STRUCTURE & FUNCTION OF TRANSFER RIBONUCLEIC ACIDS

这项建议的主要目的是：(1)了解 独特的结构特征与高度的 真核和原核启动子的特性 蛋氨酸tRNAs和(2)利用tRNA基因的突变来 分离出各种携带废话的稳定的动物细胞系 抑制者。 我们的方法是使用定点突变来产生 不同类别的突变tRNA：(I)源自 真核和原核启动子tRNAs已丢失一个、两个 或其全部三个独特特征，以及(Ii)源自 具有一个或多个特征的延伸物蛋氨酸tRNA 启动子tRNA所特有？这些和它们的结构和性能 其他突变的tRNA将被研究。具体问题有：威尔 启动子tRNA突变体仍在启动中起作用吗？会不会有些人 它们现在起延长器的作用？会不会有一些突变体来自于 延长子tRNA获得了一种或多种特定于 启动子tRNA？为了实现这一目标，九种不同的突变体 人类启动子tRNA缺乏一个、两个或全部三个独特特征 已经被分离出来，目前正在进行分析。与大肠杆菌 启动子tRNA，其中一个独特的功能已被证明是 重要的是印心，而另一个重要的是 阻止tRNA作为延伸器工作。再加上这些 有趣的功能变化在结构上的变化 突变的tRNA。我们建议确定这些问题的性质 结构变化及其与经济结构变化的关系 功能。 利用诱变方法研究高度特异的 Met-tRNA甲酰基酶对大肠杆菌启动子tRNA的识别 表明该酶在tRNA上的识别位点可能 定位于受体茎的末端。我们将进行调查 这是通过进一步诱变启动子和延伸子来实现的 大肠杆菌甲硫氨酸tRNA的种类。该酶将被提纯 用于研究酶-MET-tRNA的拓扑结构 互动。还将尝试克隆该基因以用于 酶，使酶结晶。 最后，我们建议扩展我们最近关于生成 稳定的猴肾细胞系携带可诱导琥珀 抑制tRNA基因，包括鸵鸟和蛋白石抑制基因 也可以获得携带这种抑制子的小鼠细胞系。

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.