Ribosomes accurately decode mRNA by proofreading each aminoacyl-tRNA that is delivered by the elongation factor EF-Tu1. To understand the molecular mechanism of this proofreading step it is necessary to visualize GTP-catalysed elongation, which has remained a challenge2-4. Here we use time-resolved cryogenic electron microscopy to reveal 33 ribosomal states after the delivery of aminoacyl-tRNA by EF-Tu•GTP. Instead of locking cognate tRNA upon initial recognition, the ribosomal decoding centre dynamically monitors codon-anticodon interactions before and after GTP hydrolysis. GTP hydrolysis enables the GTPase domain of EF-Tu to extend away, releasing EF-Tu from tRNA. The 30S subunit then locks cognate tRNA in the decoding centre and rotates, enabling the tRNA to bypass 50S protrusions during accommodation into the peptidyl transferase centre. By contrast, the decoding centre fails to lock near-cognate tRNA, enabling the dissociation of near-cognate tRNA both during initial selection (before GTP hydrolysis) and proofreading (after GTP hydrolysis). These findings reveal structural similarity between ribosomes in initial selection states5,6 and in proofreading states, which together govern the efficient rejection of incorrect tRNA.

Central European Institute of Technology Masaryk University Brno Czech Republic

RNA Therapeutics Institute Department of Biochemistry and Molecular Pharmacology University of Massachusetts Medical School Worcester MA USA

See more in PubMed

Hopfield JJ Kinetic proofreading: a new mechanism for reducing errors in biosynthetic processes requiring high specificity. Proc Natl Acad Sci U S A 71, 4135–4139 (1974). PubMed PMC

Voorhees RM & Ramakrishnan V Structural basis of the translational elongation cycle. Annu Rev Biochem 82, 203–236, doi:10.1146/annurev-biochem-113009-092313 (2013). PubMed DOI

Pavlov MY & Ehrenberg M Substrate-Induced Formation of Ribosomal Decoding Center for Accurate and Rapid Genetic Code Translation. Annu Rev Biophys 47, 525–548, doi:10.1146/annurev-biophys-060414-034148 (2018). PubMed DOI

Rodnina MV, Fischer N, Maracci C & Stark H Ribosome dynamics during decoding. Philos Trans R Soc Lond B Biol Sci 372, doi:10.1098/rstb.2016.0182 (2017). PubMed DOI PMC

Loveland AB, Demo G, Grigorieff N & Korostelev AA Ensemble cryo-EM elucidates the mechanism of translation fidelity. Nature 546, 113–117, doi:10.1038/nature22397 (2017). PubMed DOI PMC

Fislage M et al. Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-TuH84A. Nucleic Acids Res 46, 5861–5874, doi:10.1093/nar/gky346 (2018). PubMed DOI PMC

Moazed D & Noller HF Intermediate states in the movement of transfer RNA in the ribosome. Nature 342, 142–148, doi:10.1038/342142a0 (1989). PubMed DOI

Yusupov MM et al. Crystal structure of the ribosome at 5.5 A resolution. Science 292, 883–896 (2001). PubMed

Stark H et al. Visualization of elongation factor Tu on the Escherichia coli ribosome. Nature 389, 403–406, doi:10.1038/38770 (1997). PubMed DOI

Ehrenberg M & Blomberg C Thermodynamic constraints on kinetic proofreading in biosynthetic pathways. Biophys J 31, 333–358, doi:10.1016/S0006-3495(80)85063-6 (1980). PubMed DOI PMC

Pape T, Wintermeyer W & Rodnina M Induced fit in initial selection and proofreading of aminoacyl-tRNA on the ribosome. Embo J 18, 3800–3807 (1999). PubMed PMC

Fischer N et al. Structure of the E. coli ribosome-EF-Tu complex at <3 A resolution by Cs-corrected cryo-EM. Nature 520, 567–570, doi:10.1038/nature14275 (2015). PubMed DOI

Schmeing TM et al. The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA. Science 326, 688–694 (2009). PubMed PMC

