VACCINIA VIRUS MRNA CAPPING ENZYME

这个项目的长期目标是了解所采用的机制。 在早期和晚期牛痘病毒基因的表达中，以及在 对它们的表达进行调控。在这项提案中，我们将注意力集中在 在一种病毒酶上，牛痘病毒封顶酶复合体。这种酶有 已被证明催化了CAP途径的前三步 队形。此外，DNA还需要这种非凡的复合体 早期基因的序列特异性转录终止。在这 建议我们将首先使用免疫学和生化技术来 研究病毒封端酶复合体的合成和组装， 并确定封闭酶是否在感染后期使用 与病毒粒子包裹的酶相同。第二，我们将结合 用遗传学和生物化学方法研究这种酶复合体 体内和体外。我们将首先对封闭酶进行特性分析，然后 S-腺苷抗性突变体体内形成的研究 蛋氨酸类似物辛芬净。接下来，我们将分离条件致死 突变决定mRNA帽子形成和早期基因的作用 痘苗病毒生命周期中的转录终止。最后，我们会 构建表达载体以产生大量的每个封顶 大肠杆菌中的酶亚基。我们将开发提纯亚基的方法 并重建封顶活性和转录终止活性 在试管中。我们将每种酶的活性部位定位到一个 亚基，然后使用特定底物类似物来鉴定氨基 存在于每个活性部位的酸。

The long term goal of this project is to understand the mechanisms employed in the expression of early and late vaccinia virus genes, and in the regulation of their expression. In this proposal, we focus our attention on one viral enzyme, the vaccinia capping enzyme complex. This enzyme has been shown to catalyze the first three steps in the pathway of cap formation. In addition, this remarkable complex is required for DNA sequence specific transcription termination of early genes. In this proposal we will first employ immunological and biochemical techniques to investigate the synthesis and assembly of the virus capping enzyme complex, in vivo, and to determine if the capping enzyme employed late in infection is the same as the virion encapsidated enzyme. Secondly, we will combine genetic and biochemical approaches to study this enzyme complex both in vivo and in vitro. We will first characterize the capping enzyme, and capt formation in vivo, in mutants which are resistant to the S-adenosyl methionine analogue sinefungin. Next, we will isolate conditional lethal mutations to determine the role of mRNA cap formation and early gene transcription termination in the vaccinia life cycle. Finally, we will construct expression vectors to produce large quantities of each capping enzyme subunit in E. coli. We will develop methods to purify the subunits and to reconstitute capping activity and transcription termination activity in vitro. We will localize the active site of each enzyme activity to one subunit and then employ specific substrate analogues to identify the amino acids present in each active site.