ISOLATION AND POINT OF ACTION OF A FACTOR FROM ESCHERICHIA-COLI REQUIRED TO RECONSTRUCT TRANSLATION
ISOLATION AND POINT OF ACTION OF A FACTOR FROM ESCHERICHIA-COLI REQUIRED TO RECONSTRUCT TRANSLATION
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DOI：
10.1073/pnas.82.6.1648
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
GREEN, RM
中科院分区：
文献类型：
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作者：
GANOZA, MC;CUNNINGHAM, C;GREEN, RM
为了研究翻译机制，尝试从纯化的成分重建该过程。蛋白质合成由噬菌体 f2 或 MS2 的野生型或琥珀突变体的 RNA 编程。由 MS2 或 f2am3 RNA 编程的翻译不会使用核糖体、预充电的氨酰基-tRNA 和参与蛋白质合成起始（起始因子：IF-1、IF-2 和 IF-3）、增殖（延伸因子；Ef-Tu、EF-Ts 和 EF-G）和终止（释放因子；RF-1 或 RF-2）的纯化蛋白质的总和发生。证明了对一种名为 W 的蛋白质的需求。蛋白 W 经纯化，不含所有翻译因子、激活酶和其他与翻译有关的蛋白，例如 RR、rescue 和 EF-P。 W对增殖的刺激取决于外壳蛋白的NH2末端六肽合成中要添加的氨基酸残基的位置。在重建的系统中，在所有翻译因子的总和但没有 W 的情况下，在 f2am3 RNA 的指导下仅合成二肽和少量的三肽。 W 刺激由该 RNA 指导的六肽 fMet-Ala-Ser-AspNH2-Phe-Thr 的合成。 W 还刺激与核糖体颗粒结合的非同源 tRNA 的喷射。
To study the mechanism of translation, reconstruction of the process from purified components was attempted. Protein synthesis was programmed by the RNA of wild-type or amber mutants of bacteriophages f2 or MS2. Translation programmed by MS2 or f2am3 RNA does not occur using ribosomes, precharged aminoacyl-tRNA and the sum of the purified proteins involved in initiation (initiation factors: IF-1, IF-2 and IF-3), propagation (elongation factors; Ef-Tu, EF-Ts and EF-G) and termination (release factors; RF-1 or RF-2) of protein synthesis. The requirement for a protein called W was demonstrated. Protein W was purified free of all translation factors, activating enzymes, and other proteins such as the RR, rescue, and EF-P implicated in translation. The stimulation of propagation by W depended on the position of the amino acid residue to be added in the synthesis of the NH2-terminal hexapeptide of the coat protein. In the reconstructed system, with the sum of all translation factors but in the absence of W, only dipeptides and smaller quantities of tripeptides were synthesized under the direction of f2am3 RNA. W stimulated the synthesis of the hexapeptide, fMet-Ala-Ser-AspNH2-Phe-Thr directed by this RNA. W also stimulated ejection of non-cognate tRNA that bind to ribosomal particles.