Noel JK & Whitford PC How EF-Tu can contribute to efficient proofreading of aa-tRNA by the ribosome. Nat Commun 7, 13314, doi:10.1038/ncomms13314 (2016). PubMed DOI PMC

Sanbonmatsu KY, Joseph S & Tung CS Simulating movement of tRNA into the ribosome during decoding. Proc Natl Acad Sci U S A 102, 15854–15859, doi:0503456102 [pii] 10.1073/pnas.0503456102 (2005). PubMed DOI PMC

Abeyrathne PD, Koh CS, Grant T, Grigorieff N & Korostelev AA Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome. Elife 5, doi:10.7554/eLife.14874 (2016). PubMed DOI PMC

Thompson RC, Dix DB, Gerson RB & Karim AM A GTPase reaction accompanying the rejection of Leu-tRNA2 by UUU-programmed ribosomes. Proofreading of the codon-anticodon interaction by ribosomes. J Biol Chem 256, 81–86 (1981). PubMed

Dunkle JA et al. Structures of the bacterial ribosome in classical and hybrid states of tRNA binding. Science 332, 981–984, doi:332/6032/981 [pii] 10.1126/science.1202692 (2011). PubMed DOI PMC

Korostelev A, Trakhanov S, Laurberg M & Noller HF Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements. Cell 126, 1065–1077 (2006). PubMed

Ieong KW, Uzun U, Selmer M & Ehrenberg M Two proofreading steps amplify the accuracy of genetic code translation. Proc Natl Acad Sci U S A 113, 13744–13749, doi:10.1073/pnas.1610917113 (2016). PubMed DOI PMC

Yang H, Perrier J & Whitford PC Disorder guides domain rearrangement in elongation factor Tu. Proteins 86, 1037–1046, doi:10.1002/prot.25575 (2018). PubMed DOI

Kothe U & Rodnina MV Delayed release of inorganic phosphate from elongation factor Tu following GTP hydrolysis on the ribosome. Biochemistry 45, 12767–12774, doi:10.1021/bi061192z (2006). PubMed DOI

Kavaliauskas D et al. Structural dynamics of translation elongation factor Tu during aa-tRNA delivery to the ribosome. Nucleic Acids Res 46, 8651–8661, doi:10.1093/nar/gky651 (2018). PubMed DOI PMC

Berchtold H et al. Crystal structure of active elongation factor Tu reveals major domain rearrangements. Nature 365, 126–132, doi:10.1038/365126a0 (1993). PubMed DOI

Polekhina G et al. Helix unwinding in the effector region of elongation factor EF-Tu–GDP. Structure 4, 1141–1151, doi:10.1016/S0969-2126(96)00122-0 (1996). PubMed DOI

Kjeldgaard M, Nissen P, Thirup S & Nyborg J The crystal structure of elongation factor EF-Tu from Thermus aquaticus in the GTP conformation. Structure 1, 35–50 (1993). PubMed

Voorhees RM, Schmeing TM, Kelley AC & Ramakrishnan V The mechanism for activation of GTP hydrolysis on the ribosome. Science 330, 835–838, doi:330/6005/835 [pii] 10.1126/science.1194460 (2010). PubMed DOI PMC

Kothe U, Wieden HJ, Mohr D & Rodnina MV Interaction of helix D of elongation factor Tu with helices 4 and 5 of protein L7/12 on the ribosome. J Mol Biol 336, 1011–1021, doi:10.1016/j.jmb.2003.12.080 (2004). PubMed DOI

Schuette JC et al. GTPase activation of elongation factor EF-Tu by the ribosome during decoding. EMBO J 28, 755–765, doi:10.1038/emboj.2009.26 (2009). PubMed DOI PMC

Villa E et al. Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis. Proc Natl Acad Sci U S A 106, 1063–1068, doi:0811370106 [pii] 10.1073/pnas.0811370106 (2009). PubMed DOI PMC

Pape T, Wintermeyer W & Rodnina MV Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome. Embo J 17, 7490–7497 (1998). PubMed PMC

