Molecular Dissection of Functional Structures in the Ribosome

核糖体GTPase相关中心由部分23S rRNA和少量蛋白质组成，它们聚集在rRNA区域，形成一个特征性的复合体，称为茎。这种rRNA-蛋白质复合体在核糖体与GTP结合翻译因子的相互作用、GTP水解和翻译速率的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，获得了以下新的信息，特别是关于茎复合体的新信息1)功能分析系统的建立，“杂交核糖体”我们建立了体外重组大肠杆菌核糖体中的茎复合体的条件，并将其替换为其他物种的茎复合体。这为研究真核生物PO的茎复合体的功能结构提供了一个有用的系统。我们证明了真核蛋白P1和P2形成一个异二聚体，两个二聚体结合到P0的C-末端区域形成五聚体。3)考古细菌柄复合体的结构和功能鉴定我们证明了考古菌L12蛋白形成一个同源二聚体，三个二聚体与锚蛋白P0的C-末端区域结合，所得到的七聚体复合体不仅可以与真核细胞结合，也可以与考古细菌的翻译因子结合。4)真细菌茎复合体的结构和功能表征我们证明了两个大肠杆菌L12同源二聚体与锚定蛋白L10的C-末端区域结合，而对于嗜热细菌，三个L12二聚体与L10结合。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。这表明核糖体中L12二聚体的数量与茎复合体的稳定性有关。

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.