title: Fluctuations between multiple EF-G-induced chimeric tRNA states during translocation on the ribosome.
abstract: The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Goettingen 37077, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Adio
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Goettingen 37077, Germany.
authors.forename: Tamara
authors.initials: T
authors.lastname: Senyushkina
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Goettingen 37077, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Peske
authors.affiliation: 3D Electron Cryomicroscopy Group, Max Planck Institute for Biophysical Chemistry, Goettingen 37077, Germany.
authors.forename: Niels
authors.initials: N
authors.lastname: Fischer
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Goettingen 37077, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wintermeyer
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Goettingen 37077, Germany.
authors.forename: Marina V
authors.initials: MV
authors.lastname: Rodnina
mesh_terms: D018360:Crystallography, X-Ray; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D031541:Fluorescence Resonance Energy Transfer; D020653:Peptide Elongation Factor G / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D012269:Ribosomal Proteins; D054681:Ribosome Subunits, Large, Bacterial / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D020653:Peptide Elongation Factor G; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C525862:rplK protein, E coli; D012343:RNA, Transfer