Evidence for the Role of Aminoacyltransferase II in Peptidyl Transfer Ribonucleic Acid Translocation

Abstract

Abstract When ribosomes are pretreated with transferase II and guanosine triphosphate, the binding of aminoacyl transfer ribonucleic acid to ribosomes in the presence of transferase I and the GTP analogue 5'-guanylyl methylenediphosphonate is markedly increased. Binding of aminoacyl-tRNA to ribosomes is also obtained in the absence of transferase I and nucleotide when incubations are carried out in solutions containing high concentrations of MgCl2 (20 mm). The addition of nonenzymatically bound aminoacyl-tRNA or of puromycin to endogenous peptides is carried out in the absence of transfer factors or nucleotide by a ribosome-associated activity. When ribosomes are pretreated with transferase II and GTP, the amount of aminoacyl-tRNA that is bound nonenzymatically and then incorporated into a peptide-bound form is markedly increased. Concomitantly, the reaction of aminoacyl-tRNA with endogenous peptidyl-tRNA leads to a decrease in the amount of peptidyl-tRNA that can react with puromycin. These results suggest that (a) when transferase II and GTP interact with ribosomes additional sites for the enzymatic or nonenzymatic binding of aminoacyl-tRNA are made available because of the translocation of peptidyl-tRNA from the aminoacyl to the peptidyl site; consistent with this interpretation is the observation that puromycin incorporation is increased when ribosomes are incubated with transferase II and GTP; (b) binding of aminoacyl-tRNA and formation of a new peptide bond lead to a decrease in peptidyl-puromycin formation because the newly synthesized peptidyl-tRNA, one amino acid longer than the pre-existing endogenous one, remains at the site at which the incoming aminoacyl-tRNA was present. Thus, ribosome-bound peptidyl-tRNA occurs at the aminoacyl site as the product of peptide bond synthesis with aminoacyl-tRNA, or at the peptidyl site as the product of translocation. Transferase I and GTP are required for the binding of aminoacyl-tRNA, peptide bond synthesis is a ribosomal activity, and transferase II and GTP are required for translocation.