Development of a new translation system for animal proteins: the engineering strategy of ribosome function

在本项目中，我们分析了翻译机器中的功能结构，并通过分子工程对其进行改造，以开发一种新的翻译系统，用于真核蛋白质的合成。以下是该项目的主要成果。在真核生物中，一对P1-P2异二聚体与PO的C-末端结合形成五聚体复合物。在真细菌中，2 × 3个L12同源二聚体（因物种而异）与L10的C-末端结合。在古细菌中，3 L12同二聚体结合PO，并形成一个七聚体复合物，通过生化分析和X-射线晶体学显示。通过从每个复合物中去除一个或两个二聚体，降低了GT3和多肽合成的活性.昆虫病毒IRES介导的翻译起始机制：1）IRES通过与小亚基蛋白S25的相互作用与核糖体结合，2）诱导核糖体的某些构象变化，3）激活核糖体对延伸因子eEF 2的可接近性，4）增强第一个氨酰-tRNA与核糖体P位点的结合。我们还成功地使用IRES-荧光素酶融合mRNA翻译IRES介导的荧光素酶。利用该系统，我们阐明了PSIV IRES基因的3 '端非翻译区参与了翻译效率.一种新的体内翻译系统的开发大肠杆菌突变株dL 11缺乏L11核糖体蛋白，而L11核糖体蛋白是GTP酶相关中心的组成部分。该菌株显示出非常低的生长速率（野生型的114 × 115），并用于蛋白质合成的宿主细胞。我们证明了这个E.大肠杆菌菌株可用于合成通常菌株不能以可溶状态获得的真核蛋白。

In this project, we analyzed functional structures in the translational machinery and modified them by molecular engineering in order to develop a new translation system useful for synthesis of eukaryotic proteins. The following are major results of this project.1. The functional structure of protein complex located in the ribosomal GTPase-associated center.In eukaryotes, a couple of P1-P2 heterodimers bound to the C-terminal regions of PO and formed a pentameric complex. In eubacteria, 2〜3 L12 homodimers (differing with species) bound to the C-terminus of L10. In archaebacteria, 3 L12 homodimers bound to PO and formed a heptameric complex, as revealed by biochemical analysis and X-ray crystallography. By removing one or two dimers from each the complex, the activity of GTPase and polypeptide synthesis was reduced.2. Mechanism of translation initiation mediated with the IRES of an insect virus (PSIV).We elucidated that 1) the IRES binds to the ribosome through interaction of the small subunit protein S25, 2) induces some conformational changes in the ribosome, 3) activates the ribosomal accessibility to elongation factor eEF2, and then 4) enhances binding of the first aminoacyl-tRNA to the ribosomal P site. We also succeeded translation of luciferase mediated by the IRES using an IRES-luciferase fusion mRNA. By using this system, we clarified that the 3'-untranlated region of the PSIV IRES-carrying gene participates in the translation efficiency.3. Development of a new in vivo translation system.The E.coli mutant strain dL11 lacks L11 ribosomal protein that is a component of the GTPase-associated center. This strain showed very low growing rate (114〜115 of wild type) and was used for a host cell of protein synthesis. We demonstrated that this E. coli strain is useful for synthesis of eukatyotic proteins that cannot be obtained as a soluble state by usual strains.