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.
D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D010441:Peptide Chain Elongation, Translational; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000378:metabolism; D010455:Peptides / Q000235:genetics; D011072:Poly U / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism*
D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D010455:Peptides; D012346:RNA, Transfer, Amino Acyl; D011072:Poly U; C012001:polyphenylalanine