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Molecular Dissection of Functional Structures in the Ribosome

核糖体功能结构的分子解剖

基本信息

批准号：
14035222
负责人：
UCHIUMI Toshio
金额：
$ 40.06万
依托单位：
Niigata University (2003-2006)Shinshu University (2002)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14035222/
关键词：
Ribosome GTPase Ribosomal protein rRNA Translation Elongation factor RNA-protein interaction Archaebacteria RNA-タンバク質相互作用 GTPase IRES 翻訳蚕リボソームRNA リボソームPタンパク質 L7 L12 L10

项目摘要

The ribosomal GTPase-associated center is composed of a part of 23S rRNA and a few proteins, which assemble onto the rRNA region and form a characteristic complex, termed the stalk. This rRNA-protein complex plays a crucial role in ribosomal interaction with GTP binding translation factors, GTP hydrolysis, and regulation of translation rate. In this project, I analyzed the complex structure by biochemical approaches, and obtained the following new information, particularly on the stalk complex.1) Establishment of a functional assay system, "hybrid ribosome"We established conditions for in vitro reconstitution of the stalk complex in Escherichia coli ribosomes and its replacement with its counterparts from other species. This provided a useful system to investigate functional structure of the stalk complex.2) Assembly mode of eukaryotic PO. P1-P2 stalk complex and its functional characterizationWe demonstrated that eukaryotic proteins P1 and P2 form a heterodimer and two dimes bind to the C-terminal region of P0 to form pentameric complex. We also clarified that the stalk complex modulates functional structure of 23S rRNA in the GTPase-associated center.3) Structural and functional characterization of the archaebacterial stalk complexWe demonstrated that archaebacterial L12 protein forms a homodimer and three dimers bind to the C-terminal region of the anchor protein P0, and that the resultant heptameric complex shows accessibility to eukaryotic as well as archaebacterial translation factors.4) Structural and functional characterization of the eubacterial stalk complexWe showed that two E. coli L12 homodimers bind to the C-terminal region of the anchor protein L10, whereas three L12 dimers bind to L10 in case of thermophilic eubacteria. We also clarified that a complex variant composed of L10 and one L12 dimer is unstable on the ribosome. This suggests a relationship between number of L12 dimer and stability of the stalk complex in the ribosome.

核糖体GTP酶相关中心由23 S rRNA的一部分和少数蛋白质组成，它们组装到rRNA区域并形成特征性复合物，称为柄。这种rRNA-蛋白质复合物在核糖体与GTP结合翻译因子的相互作用、GTP水解和翻译速率调节中起着至关重要的作用。在本课题中，通过生物化学方法分析了复合物的结构，特别是茎复合物，获得了以下新的信息。1）功能性测定系统“杂合核糖体“的建立我们建立了大肠杆菌核糖体中茎复合物的体外重组条件，并将其替换为其他物种的对应物。这为研究茎复合物的功能结构提供了一个有用的系统。2）真核PO的组装模式。P1-P2柄复合体及其功能研究我们发现真核生物蛋白质P1和P2形成异二聚体，两个二聚体与P0的C端结合形成五聚体复合体。3）古细菌柄复合物的结构和功能表征我们证明古细菌L12蛋白形成同源二聚体，并且三个二聚体结合到锚蛋白P0的C-末端区域，并且所得到的七聚体复合物显示出对真核生物以及古细菌翻译因子的可接近性。4）真细菌柄复合物的结构和功能表征我们发现两个E.大肠杆菌L12同源二聚体与锚蛋白L10的C-末端区域结合，而在嗜热真细菌的情况下，三个L12二聚体与L10结合。我们还阐明了由L10和一个L12二聚体组成的复杂变体在核糖体上不稳定。这表明L12二聚体的数量与核糖体中柄复合物的稳定性之间的关系。