Testing an alternative model for the ribosomal peptide elongation cycle.

A kinetic analysis of poly(U)-dependent poly(Phe) synthesis with [14C]tRNAPhe and [3H]phenylalanine demonstrated that, in the course of efficient poly(Phe) synthesis, two tRNAs are present per 70S ribosome at all times, although at least 70% of the poly(Phe)-tRNAPhe is found at the peptidyl-tRNA (P) site. Together with our recent observation of a third tRNA-binding site on Escherichia coli ribosomes, these findings suggest a model for the peptide elongation cycle in which two tRNA molecules are present on the ribosome at both the pre- and the post-translocational state. This model predicts that deacylated tRNA is not released from the P site but translocated to the exit (E) site before release occurs. A series of translocation experiments with deacylated [14C]tRNAPhe at the P site and oligo [( 3H]Phe)-tRNA at the aminoacyl-tRNA (A) site proved that efficient elongation factor G-dependent translocation is not accompanied by a corresponding [14C]tRNAPhe release. However, significant [14C]tRNAPhe release was observed after translocation when an aminoacyl-tRNA was bound to the A site. Thus, deacylated tRNA is not released from the P site but is translocated to the E site, which therefore must be located "upstream" adjacent to the P site. Furthermore, the trigger for the release of deacylated tRNA from the E site is the binding of aminoacyl-tRNA to the A site.