcription elongation in eucaryotes in general. The goal of this project is to understand the regulation of vaccinia virus postreplicative (intermediate and late) gene transcription elongation. The project centers on two vaccinia genes, G2R and A18R, which seem to regulate postreplicative gene transcription elongation with complementing activities. Genetic experiments indicate that the A18R gene product, a DNA helicase, restricts the processivity of the viral RNA polymerase and therefore acts as negative transcription elongation factor, while the G2R gene product enhances the processivity of the viral RNA polymerase and therefore acts as a positive transcription elongation factor. Biochemical and genetic experiments implicate several other viral gene products in the regulation of postreplicative transcription elongation, including a viral late transcription initiation factor (H5R), a viral poly A polymerase subunit (J3R), and two viral RNA polymerase subunits (J4R and A24R). Our working hypothesis is that these gene products work together, perhaps as a transcription elongation complex, to regulate formation of 3' ends of intermediate and late vaccinia viral mRNAs. We propose to test, refine, and extend this hypothesis through 1) an in vitro transcription elongation assay, 2) phenotypic analysis of existing virus mutants defective in transcription elongation, and 3) isolation and characterization of additional elongation defective mutants.