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Reactions of N-acetylphenylalanyl transfer RNA with rat-liver ribosomes

1969; Elsevier BV; Volume: 195; Issue: 1 Linguagem: Inglês

10.1016/0005-2787(69)90609-1

1879-3002

Jack Siler, Kivie Moldave,

DNA and Nucleic Acid Chemistry

Rat-liver ribosomes, prepared free of peptidyl-tRNA and mRNA, bind phenylalanyl-tRNA at low Mg2+ concentrations, in the presence of transferase I (aminoacyl-tRNA-binding factor), GTP, and poly U. At higher Mg2+ concentrations, binding requires poly U but not transferase I and GTP. N-Acetylphenylalanyl-tRNA is bound to ribosomes in poly U- and high Mg2+-containing solutions, but the reaction is not affected by GTP, transferase I or other soluble proteins. The N-acetylphenylalanyl-tRNA bound to ribosomes does not react with puromycin, unless transferase II and GTP are present or are added to the incubation subsequent to binding. When ribosomes are isolated from binding-reaction mixtures containing transferase II and GTP, the reaction with puromycin is rapid and occurs in the absence of additional transferase II and GTP. The extent to which a puromycin-reactive intermediate is formed is dependent on the concentration of transferase II (and GTP) and the time of incubation. The results suggest that N-acetylphenylalanyl-tRNA binds to the aminoacyl (puromycin-insensitive) ribosomal site and is then translocated with transferase II and GTP to the peptidyl (puromycin-reactive) ribosomal site. Data obtained are consistent with a role of transferase II in translocation and rule out a direct participation of this factor in binding of substrate tRNA's to ribosomes and in the formation of the peptide bond.