STRUCTURE AND FUNCTION OF TRANSFER RIBONUCLEIC ACIDS

这项建议的主要目标是：(1)调查 TRNAs的结构-功能关系和生物合成 扩大我们对tRNA基因定点突变的研究，以产生 在哺乳动物细胞中起作用的各种抑制子。我们的方法是 体内tRNA功能的变化。原核生物和 将研究真核tRNAs。如果tRNA基因有缺陷 在体内的功能我们将确定它们的生物合成是否 受伤了。 并通过体外研究确定它们在哪一步有缺陷 功能(氨基酰化、与延伸因子结合、核糖体等)。 我们将用这种方法来研究(A)不变量的作用 核苷酸在tRNA生物合成和功能中的作用，以及(B)关系 启动子tRNA中存在的独特结构特征与其 特殊属性。高度保守的TPsiC-序列在蛋白质合成中的作用 还将确定tRNA的功能。最后，我们建议延长我们的 黄瓜琥珀抑制因子tRNA基因的克隆研究进展 哺乳动物细胞包括各种无意义的抑制子，这将 在哺乳动物细胞中的功能。最终目标是建立稳定的细胞 细胞DNA。在我们对tRNAs和tRNA基因的继续研究中 ，我们建议研究与其线粒体相关的酶。 生物合成。我们将确定高度保守的回文 粗枝线虫线粒体中tRNA基因两侧的DNA序列起着一定的作用 在RNA处理中。此外，我们还将提纯tRNA加工酶 “核糖核酸酶P”，并确定它是否具有 基本的RNA成分，如大肠杆菌酶，如果是，这种RNA是否 是线粒体编码的。

The major objectives of this proposal are: (1) to investigate structure-function relationships and biosynthesis of tRNAs and (2) to extend our studies on site-specific mutagenesis of tRNA genes to generate a variety of suppressors which function in mammalian cells. Our approach is to alter single nucleotides at predetermined sites in tRNA genes, introduce them into the appropriate organism and examine the effect of these alterations on the function of the tRNA in vivo. Both prokaryotic and eukaryotic tRNAs will be studied. Should the tRNA genes be defective in function in vivo we shall determine whether their biosynthesis is impaired. If biosynthesis is unaffected, we shall purify the mutant tRNAs and by in vitro studies identify the step at which they are defective in function (aminoacylation, binding to elongation factors, ribosome etc.). We shall use this approach to study (a) the role of the invariant nucleotides in tRNA biosynthesis and in function, and (b) the relationship between the unique structural features present in initiator tRNAs and their special properties. The role of the highly conserved -TPsiC- sequence in tRNA function will also be determined. Finally, we propose to extend our recent work on generation and cloning of an amber suppressor tRNA gene in mammalian cells to include a variety of nonsense suppressors which will function in mammalian cells. The eventual goal is to establish stable cell lines which contain different suppressor tRNA genes integrated into their cellular DNA. In a continuation of our studies of tRNAs and tRNA genes in N.crassa mitochondria, we propose to study enzymes involved in their biosynthesis. We shall determine whether the highly conserved palindromic DNA sequences which flank tRNA genes in N.crassa mitochondria play a role in RNA processing. In addition, we shall purify the tRNA processing enzyme "RNase P" from N.crassa mitochondria and determine whether it has an essential RNA component like the E.coli enzyme and, if so, whether this RNA is mitochondrially coded.