Molecular mechanism of the animal mitochondrial translation systems

本研究旨在通过(1)动物线粒体tRNAs的结构分析和(2)利用线粒体体外翻译系统验证这些tRNAs的翻译能力，从而阐明动物线粒体(Mt)翻译系统的分子机制。动物MT翻译系统具有与原核和真核细胞质系统不同的特点：tRNA具有不寻常的二级结构和非通用的遗传密码，线粒体中只有22种tRNA，单个tRNA同时用作启动子和延长子，核糖体含有更短的rRNA和更多的蛋白质。我们对翻译系统中涉及的各个因素进行了分离和表征，得到了以下新的发现。1)我们在不同的动物NIT系统中发现了几个非通用的遗传密码，并分析了解码这些密码的tRNAs的一级结构。结果表明，遗传密码的变异主要是由反密码子第一位置(摆动位置)的碱基(主要是修饰碱基)引起的，从而导致了mt特异性摆动规则的建立。2)通过生化实验和核磁共振氢谱分析相结合的方法，验证了即使是缺乏D臂和T臂的横断二级结构的tRNA也可以折叠成L形状的三级结构。3)我们成功地构建了仅由MT翻译器组成的体外翻译系统。利用这个系统，我们证明了AUA异亮氨酸密码子通过tRNA^&lt；Met&gt；被修饰的碱基f^6C在反密码子第一位被翻译为蛋氨酸。4)从牛线粒体中分离纯化了氨基酰基-tRNA合成酶、延伸因子、释放因子和甲酰基转移酶，并对其进行了鉴定，建立了它们在大肠杆菌中的表达系统。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.