Evidence for aminoacyl-tRNA binding, peptide bond synthesis, and translocase activities in the aminoacyl transfer reaction

Abstract

Incubation of ribosomes with puromycin leads to the formation of peptidyl puromycin. The synthesis of the peptide bond between endogenous peptides and puromycin does not require transferases I and II or guanosine 5′-triphosphate (GTP). When the transferase I- and nucleotide-dependent binding of aminoacyl-tRNA to ribosomes is carried out with GTP, some of the bound aminoacyl-tRNA is incorporated into a peptide-bonded form in the absence of transferase II. When the binding reaction is carried out with the GTP analogue, 5′-guanylyl methylenediphosphonate (GDPCP), aminoacyl-tRNA is bound to the ribosomes, but it: does not react with endogenous peptidyl-tRNA. Thus, the aminoacyl-tRNA binding reaction requires transferase I and GTP but does not involve hydrolysis of the nucleotide; then, a reaction occurs subsequent to binding, but prior to peptide bond synthesis, which requires GTP specifically. The reaction of endogenous peptidyl-tRNA with the bound aminoacyl-tRNA is catalyzed by the ribosome preparation and results in the formation of a new peptidyl-tRNA, one amino acid longer, attached through tRNA to the site that was occupied by the incoming aminoacyl-tRNA. In this position, it does not react readily with low levels of puromycin. Incubation of ribosomes with transferase II and GTP leads to a conversion of peptidyl-tRNA from the aminoacyl to the peptidyl (puromycin-reactive) site. This translocation reaction requires GTP specifically; it does not occur with GDPCP. These results suggest that transferase I is the aminoacyl-tRNA binding factor, that peptide bond-synthetase is a ribosomal activity, that transferase II is involved in translocation, and that GTP has multiple sites of action, two of which appear to require GTP specifically.