title: A fast dynamic mode of the EF-G-bound ribosome.
abstract: A key intermediate in translocation is an 'unlocked state' of the pre-translocation ribosome in which the P-site tRNA adopts the P/E hybrid state, the L1 stalk domain closes and ribosomal subunits adopt a ratcheted configuration. Here, through two- and three-colour smFRET imaging from multiple structural perspectives, EF-G is shown to accelerate structural and kinetic pathways in the ribosome, leading to this transition. The EF-G-bound ribosome remains highly dynamic in nature, wherein, the unlocked state is transiently and reversibly formed. The P/E hybrid state is energetically favoured, but exchange with the classical P/P configuration persists; the L1 stalk adopts a fast dynamic mode characterized by rapid cycles of closure and opening. These data support a model in which P/E hybrid state formation, L1 stalk closure and subunit ratcheting are loosely coupled, independent processes that must converge to achieve the unlocked state. The highly dynamic nature of these motions, and their sensitivity to conformational and compositional changes in the ribosome, suggests that regulating the formation of this intermediate may present an effective avenue for translational control.
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, NY 10021, USA.
authors.forename: James B
authors.initials: JB
authors.lastname: Munro
authors.forename: Roger B
authors.initials: RB
authors.lastname: Altman
authors.forename: Chang-Shung
authors.initials: CS
authors.lastname: Tung
authors.forename: Kevin Y
authors.initials: KY
authors.lastname: Sanbonmatsu
authors.forename: Scott C
authors.initials: SC
authors.lastname: Blanchard
mesh_terms: D001665:Binding Sites; D031541:Fluorescence Resonance Energy Transfer; D046911:Macromolecular Substances / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D020653:Peptide Elongation Factor G / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D055550:Protein Stability; D020871:RNA Stability; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D046911:Macromolecular Substances; D020653:Peptide Elongation Factor G; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C028546:ribosomal protein L1; D012343:RNA, Transfer