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

用结构生物学阐明线粒体翻译系统的构建原理

• 批准号: 11308024
• 负责人: WATANABE Kimitsuna
• 金额: $ 22.22万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11308024/
• 关键词: Translation System; mitochondria; EF-Tu; tRNA; Seryl-tRNA synthetase; Nematode; ribosome; X-ray analysis; in vitro翻訳システム; tRNA^<Ser>GCU; tRNA^<Ser>UGA; セリルtRNAシンテータゼ; X線構造解析; 機能構造; ミトコンドリアtRNA; トランスホルミラーゼ; リボソーム蛋白質; X線結晶解析; SerRS; 異常構造

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.

本研究的目的是阐明线粒体的结构原理！翻译系统，通过使用X射线分析和NMR光谱分析动物线粒体翻译系统的每个组件的功能结构，重点关注组件的功能特征和结构异常。这三年来取得了以下成果。(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系统，其效率与“。co//系统中的poly（U）-依赖的poly（Phe）合成。(2)核糖体在E. coli和线粒体系统，就使用相同来源的tRNA和翻译因子而言，E.使用大肠杆菌来源。因此，E.大肠杆菌和线粒体系统。(3)证实了2种具有不寻常二级结构的丝氨酸tRNA具有功能。缺乏D臂的丝氨酸tRNA的低效率源自其与核糖体A位点结合的缺陷。(4)这两个丝氨酸tRNA被一个单一的丝氨酸tRNA合成酶（serRS）识别，并确定了其识别位点。(5)发现了一个独特的EF-Tu，它识别线虫线粒体中几乎所有T臂缺失的tRNA。它在C末端有57个氨基酸的延伸，被认为是对tRNA 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，对丝氨酸具有特异性。(6)这些翻译因素的具体化正在进行之中。我们获得了一个针状晶体。X射线分析将在未来进行。

项目成果

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Hanada, T., Suzuki, T., Yokogawa, T., Takemoto-Hori, C., Sprinzl, M. and Watanabe, K.: "Translation ability of mitochondrial tRNAs^<Ser> with unusual secondary structures in an in vitro translation system of bovine mitochondria"Genes Cells. 6. 1019-1030 (

Hanada, T.、Suzuki, T.、Yokokawa, T.、Takemoto-Hori, C.、Sprinzl, M. 和 Watanabe, K.：“具有不寻常二级结构的线粒体 tRNAs^<Ser> 在体外的翻译能力

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Okawa T. 等人：“一种针对特定转移 RNA 反密码子的细胞毒性核糖核酸酶”《科学》。

Kondou A. et al.: "An extra tRNA^<Gly>(V^*CU) found in ascidian mitochondria responsible for decoding non-universal codons AGA/AGG as Gly"Nucleic Acido Res.. 2T12. 2554-2559 (1999)

Kondou A. 等人：“在海鞘线粒体中发现了一种额外的 tRNA^<Gly>(V^*CU)，负责将非通用密码子 AGA/AGG 解码为 Gly”Nucleic Acido Res.. 2T12。