Translocation Reaction Promoted by Polypeptide Chain Elongation Factor-2 from Pig Liver

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Abstract
Translocation of peptidyl-tRNA from the ribosomal A- to the P-site in the eukaryotic system was extensively investigated using a model system, in which translocation of Phe-tRNA from the A- to the P-site was examined by using the puromycin reaction, and the following results were obtained. 1) The puromycin reaction but not the translocation reaction proceeded at 0°C. Since the latter could be demonstrated at 30°C, it was possible to analyze translocation per se separately from the puromycin reaction. 2) Translocation was completely dependent on the elongation factor-2 (EF-2) and required the presence of GTP, which could be replaced by GMP-P(NH)P provided that the stoichiometric amount of EF-2 with respect to the amount of ribosomes was present. It was further demonstrated that translocation observed in the presence of GTP was catalytic, while that in the presence of GMP-P(NH)P was stoichiometric, indicating that hydrolysis of GTP was required for the catalytic reutilization of EF-2. 3) Translocation promoted by EF-2 in the presence of GMP-P(NH)P could be reversed, which suggests that hydrolysis of GTP is indispensable of the translocation reaction to proceed catalytically and unidirectionally forward.

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