Hausner TP, Atmadja J & Nierhaus KH Evidence that the G2661 region of 23S rRNA is located at the ribosomal binding sites of both elongation factors. Biochimie 69, 911–923 (1987). PubMed

Moazed D, Robertson JM & Noller HF Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA. Nature 334, 362–364, doi:10.1038/334362a0 (1988). PubMed DOI

Daviter T, Wieden HJ & Rodnina MV Essential role of histidine 84 in elongation factor Tu for the chemical step of GTP hydrolysis on the ribosome. J Mol Biol 332, 689–699 (2003). PubMed

Maracci C, Peske F, Dannies E, Pohl C & Rodnina MV Ribosome-induced tuning of GTP hydrolysis by a translational GTPase. Proc Natl Acad Sci U S A 111, 14418–14423, doi:10.1073/pnas.1412676111 (2014). PubMed DOI PMC

Koripella RK et al. A conserved histidine in switch-II of EF-G moderates release of inorganic phosphate. Sci Rep 5, 12970, doi:10.1038/srep12970 (2015). PubMed DOI PMC

Ogle JM et al. Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science 292, 897–902 (2001). PubMed

Ogle JM & Ramakrishnan V Structural insights into translational fidelity. Annu Rev Biochem 74, 129–177, doi:10.1146/annurev.biochem.74.061903.155440 (2005). PubMed DOI

Demeshkina N, Jenner L, Westhof E, Yusupov M & Yusupova G A new understanding of the decoding principle on the ribosome. Nature 484, 256–259, doi:10.1038/nature10913 (2012). PubMed DOI

Whitford PC et al. Accommodation of aminoacyl-tRNA into the ribosome involves reversible excursions along multiple pathways. RNA 16, 1196–1204, doi:rna.2035410 [pii] 10.1261/rna.2035410 (2010). PubMed DOI PMC

Jenner L, Demeshkina N, Yusupova G & Yusupov M Structural rearrangements of the ribosome at the tRNA proofreading step. Nat Struct Mol Biol 17, 1072–1078 (2010). PubMed

Frank J & Agrawal RK A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature 406, 318–322 (2000). PubMed

Noller HF, Lancaster L, Zhou J & Mohan S The ribosome moves: RNA mechanics and translocation. Nat Struct Mol Biol 24, 1021–1027, doi:10.1038/nsmb.3505 (2017). PubMed DOI PMC

Zhang J, Pavlov MY & Ehrenberg M Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions. Nucleic Acids Res 46, 1362–1374, doi:10.1093/nar/gkx1256 (2018). PubMed DOI PMC

Zhang J, Ieong KW, Johansson M & Ehrenberg M Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome. Proc Natl Acad Sci U S A 112, 9602–9607, doi:10.1073/pnas.1506823112 (2015). PubMed DOI PMC

Gromadski KB & Rodnina MV Kinetic determinants of high-fidelity tRNA discrimination on the ribosome. Mol Cell 13, 191–200 (2004). PubMed

Johansson M, Bouakaz E, Lovmar M & Ehrenberg M The kinetics of ribosomal peptidyl transfer revisited. Mol Cell 30, 589–598, doi:10.1016/j.molcel.2008.04.010 (2008). PubMed DOI

Fu Z et al. Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy. Structure 24, 2092–2101, doi:10.1016/j.str.2016.09.014 (2016). PubMed DOI PMC

Kaledhonkar S et al. Late steps in bacterial translation initiation visualized using time-resolved cryo-EM. Nature 570, 400–404, doi:10.1038/s41586-019-1249-5 (2019). PubMed DOI PMC

Nikolay R et al. Structural Visualization of the Formation and Activation of the 50S Ribosomal Subunit during In Vitro Reconstitution. Mol Cell 70, 881–893 e883, doi:10.1016/j.molcel.2018.05.003 (2018). PubMed DOI

