REGULATION OF GENE EXPRESSION IN BACTERIOPHAGE T4

噬菌体T4感染大肠杆菌后，宿主RNA聚合酶 获得了几个小的噬菌体诱导的多肽，其α亚基是 ADP-核糖化。这些修饰在转录调控中的作用 将使用生化、遗传学和生物化学的组合进行研究 生理学方法。我们已经提纯了四个相关的 多肽(15K、19K、25K和29K蛋白质)以及RNA聚合酶 不同的ADP-核糖化状态，并建议研究其 直接结合分析中的相互作用。体内形成的动力学 以及它们与其他细胞内蛋白质的相互作用 将分析组件，以了解组件之间的协调 转录和T4发育的其他事件，如DNA复制。 我们将识别参与修饰的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 建议研究正常和修饰的RNA聚合酶之间的相互作用 与个别发起人合作，强调特异性的变化 从早期到晚期的启动子识别。我们已经展示了 25K蛋白诱导晚期启动子特异性，而15K蛋白诱导晚期启动子特异性 减少早期推动者的使用率。要了解分子 根据特异度的变化，我们将确定动力学参数 启动子与不同形式的RNA聚合酶一起发挥作用。实验 针对T4诱导的抗流产的生化鉴定 并对其机理进行了探讨。体外实验将得到以下支持 同一转录位点在体内的功能分析 以新近测序的T4株tRNA基因区为实验系统。

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.