title: Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation.
abstract: Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G-GDP-bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Wasserman
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA.
authors.forename: Jose L
authors.initials: JL
authors.lastname: Alejo
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA.
authors.forename: Roger B
authors.initials: RB
authors.lastname: Altman
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA.|Tri-Institutional Training Program in Chemical Biology, Weill Cornell Medical College, New York, New York, USA.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Blanchard
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006153:Guanosine Diphosphate / Q000032:analysis / Q000378:metabolism; D008958:Models, Molecular; D020653:Peptide Elongation Factor G / Q000032:analysis / Q000378:metabolism*; D021381:Protein Transport; D012329:RNA, Bacterial / Q000032:analysis / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000032:analysis / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D020653:Peptide Elongation Factor G; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; C019037:tRNA, peptidyl-; D006153:Guanosine Diphosphate