Elucidation of basic principle in genetic information translation system by using the specialty of animal mitochondria

本课题旨在利用动物线粒体的特性，阐明遗传信息翻译系统的基本原理。这可以通过将大肠杆菌翻译系统与动物线粒体的特殊翻译系统相结合来实现。(1) tRNA的最小先决结构：利用核磁共振（NMR）检测了线虫线粒体（mt） tRNA^<Ser>_<UCU>的高阶结构（54个核苷酸，无D臂，小T臂由3个碱基对和一个4个碱基的环组成），是现存生物体中最小的tRNA。结果表明，根据其三叶草结构预测的碱基对几乎全部存在于溶液中，由截断的D环和额外环组成的连接区域具有相当灵活的结构。这种迁移性对于tRNA^<Ser>_<UCU>保持与通常tRNA相同的3'端和反密码子的相对排列是必不可少的。(2)最小的先决结构…更多的核糖体：与大肠杆菌核糖体相比，在动物核糖体中，rRNA的含量约为一半，但蛋白质的含量增加了一倍。mt rRNA的功能区域被严格保守在两个核糖体亚基的中心部分，mt核糖体中缺乏的rRNA的外周区域被蛋白质包裹，从而使mt和大肠杆菌之间的核糖体的整体形状几乎相等。结果表明，mt核糖体外周区域的一些茎环结构是可有可无的。(3)翻译系统中作为能量来源的GTP水解反应的触发器是什么：根据EF-G的GTP酶活性是由L7/L12激活的，认为GTP水解反应的触发器是50S核糖体亚基中的L7/L12蛋白。我们通过阐明大肠杆菌EF-G可以作用于mt核糖体，其中只有L7/L12蛋白被大肠杆菌核糖体的对应物取代，尽管大肠杆菌EF-G不能作用于天然mt核糖体，从而证实了这一建议。少

The aim of this project is to elucidate the basic principle in genetic information translation system by using the specialty of animal mitochondria. This can be done by combining the well-studied E.coli translation system with the special translation system of animal mitochondria. (1)The minimal prerequisite structure of tRNA : The higher-order structure of nematode mitochondrial (mt) tRNA^<Ser>_<UCU>, which is the smallest tRNA in the extant organisms (54 nucleotides, having no D arm and small T arm consisting of 3 base pairs and a loop with 4 bases), was examined by using NMR. It turned out that almost all the base-pairs predicted from its clover-leaf structure existed in solution and the connector region consisting of the truncated D loop and extra loop had a rather flexible structure. This mobility is indispensable for the tRNA^<Ser>_<UCU> to maintain the relative arrangement of the 3'-terminus and the anticodon equal to that of the usual tRNAs. (2)The minimal prerequisite structur … More e of ribosome : In the animal mt ribosome, the content of rRNA is about a half, but that of proteins is doubled, as compared with the counterparts of E.coli ribosome. The functional regions of mt rRNA are strictly conserved in the central parts of two ribosomal subunits and the peripheral regions of rRNA lacking in the mt ribosome are embedded by proteins so as to maintain the whole shapes of ribosomes almost equal between mt and E.coli. It turned out that some of the stem/loop structures in the peripheral regions of mt ribosome are dispensable. (3)What is the trigger in the hydrolysis reaction of GTP which is an energy source in the translation system : It was suggested that the trigger in the hydrolysis reaction of GTP is L7/L12 protein in 50S ribosomal subunit, on the basis of the fact that GTPase activity of EF-G is activated by L7/L12. We have confirmed this proposal by elucidating that E.coli EF-G can function on the mt ribosome in which only the L7/L12 protein is replaced by the counterpart of E.coli ribosome, although E.coli EF-G cannot work on the native mt ribosome. Less