Translocation reaction promoted by polypeptide chain elongation factor-2 from pig liver

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 degrees C. Since the latter could be demonstrated at 30 degrees 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 or 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.