Graf M et al. Visualization of translation termination intermediates trapped by the Apidaecin 137 peptide during RF3-mediated recycling of RF1. Nat Commun 9, 3053, doi:10.1038/s41467-018-05465-1 (2018). PubMed DOI PMC

Svidritskiy E & Korostelev AA Conformational control of translation termination on the 70S ribosome. Structure 26, 821–828 (2018). PubMed PMC

Lancaster L & Noller HF Involvement of 16S rRNA nucleotides G1338 and A1339 in discrimination of initiator tRNA. Mol Cell 20, 623–632, doi:S1097–2765(05)01676-X [pii] 10.1016/j.molcel.2005.10.006 (2005). PubMed DOI

Walker SE & Fredrick K Preparation and evaluation of acylated tRNAs. Methods 44, 81–86, doi:10.1016/j.ymeth.2007.09.003 (2008). PubMed DOI PMC

Mastronarde DN Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol 152, 36–51, doi:10.1016/j.jsb.2005.07.007 (2005). PubMed DOI

Kremer JR, Mastronarde DN & McIntosh JR Computer Visualization of Three-Dimensional Image Data Using IMOD. J. Struct. Biol 116, 71–76, doi:10.1006/jsbi.1996.0013 (1996). PubMed DOI

Rohou A & Grigorieff N CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol 192, 216–221, doi:10.1016/j.jsb.2015.08.008 (2015). PubMed DOI PMC

Chen JZ & Grigorieff N SIGNATURE: A single-particle selection system for molecular electron microscopy. J. Struct. Biol 157, 168–173, doi:10.1016/j.jsb.2006.06.001 (2007). PubMed DOI

Gabashvili IS et al. Solution Structure of the E. coli 70S Ribosome at 11.5 Å Resolution. Cell 100, 537–549, doi:10.1016/S0092-8674(00)80690-X (2000). PubMed DOI

Tang G et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol 157, 38–46, doi:10.1016/j.jsb.2006.05.009 (2007). PubMed DOI

Lyumkis D, Brilot AF, Theobald DL & Grigorieff N Likelihood-based classification of cryo-EM images using FREALIGN. J. Struct. Biol 183, 377–388, doi:10.1016/j.jsb.2013.07.005 (2013). PubMed DOI PMC

Wieden HJ, Wintermeyer W & Rodnina MV A common structural motif in elongation factor Ts and ribosomal protein L7/12 may be involved in the interaction with elongation factor Tu. J Mol Evol 52, 129–136 (2001). PubMed

Cornish PV, Ermolenko DN, Noller HF & Ha T Spontaneous intersubunit rotation in single ribosomes. Mol Cell 30, 578–588, doi:S1097–2765(08)00334–1 [pii] 10.1016/j.molcel.2008.05.004 (2008). PubMed DOI PMC

Jenner LB, Demeshkina N, Yusupova G & Yusupov M Structural aspects of messenger RNA reading frame maintenance by the ribosome. Nat Struct Mol Biol 17, 555–560 (2010). PubMed

Nierhaus KH The allosteric three-site model for the ribosomal elongation cycle: features and future. Biochemistry 29, 4997–5008 (1990). PubMed

Dinos G, Kalpaxis DL, Wilson DN & Nierhaus KH Deacylated tRNA is released from the E site upon A site occupation but before GTP is hydrolyzed by EF-Tu. Nucleic Acids Res 33, 5291–5296, doi:10.1093/nar/gki833 (2005). PubMed DOI PMC

Semenkov YP, Rodnina MV & Wintermeyer W The “allosteric three-site model” of elongation cannot be confirmed in a well-defined ribosome system from Escherichia coli. Proc Natl Acad Sci U S A 93, 12183–12188, doi:10.1073/pnas.93.22.12183 (1996). PubMed DOI PMC

Petropoulos AD & Green R Further in vitro exploration fails to support the allosteric three-site model. J Biol Chem 287, 11642–11648, doi:10.1074/jbc.C111.330068 (2012). PubMed DOI PMC

Uemura S et al. Real-time tRNA transit on single translating ribosomes at codon resolution. