A Novel Cell-Free Peptide Synthesis Driven by Pyridine

吡啶驱动的新型无细胞肽合成

• 批准号: 08555200
• 负责人: WATANABE Kimitsuna
• 金额: $ 6.02万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08555200/
• 关键词: aminoglycoside; in-vitro translation; non-enzymatic tRNA binding; pre-translocational ribosome; puromycin reaction; ribosome-inactivating protein; トランスロケーション; 抗生物質; リボソーム; テンブレート重合; 坑生物質; rRNA; アミノピリジン; タンパク質合成系

We have already demonstrated that high concentrations (40-60%) of pyridine was able to promote polypeptide synthesis on ribosomes from various organisms without any protein factors. For better understanding the mechanism of this pyridine system, association of ribosomal subunits, assembly of ribosomal proteins, as well as translocation reaction in 60% pyridine were investigated using E.coli ribosomes. Sucrose density gradient profiles showed that while the subunits once exposed to pyridine failed to re-associate themselves in the aqueous solution even containing 20 mM Mg^<2+>, they could form the total couple in 60% pyridine. Two-dimensional gel electrophoresis of ribosomal proteins revealed that some proteins composing the three features of the large subunit, especially those included in the elongation factor-binding domain (the L7/L12 atalk and L11), were released from the reconstituted particle. Both of polyphenylalanine synthesis and translocation activities in 60% pyridine were completely abolished by neomycin, a strong inhibitor against factor-independent translocation, whereas both of the reactions were not affected at all by gypsophilin, a ribotoxin to inhibit factor-dependent translocation. A similar antibiotic inhibitor, thiostrepton, affected neither of the reactions as well, which is consistent with the fact that the antibiotics requires L11 for its binding to the ribosome. These results suggest that translocation actually occurs in the pyridine system, but in a factors-independent (spontaneous) manner.

我们已经证明，高浓度（40-60%）的吡啶能够促进来自各种生物体的核糖体上的多肽合成，而无需任何蛋白质因子。为了更好地理解这种吡啶系统的机制，使用大肠杆菌核糖体研究了核糖体亚基的缔合、核糖体蛋白的组装以及在60%吡啶中的易位反应。蔗糖密度梯度曲线显示，尽管一旦暴露于吡啶，即使在含有20 mM Mg^<2+>的水溶液中，亚基也不能重新结合，但它们可以在60%的吡啶中形成总偶联。核糖体蛋白质的二维凝胶电泳显示，一些蛋白质组成的大亚基的三个功能，特别是那些包括在延伸因子结合域（L7/L12 atalk和L11），从重组颗粒释放。在60%吡啶溶液中，新霉素能完全抑制多聚苯丙氨酸的合成和转运活性，而丝石竹素对多聚苯丙氨酸的合成和转运活性没有影响。一种类似的抗生素抑制剂，硫链丝菌素，也不影响这两种反应，这与抗生素需要L11才能与核糖体结合的事实一致。这些结果表明，易位实际上发生在吡啶系统中，但在一个因素独立（自发）的方式。

项目成果

S.Watanabe et al.: "An RNA fragment consisting of the P7 and P9.0 stems and the 3'-terminal guanosine of the tetrahymene group I infnon" Nucl.Acids Res.24,7. 1337-1344 (1996)

S.Watanabe 等人：“由 P7 和 P9.0 茎以及四膜虫 I 组 infnon 的 3 末端鸟苷组成的 RNA 片段”Nucl.Acids Res.24,7。

Itaru, Nitta: "A novel factor-and energy-free translation system promoted by tertiary amines and its implications toward the origin of the translation system." Vira Origino. 23. 209-222 (1995)

Itaru, Nitta：“一种由叔胺促进的新型无因子和无能量翻译系统及其对翻译系统起源的影响。”

Itaru, Nitta: "A novel cell-free system for peptide synthesis driven by pyridine." Bio1.Chem.(in press). (1998)

Itaru, Nitta：“一种由吡啶驱动的新型无细胞肽合成系统。”

新田 至, 他: "A novel cell-free system for peptide synthesis driven by pyridine." Biol.Chem.(印刷中). (1998)

Itaru Nitta 等人：“一种由吡啶驱动的新型无细胞肽合成系统。”（正在出版）。

新田 至、他: "A novel cell-free system for peptide synthesis driven by pyridine." Biol.Chem.(印刷中). (1998)

Itaru Nitta 等人：“一种由吡啶驱动的新型无细胞肽合成系统”（正在出版）。