Molecular Dissection of Functional Structures in the Ribosome

核糖体gtpase相关中心由一部分23S rRNA和一些蛋白质组成，这些蛋白质聚集在rRNA区域并形成一个特征复合物，称为柄。这种rrna -蛋白复合物在核糖体与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。在这个项目中，我用生化方法分析了复合体的结构，得到了以下新的信息，特别是关于茎复合体。1)“杂交核糖体”功能检测体系的建立我们建立了大肠杆菌核糖体中茎状复合体的体外重建条件，并将其替换为其他物种的对应物。这为研究茎复合体的功能结构提供了一个有用的系统。2)真核生物PO的组装方式。我们证实真核蛋白P1和P2形成异二聚体，两个二聚体结合到P0的c端形成五聚体复合物。我们还阐明了茎复合体调节gtpase相关中心23S rRNA的功能结构。我们证明了古细菌L12蛋白形成一个同型二聚体，三个二聚体结合到锚蛋白P0的c端区域，并且所得到的七聚体复合物显示真核生物和古细菌翻译因子的可及性。4)真细菌柄复合物的结构和功能表征我们发现，大肠杆菌L12有2个同型二聚体结合到锚蛋白L10的c端，而嗜热真细菌的L12有3个二聚体结合到L10。我们还澄清了由L10和一个L12二聚体组成的复杂变体在核糖体上是不稳定的。这表明L12二聚体的数量与核糖体中柄复合物的稳定性有关。

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.