Analysis of functional structure of ribosomal RNA

本课题旨在通过分析核糖体和核糖体RNA （rRNA）的功能结构，阐明翻译系统构成和功能的基本原理。翻译系统是生命中最基本的系统，阐明其构成原则也就意味着阐明生命的起源。在本研究开始时，为了阐明大肠杆菌核糖体和rRNA的功能结构，我们试图利用吡啶处理的蛋白剥夺核糖体来阐明翻译反应的每个步骤。结果表明，这种核糖体具有非酶（自发）易位活性。然而，在这两年中，核糖体晶体的x射线分析有了很大的改进，通过在原子水平上检查核糖体的三维结构来阐明核糖体的反应机制成为可能。因此，我们将研究目标从大肠杆菌转移到动物线粒体肋骨，因为线粒体核糖体具有大约一半缩短的rRNA，而不是加倍放大的蛋白质，而分子量和总大小相似，与大肠杆菌核糖体相比，这表明rRNA功能位点的识别要容易得多，而且RNA的作用可能在线粒体进化过程中转移到蛋白质上。我们已经从牛线粒体核糖体（mitribosome）的约70种蛋白质中鉴定出55种核糖体蛋白，通过二维凝胶电泳分离每个核糖体蛋白，然后用LC/MS技术对其进行鉴定。我们现在正在描述这些蛋白质。研究还发现，只有L7/L12蛋白被大肠杆菌对应物取代的杂交型核糖体既可以与一组线粒体tRNA和翻译因子一起工作，也可以与一组大肠杆菌tRNA和翻译因子一起工作，这表明尽管rna -蛋白比例相反，但大肠杆菌和线粒体核糖体的大部分功能是相同的。我们也在研究大肠杆菌和线粒体核糖体之间这种功能互换性的起源。少

This research project aims to elucidate the basal principle by which the translation system is constituted and functions, by analyzings the, functional structures of the ribosome and ribosomal RNA (rRNA). Since the translation system is the most basic system in life, elucidation of its constitution principle would lead to elucidation of origin of life.In the beginning of this research, we tried to elucidate each step in the translation reaction using protein-deprived ribosomes treated with pyridine for the, purpose of elucidating the functional structure of E. coli ribosomes and rRNA. It turned out that such ribosomes possess non-enzymatic (spontaneous) translocation activity.However, in these two years X-ray analysis of ribosome crystals has been greatly improved and it became possible to elucidate the reaction mechanism of ribosomes by inspection of the three-dimensional structure of ribosomes at atomic level. We thus moved our research target from E. coli to animal mitochondrial rib … More osomes, because mitochondrial ribosomes possess about a half-shortened rRNA and instead, doubly enlarged proteins, while the molecular weight and whole size being similar, as compared to those of E. coli ribosomes, suggesting that the identification of functional sites in rRNA is much easier and moreover, the roles of RNA could have transferred to proteins during evolution of mitochondria.We have already identified 55 ribosomal proteins out of about 70 proteins in bovine, mitochondrial ribosome (mitoribosome) by separating each ribosomal, protein, on the two-dimensional gel electrophoresis followed by its identification by LC/MS technique. We are now characterizing these proteins. It also turned out that a hybrid-type mitoribosome in which only L7/L12 protein was replaced by the E. coli counterpart can function either with a set of mitochondrial tRNA and translation factors or with a set of E. coli tRNA and translation factors, suggesting that the most part of the ribosome is functionally equivalent between E. coli and mitochondrial ribosomes, notwithstanding that the RNA-protein ratio is reversed. We are also examining the origin of this functional interchangeability between E. coli and mitochondrial ribosomes. Less