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

本研究的目的是阐明线粒体的构造原理！通过对动物线粒体翻译系统各组成部分的功能结构进行x射线分析和核磁共振波谱分析，重点研究各组成部分的功能特征和结构异常。这三年的研究结果如下：(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，其效率与“。co//体系中依赖聚(U)的聚（Phe）合成。(2)核糖体在大肠杆菌和线粒体系统之间是可交换的，只要使用相同来源的tRNA和翻译因子，并使用大肠杆菌来源的EF-Tu。因此，核糖体在大肠杆菌和线粒体系统之间具有相同的功能。(3)验证了2种具有特殊二级结构的丝氨酸trna可能具有功能。D臂缺失丝氨酸tRNA的低效率源于其与核糖体a位点的结合缺陷。(4)这两个丝氨酸trna被单个seryl-tRNA合成酶（serRS）识别，并在两个trna上确定了其识别位点。(5)发现了一个独特的EF-Tu，可以识别线虫线粒体中几乎所有缺乏T臂的trna。它在C端有57个氨基酸的延伸，被认为是对trna缺乏T臂部分的补充。另一个EF-Tu识别两个缺乏D臂的丝氨酸trna是对丝氨酸特异性的。这些转译因素的结晶正在进行中。我们获得了SerRS的针状晶体。x射线分析将在未来进行。

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.