REGULATION OF GENE EXPRESSION IN BACTERIOPHAGE T4

噬菌体 T4 中基因表达的调控

• 批准号: 3278544
• 负责人: Alexander Goldfarb
• 金额: $ 20.79万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-03-01 至 1991-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3278544
• 关键词: ADP ribosylation; DNA binding protein; DNA directed RNA polymerase; Escherichia coli; bacteriophage T4; binding proteins; centrifugation; endonuclease; enzyme mechanism; gel electrophoresis; gene expression; genetic mapping; genetic promoter element; genetic transcription; molecular cloning; mutant; nucleic acid sequence; protein structure; radiotracer; scintillation counter; transcription termination; virus RNA; virus diseases; virus genetics

After infection of E. coli by bacteriophage T4, the host RNA polymerase acquires several small phage-induced polypeptides and its alfa subunits are ADP-ribosylated. The role of these modifications in transcription control will be studied using a combination of biochemical, genetic and physiological approaches. We have purified four of the associated polypeptides (15K, 19K, 25K and 29K proteins) as well as RNA polymerases differing in the state of ADP-ribosylation and propose to study their interactions in direct binding assays. The kinetics of in vivo formation of these proteins and their interactions with other intracellular components will be analysed in order to understand the coordination between transcription and other events of T4 development such as DNA replication. We will identify T4 genes involved in the modifications and use their mutants to determine their role in phage development. In vitro experiments are proposed to study the interaction of normal and modified RNA polymerase with individual promoters with emphasis given to the change of specificity of promoter recognition from early to late sites. We have already shown that 25K protein induces late promoter specificity while 15K protein decreases the utilization of early promoters. To understand the molecular basis of the specificity change we will determine the kinetic parameters of promoter functioning with different forms of RNA polymerase. Experiments directed at biochemical identification of T4-induced antitermination mechanism are also proposed. The in vitro experiments will be backed up by the analysis of in vivo functioning of the same transcription sites using recently sequenced tRNA gene region of T4 as the experimental system.

感染E. T4噬菌体，宿主RNA聚合酶 获得了几种小噬菌体诱导的多肽，其α亚基， ADP-核糖基化。 这些修饰在转录控制中的作用 将使用生物化学，遗传学和 生理学方法。 我曾以四种不同的方式， 多肽（15 K、19 K、25 K和29 K蛋白）以及RNA聚合酶 不同的ADP-核糖基化状态，并建议研究他们的 直接结合试验中的相互作用。 体内形成的动力学 这些蛋白质及其与其他细胞内 将对各组成部分进行分析，以了解 转录和T4发育的其他事件，如DNA复制。 我们将确定参与修饰的T4基因，并使用它们的 突变体以确定它们在噬菌体发育中的作用。 体外实验 研究正常和修饰的RNA聚合酶的相互作用 用单个启动子，强调特异性的改变 从早期到晚期的启动子识别。 我们已经表明 25 K蛋白诱导晚期启动子特异性，而15 K蛋白 降低了早期启动子的利用率。 为了了解分子 根据特异性变化，我们将确定动力学参数 启动子与不同形式的RNA聚合酶一起起作用。 实验 针对T4诱导的抗终止的生物化学鉴定 机制也提出了。 体外实验将得到以下支持： 分析相同转录位点的体内功能， 以T4的tRNA基因区序列为实验系统。