Ganoza M C, Cunningham C, Green R M
Proc Natl Acad Sci U S A. 1985 Mar;82(6):1648-52. doi: 10.1073/pnas.82.6.1648.
To study the mechanism of translation we have attempted to reconstruct the process from purified components. Protein synthesis was programmed by the RNAs 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-tRNAs, 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. In addition, W stimulated ejection of non-cognate tRNAs that bind to ribosomal particles.
为了研究翻译机制，我们尝试从纯化的组分中重建这一过程。蛋白质合成由噬菌体f2或MS2的野生型或琥珀突变体的RNA进行编程。使用核糖体、预负载的氨酰-tRNA以及参与蛋白质合成起始（起始因子；IF-1、IF-2和IF-3）、延伸（延伸因子；EF-Tu、EF-Ts和EF-G）和终止（释放因子；RF-1或RF-2）的纯化蛋白质总和，由MS2或f2am3 RNA编程的翻译不会发生。证明了对一种名为W的蛋白质的需求。蛋白质W被纯化，不含所有翻译因子、激活酶以及其他与翻译相关的蛋白质，如RR、“拯救”蛋白和EF-P。W对延伸的刺激取决于在外壳蛋白NH2末端六肽合成中要添加的氨基酸残基的位置。在重建系统中，使用所有翻译因子总和但不存在W时，在f2am3 RNA的指导下仅合成二肽和少量三肽。W刺激了由该RNA指导的六肽fMet-Ala-Ser-AspNH2-Phe-Thr的合成。此外，W刺激了与核糖体颗粒结合的非同源tRNA的排出。
