STRUCTURE AND FUNCTION OF TRANSFER RIBONUCLEIC ACIDS

本提案的目标是（1）确定序列和/或 原核生物起始物tRNA的结构特征， （2）了解分子机制， 蛋白质合成机制的不同组成部分 从其它tRNA起始的tRNA和（3）产生动物细胞系 和携带可诱导抑制tRNA基因的果蝇菌株。 该方法是产生突变的tRNA，并分析它们的功能， 体外和体内启动途径上的每一步。 的 启动子tRNA的两个独特特征的作用已经被 鉴定 我们的目标是确定第三个特征的作用。 同时，尝试将延伸体tRNA转化为有效的 通过引入在起始物tRNA中发现的序列特征， 继续 已经获得了在起始的特定步骤有缺陷的突变体。 一些是无活性的，因为它们不能被甲酰化，另一些是因为它们不能被甲酰化。 不能与核糖体上的P位点结合。 我们将调查是否 任何E. coli基因会抑制这些 缺陷 我们将鉴定相关基因并分析其表达模式。 镇压 这项工作可以导致识别组件， 与起始物tRNA相互作用，并了解这种相互作用如何 相互作用有助于蛋白质合成。 还可能导致 鉴定新基因。 高度特异性的分子机制研究 Met-tRNA转化酶对起始tRNA的识别将继续。 其关键元件似乎聚集在受体茎中， 受体茎末端的错配或弱碱基对 重要. 我们将研究这些结构要求。 我们将 探索溶液中酶与tRNA相互作用的拓扑结构， 使酶和tRNA复合物结晶。 应促进这种工作 通过我们开发的用于大规模制备类似物的方法， 其中甲硫氨酸通过稳定的 酰胺键 最后，我们建议利用目前的知识，tRNA和氨酰-tRNA 合成酶来调节抑制性tRNA基因或其 在真核细胞中的功能。 我们的目标是开发方法， 通常适用于产生哺乳动物细胞系和果蝇 携带可诱导抑制基因的菌株。

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.