Elucidation of the construction principle of the mitochondria translation system by means of structural biology

本研究旨在阐明线粒体的结构原理！翻译系统，通过X射线分析和核磁共振波谱分析动物线粒体翻译系统各组成部分的功能结构，重点分析各组成部分的功能特点和结构异常。三年来取得了以下研究成果：(1)通过优化各组分的浓度和反应条件，建立了牛线粒体体外翻译体系，该体系的效率与“。(2)核糖体可在大肠杆菌和线粒体系统之间交换，只要使用相同来源的tRNA和翻译因子，并使用来源于大肠杆菌的EF-Tu。因此，在大肠杆菌和线粒体系统中，核糖体在功能上是等价的。(3)验证了两种具有异常二级结构的丝氨酸tRNAs具有功能。缺乏D臂的丝氨酸tRNA的低效率源于它与核糖体A位点的结合缺陷。(4)这两个丝氨酸tRNA被单一的丝氨酸tRNA合成酶识别，并确定了它在这两个tRNA上的识别位置。(5)发现了一个独特的EF-Tu，它识别线虫线粒体中几乎所有缺乏T臂的tRNA。它在C端有57个氨基酸延伸，被认为是对tRNAs T臂缺失部分的补充。另一个EF-Tu识别两个D臂缺失的丝氨酸tRNA是丝氨酸所特有的。(6)这些翻译因子的结晶正在进行中。我们已经获得了一种针状的SERRS晶体。今后将进行X射线分析。

The aim of this research is to elucidate the construction principle of the mitochondria! translation system, by analyzing the functional structures of each component of the animal mitochondrial translation system using X-ray analysis and NMR spectroscopy, focusing on the functional characteristics and structural abnormalities of the components. The following results have been obtained for these three years.(1) Bovine mitochondrial in vitro translation system has been established by optimizing concentration of each component and reaction conditions, which has similar efficiency to that of 」. co// system in terms of poly(U)-dependent poly(Phe) synthesis.(2) Ribosomes are exchangeable between E. coli and mitochondrial systems, as far as tRNA and translation factors of the same origins are used, and EF-Tu of E. coli origin is used. Thus, the ribosomes are functionally equivalent between E. coli and mitochondrial systems.(3) It was verified that 2 species of serine tRNAs with unusual secondary structures can be functional. The low efficiency of the D arm-lacking serine tRNA is derived from a defect in its binding to the ribosomal A site.(4) These two serine tRNAs were recognized by a single seryl-tRNA synthetase (serRS) and its recognition sites on the two tRNAs were identified.(5) A unique EF-Tu was found which recognized almost all the T arm-lacking tRNAs in nematode mitochondria. It had 57 amino acid extension at the C terminus which was considered to complement the T arm-lacking part of tRNAs. Another EF-Tu recognizing two D arm-lacking serine tRNAs was specific for serine.(6) Crystallization of these translation factors are being under way. We have obtained a needle-like srystal for SerRS. The X-ray analysis will be made in future.