Translation Elongation by a Hybrid Ribosome in Which Proteins at the GTPase Center of the Escherichia coli Ribosome Are Replaced with Rat Counterparts*
Translation Elongation by a Hybrid Ribosome in Which Proteins at the GTPase Center of the Escherichia coli Ribosome Are Replaced with Rat Counterparts*
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DOI：
10.1074/jbc.m107730200
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发表时间：
2002-02
期刊：
影响因子：
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通讯作者：
T. Uchiumi;S. Honma;Takao Nomura;E. Dabbs;A. Hachimori
中科院分区：
文献类型：
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作者：
T. Uchiumi;S. Honma;Takao Nomura;E. Dabbs;A. Hachimori
核糖体L10·L7/L12蛋白复合物和L11结合于23 S rRNA结构域II的1070位附近的高度保守的RNA区域，构成大肠杆菌核糖体中GTP酶相关中心的一部分。我们在体外用大鼠对应物P0·P1/P2复合物和RL 12替换这些核糖体蛋白，并测试它们的核糖体活性。在温和条件下从核糖体蛋白L11缺陷的突变体制备缺乏1070 RNA结构域上的蛋白质的核心50 S亚基。大鼠蛋白通过与1070 RNA结构域的相互作用与核心50 S亚基结合。所得到的杂合核糖体对硫链丝菌素不敏感，并且显示聚（U）程序化的聚苯丙氨酸合成依赖于真核延伸因子1α（eEF-1α）和2（eEF-2）的作用，而不依赖于原核等效因子EF-Tu和EF-G。用大鼠蛋白替代L10·L7/L12复合物或L11的结果表明，P0·P1/P2复合物，而不是RL 12，负责真核核糖体对真核延伸因子的特异性和伴随的GT3活性。E. coliL 11或ratRL 12能显著促进杂合核糖体合成多聚苯丙氨酸，提示L11/RL 12蛋白在GT3后翻译延伸过程中起重要作用。
Ribosomal L10·L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0·P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1α (eEF-1α) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10·L7/L12 complex or L11 with rat protein showed that the P0·P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.