STRUCTURE AND FUNCTION OF TRANSFER RIBONUCLEIC ACIDS

转移核糖核酸的结构和功能

• 批准号: 3269060
• 负责人: UTTAM L RAJBHANDARY
• 金额: $ 36.8万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-06-01 至 1993-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3269060
• 关键词: Escherichia coli; Neurospora; Pongidae; acyltransferase; cell free system; chemical structure function; enzyme mechanism; enzyme structure; eukaryote; genetic manipulation; human tissue; laboratory mouse; methionine; molecular cloning; mutant; nucleic acid sequence; point mutation; prokaryote; tissue /cell culture; transfer RNA; translation factor; yeasts

The major objectives of this proposal are: (1) to understand the relationship between the unique structural features and the highly specific properties of eukaryotic and prokaryotic initiator methionine tRNAs and (2) to use mutagenesis of tRNA genes to isolate a variety of stable animal cell lines which carry nonsense suppressors. Our approach is to use site-specific mutagenesis to generate different classes of mutant tRNAs: (i) those derived from eukaryotic and prokaryotic initiator tRNAs which have lost one, two or all three of their unique features and (ii) those derived from elongator methionine tRNA which have acquired one or more features unique to initiator tRNA? The structure and properties of these and other mutant tRNAs will be studied. Specific questions are: Will the initiator tRNA mutants still act in initiation? Will some of them now act as elongators? Will some of the mutants derived from elongator tRNA acquire one or more of the properties special to initiator tRNAs? Toward this objective nine different mutants of human initiator tRNA lacking one, two or all three unique features have been isolated and are being currently analyzed. With E. coli initiator tRNA, one of the unique features has been shown to be important in initiation whereas the other is important in preventing the tRNA from working as an elongator. Along with these interesting functional changes there are structural changes in the mutant tRNAs. We propose to determine the nature of these structural changes and the relationship of these to the change in function. Use of mutagenesis approach for studying the highly specific recognition of E. coli initiator tRNA by met-tRNA formylase has indicated that the recognition site for the enzyme on the tRNA may be localized at the end of the acceptor stem. We shall investigate this by further mutagenesis of both the initiator and elongator species of E. coli methionine tRNA. The enzyme will be purified and used for studies on the topology of enzyme-met-tRNA interactions. Attempts will also be made to clone the gene for the enzyme and to crystallize the enzyme. Finally, we propose to extend our recent work on the generation of stable monkey kidney cell lines carrying an inducible amber suppressor tRNA gene to include ocher and opal suppressors and to also obtain mouse cell lines carrying such suppressors.

本提案的主要目标是： (1) 了解 独特的结构特征与高度之间的关系 真核和原核引发剂的特殊性质 蛋氨酸 tRNA 和 (2) 利用 tRNA 基因诱变 分离出多种携带无意义的稳定动物细胞系 抑制器。 我们的方法是使用位点特异性诱变来生成 不同类别的突变 tRNA：(i) 源自 真核和原核起始 tRNA 丢失 1 个、2 个 或它们的所有三个独特特征以及（ii）源自 已获得一种或多种特征的延长蛋氨酸 tRNA 起始子 tRNA 独有吗？这些的结构和性质 其他突变 tRNA 将被研究。 具体问题是： 意愿 起始tRNA突变体仍然在起始阶段起作用吗？ 会有些 他们现在充当延长器？ 一些突变体是否源自 延伸子 tRNA 获得一种或多种特殊的特性 起始tRNA？ 为了实现这一目标，九种不同的突变体 人类起始 tRNA 缺乏一种、两种或全部三种独特特征 已被隔离，目前正在分析中。 与大肠杆菌 起始子 tRNA，独特的特征之一已被证明是 在启动过程中很重要，而另一个在启动过程中很重要 防止 tRNA 发挥延伸作用。 连同这些 有趣的功能变化还有结构变化 突变 tRNA。 我们建议确定这些的性质 结构变化以及这些变化与变化的关系 功能。 使用诱变方法研究高度特异性的 met-tRNA 甲酰化酶对大肠杆菌起始子 tRNA 的识别 表明 tRNA 上酶的识别位点可能 位于受体茎的末端。 我们将调查 这是通过引发剂和延伸剂的进一步诱变来实现的 大肠杆菌蛋氨酸 tRNA 的种类。 酶将被纯化 用于酶-met-tRNA拓扑结构的研究 互动。 还将尝试克隆该基因 酶并使酶结晶。 最后，我们建议扩展我们最近在生成 携带可诱导琥珀的稳定猴肾细胞系 抑制 tRNA 基因，包括赭石和蛋白石抑制基因 还获得携带此类抑制剂的小鼠细胞系。