STRUCTURE AND FUNCTION OF TRANSFER RIBONUCLEIC ACIDS

转移核糖核酸的结构和功能

• 批准号: 2172049
• 负责人: UTTAM L RAJBHANDARY
• 金额: $ 40.83万
• 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 1997-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2172049
• 关键词: Drosophilidae; Escherichia coli; aminoacyl tRNA; chemical structure function; clone cells; conformation; crystallization; enzyme structure; enzyme substrate complex; gene expression; gene induction /repression; genetic strain; intermolecular interaction; methionine; molecular cloning; mutant; nucleic acid sequence; protein biosynthesis; protein purification; transfer RNA; Drosophilidae; Escherichia coli; aminoacyl tRNA; chemical structure function; clone cells; conformation; crystallization; enzyme structure; enzyme substrate complex; gene expression; gene induction /repression; genetic strain; intermolecular interaction; methionine; molecular cloning; mutant; nucleic acid sequence; protein biosynthesis; protein purification; transfer RNA

The objectives of this proposal are (1) to identify sequence and/or structural features in prokaryotic initiator tRNAs which specify their distinctive properties, (2) to understand the molecular mechanisms by which various components of the protein synthetic machinery distinguish initiator tRNAs from other tRNAs and (3) to generate animal cell lines and Drosophila strains carrying inducible suppressor tRNA genes. The approach is to generate mutant tRNAs and analyze their function at each of the steps on the pathway to initiation in vitro and in vivo. The role of two of the unique features of initiator tRNAs have been identified. We aim to identify the role of the third feature. Concurrently, attempts to convert an elongator tRNA into an efficient initiator by introducing sequence features found in initiator tRNA will continue. Mutants defective at specific steps in initiation have been obtained. Some are inactive because they cannot be formylated, others because they cannot bind to the P site on the ribosome. We will investigate whether overexpression or mutation of any E. coli gene will suppress these defects. We will identify the genes involved and analyze the mode of suppression. this work could lead to identification of components which interact with initiator tRNA and an understanding of how such interactions contribute to protein synthesis. It may also lead to identification of new genes. Studies on underlying molecular mechanisms in the highly specific recognition of initiator tRNA by Met-tRNA transformylase will continue. The key elements for this appear to be clustered in the acceptor stem, a mismatch or a weak base pair at the end of the acceptor stem being important. We will investigate these structural requirements. We will probe the topology of enzyme .tRNA interaction in solution and attempt to crystallize the enzyme .tRNA complex. Such work should be facilitated by a method we have developed for large scale preparation of analogues of methionyl-tRNA in which methionine is linked to tRNA through a stable amide bond. Finally, we propose to use current knowledge of tRNAs and aminoacyl-tRNA synthetases to regulate expression of suppressor tRNA genes or their function in eukaryotic cells. The goal is to develop methods that are generally applicable for generating mammalian cell lines and Drosophila strains carrying inducible suppressors.

该提案的目标是（1）确定顺序和/或 原核生物引发剂TRNA中的结构特征，该特征指定其 独特的特性，（2）了解分子机制 蛋白质合成机械的各种组成部分区分 来自其他TRNA和（3）生成动物细胞系的引发剂TRNA 果蝇菌株携带可诱导抑制器tRNA基因。 该方法是生成突变型TRNA并分析其功能 体外和体内启动途径的每个步骤。 这 启动器trnas的两个独特特征的作用已经 确定。 我们旨在确定第三个功能的作用。 同时，尝试将伸长量转换为有效 通过引入启动器tRNA中的序列特征的启动器将 继续。 在开始的特定步骤中有缺陷的突变体。 有些是不活跃的，因为它们不能大型化，而是因为他们 无法与核糖体上的P位点结合。 我们将调查是否 任何大肠杆菌基因的过表达或突变都会抑制这些 缺陷。 我们将识别涉及的基因并分析 抑制。 这项工作可能导致识别组件 与引发剂互动以及对这种方式的理解 相互作用有助于蛋白质合成。 这也可能导致 鉴定新基因。 关于高度特异性的基本分子机制的研究 Met-tRNA转化基酶对引发剂tRNA的识别将继续。 该关键元素似乎聚集在受体词干中， 受体茎末端的不匹配或弱基对是 重要的。 我们将调查这些结构性要求。 我们将 探测酶中酶的拓扑结构，并尝试 要结晶酶.TRNA复合物。 这种工作应促进 通过一种方法，我们开发了用于大规模制备类似物的方法 蛋氨酸通过稳定的蛋氨酸与tRNA相关的蛋白质基tRNA 酰胺键。 最后，我们建议使用当前的TRNA和氨基酰基-TRNA知识 调节抑制器tRNA基因或其其表达的合成酶 在真核细胞中的功能。 目标是开发方法 通常适用于产生哺乳动物细胞系和果蝇。 携带可诱导抑制器的菌株。