Isolation and point of action of a factor from Escherichia coli required to reconstruct translation.

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Abstract
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.