Studies on in vitro translation system of mitochondria

本项目的目的是研究动物线粒体(MT)翻译系统的分子机制，特别是阐明遗传密码变异之间的关系[UGA，终止到Trp密码子；AUA，Ile到Met密码子；AGA/AGG，Arg到Ser(无脊椎动物)，Gly(海鞘)和终止密码子(脊椎动物)]，与各种密码子互补，具有不寻常的一级和三级结构[缺乏D环-T环相互作用；缺乏D臂或T臂]。通过将我们团队和Spremulli团队的知识和技术结合在一起，这个项目成为可能。[1992]1)通过使用与tRNA序列互补的DNA探针的杂交方法，使得大规模制备mt tRNA成为可能。2)阐明了与UCN密码子互补的tRNA^&lt；Ser&gt；具有特殊的二级结构。3)生物活性核糖体、起始因子(IF-2)、延伸因子(EF-Tu、EF-G)和氨基酰转移核糖核酸SY…[1993]1)研究了丝氨酰-tRNA合成酶对与AGY密码子互补的tRNA的识别位点。研究表明，T环中的A44是最重要的识别位点。2)构建了牛肝线粒体聚(U)依赖的聚(Phe)合成体系，并研究了其组分与大肠杆菌组分的互换性。3)在牛mt tRNA^&lt；Met&gt；的第一反密码子上发现了一种新的修饰核苷--5-甲酰胞苷。4)用核糖核酸酶和化学试剂的敏感性研究了mttRNA^&lt；Phe&gt；的高阶结构。2)为了验证AUA是一种Met密码子，尝试了在含有AUA的mRNA存在下，Met-tRNA^&lt；Met&gt；的核糖体结合和翻译。3)用纯化的甲酰转移酶获得fMet-tRNA^&lt；Met&gt；结果表明，fMet-tRNA优先选择IF-2而不是Ef-Tu，反之亦然，说明tRNA^&lt；Met&gt；是通过甲酰化作用形成的。较少

The purpose of this project is to investigate the molecular mechanism of the animal mitochondrial (mt) translation system, especially, to elucidate the relationship between genetic code variations [UGA, termination to Trp codons ; AUA, Ile to Met codons ; AGA/AGG, Arg to Ser(invertebrates), Gly(Ascidian) and Termination codons(vertebrates)] found in animal mitochondria and tRNAs complementary to the varied codons, possessing unusual primary and tertiary structures [lack of D loop-T loop interaction ; lack of D arm or T arm]. By joining the knowledge and techniques of our group and Spremulli's group, this project was made possible. [1992] 1) A large-scale preparation of mt tRNAs was made possible by a hybridization method using DNA probes complementary to tRNA sequences. 2)It was elucidated that tRNA^<Ser> complementary to UCN codons possesses an unusual secondary structure. 3)Biologically active ribosomes, initiation factor (IF-2), elongation factors (EF-Tu, EF-G) and aminoacyl-tRNA sy … More nthetasaes were isolated and their properties investigated.[1993] 1) The recognition sites of tRNA^<Ser> complementary to AGY codons by seryl-tRNA synthetase was investigated. It was elucidated that A44 in the T loop is the most important recognition site. 2) The poly(U)-dependent poly(Phe) synthesis system was constructed for bovine liver mitochondria and the exchangeability of its components with those of E.coli was investigated. 3) A novel modified nucleoside, 5-formylcytidine was found at the first anticodon of bovine mt tRNA^<Met>.4) The higher-order structure of mt tRNA^<Phe> was investigated by the sensitivity with RNase and chemical reagents.[1994] 1) The poly(U)-dependent poly(Phe) synthesis was activated by addition of 1 mM spermine. 2) To verify that AUA is a Met codon, ribosomal binding and translation of Met-tRNA^<Met> in the presence of AUA-containing mRNA were attempted. 3) By obtaining fMet-tRNA^<Met> with the purified formyltransferase, it was elucidated that fMet-tRNA prefers IF-2 rather than EF-Tu and vice versa for Met-tRNA, indicating that the tRNA^<Met> is bifunctional by formylation of Met-tRNA. Less