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

本项目的目的是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。这可以通过将经过充分研究的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的最小先决条件结构：用核磁共振技术研究了现存生物中最小的tRNA线虫线粒体(Mt)tRNA^&lt；Ser&gt；_&lt；ucu&gt；(54个核苷酸，没有D臂和由3个碱基对组成的小T臂和4个碱基环)的高阶结构。结果表明，从其三叶草结构预测的几乎所有碱基对都存在于溶液中，由截断的D环和额外的环组成的连接区具有相当灵活的结构。这种流动性对于tRNA^&lt；Ser&gt；_&lt；UCU&gt；维持3‘-末端和反密码子的相对排列与通常的tRNAs是必不可少的。(2)最小前提结构…更多的核糖体：与大肠杆菌核糖体相比，动物线粒体核糖体中rRNA的含量约为一半，但蛋白质的含量增加了一倍。Mt rRNA的功能区在两个核糖体亚基的中心部分被严格保守，而mt核糖体缺失的rRNA的外围区域被蛋白质包埋，以保持mt和E.Coli核糖体的整体形状几乎相同。结果表明，线粒体核糖体外围区域的一些茎/环结构是必不可少的。(3)翻译系统中的能量来源GTP的水解反应的触发因素：根据EF-G的GTP酶活性被L7/L12激活的事实，推测GTP的水解反应的触发因素是50S核糖体亚基中的L7/L12蛋白。我们通过阐明E.coliEF-G可以在mt核糖体上发挥作用来证实了这一观点，在mt核糖体上，只有L7/L12蛋白被E.coliEf-G的对应物取代，尽管E.coliEF-G不能在天然mt核糖体上发挥作用。较少

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