title: EF-G-independent reactivity of a pre-translocation-state ribosome complex with the aminoacyl tRNA substrate puromycin supports an intermediate (hybrid) state of tRNA binding.
abstract: Following peptide-bond formation, the mRNA:tRNA complex must be translocated within the ribosomal cavity before the next aminoacyl tRNA can be accommodated in the A site. Previous studies suggested that following peptide-bond formation and prior to EF-G recognition, the tRNAs occupy an intermediate (hybrid) state of binding where the acceptor ends of the tRNAs are shifted to their next sites of occupancy (the E and P sites) on the large ribosomal subunit, but where their anticodon ends (and associated mRNA) remain fixed in their prepeptidyl transferase binding states (the P and A sites) on the small subunit. Here we show that pre-translocation-state ribosomes carrying a dipeptidyl-tRNA substrate efficiently react with the minimal A-site substrate puromycin and that following this reaction, the pre-translocation-state bound deacylated tRNA:mRNA complex remains untranslocated. These data establish that pre-translocation-state ribosomes must sample or reside in an intermediate state of tRNA binding independent of the action of EF-G.
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Divya
authors.initials: D
authors.lastname: Sharma
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Southworth
authors.forename: Rachel
authors.initials: R
authors.lastname: Green
mesh_terms: D002678:Chimera; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010441:Peptide Chain Elongation, Translational; D020653:Peptide Elongation Factor G / Q000378:metabolism*; D010458:Peptidyl Transferases; D014176:Protein Biosynthesis; D011691:Puromycin / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D014178:Translocation, Genetic; D014344:Trypanocidal Agents / Q000378:metabolism
chemical_list: D020653:Peptide Elongation Factor G; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; D014344:Trypanocidal Agents; C006383:tRNA, N-acetylphenylalanine-; C019037:tRNA, peptidyl-; D011691:Puromycin; D010458:Peptidyl Transferases