Molecular mechanism of the animal mitochondrial translation systems

本研究旨在通过对动物线粒体（mt）tRNA的结构分析和利用线粒体体外翻译系统验证这些tRNA的翻译能力，阐明动物mt翻译系统的分子机制。动物线粒体翻译系统与原核和真核细胞质系统相比具有明显的特点：存在具有特殊二级结构和非通用遗传密码的tRNA，线粒体中只有22种tRNA，一种tRNA<Met>同时作为起始子和延伸子，核糖体中含有链长较短的rRNA和更多的蛋白质。我们对翻译系统中的各个因素进行了分离和表征，并得到了以下新的发现。1)我们在各种动物尼特系统中发现了一些非通用的遗传密码，并分析了解码密码的tRNA的一级结构。结果表明，遗传密码变异主要是由反密码子第一位（Wobble位置）的碱基（主要是修饰碱基）引起的，这导致了mt特异性Wobble规则的建立。2)通过结合生物化学实验和^1H-NMR分析，我们证实了即使是具有缺少D臂或T臂的截断二级结构的tRNA也可以折叠成L形的三级结构。3)我们成功地构建了一个仅由mt翻译装置组成的体外翻译系统。通过使用这个系统，我们证明了AUA异亮氨酸密码子被<Met>在反密码子第一位有一个经过修饰的碱基f^6C的tRNA翻译成甲硫氨酸。4)从牛线粒体中纯化氨酰-tRNA合成酶、延伸因子、释放因子和甲酰转移酶，并对其进行表征，并在大肠杆菌中建立其表达系统。分析了这些因子与tRNA相互作用的分子机制。

This research aims to elucidate the molecular mechanism of animal mitochondrial (mt) translation system through carrying out (1) structural analysis of animal mt tRNAs, and (2) verification of translation ability of these tRNAs by using a mitochondrial in vitro translation system. The animal mt translation system possesses the distinct features as compared with those of prokaryotic and eukaryotic cytoplasmic systems ; exsistences of tRNA possessing unusual secondary structures and non-universal genetic code, only 22 species of tRNAs exist in mitochondria, a single tRNA^<Met> is used for both initiator and elongator, ribosomes contain rRNA with shorter chain lengths and much more proteins. We have isolated and characterized individual factors involved in the translation system and got the following new findings. 1) We found several non-universal genetic codes in various animal nit systems and analyzed the primary structures of tRNAs decoding the codes. it turned out that the genetic code variations are mainly induced by the base (mainly modified base) in the anticodon first position (Wobble position), which lead to the establishment of the mt specific Wobble rule. 2) By combining biochemical experiments and ^1H-NMR analyses we verified that even tRNAs possessing trancated secondary structures lacking either D arm or T arm could be folded into L-shape-like tertiary structures. 3) We succeeded to construct an in vitro translation system composed of only mt translation apparatuses. By using this system we proved that AUA isoleucine codon is translated as methionine by tRNA^<Met> having a modified base, f^6C at the anticodon first position. 4) Aminoacyl-tRNA synthetase, elongation factors, release factors, and formyltransferase were purified from bovine mitochondria, characterized, and their expression system was established in E.coli. We analyzed molecular mechanism of interaction between these factors and tRNA.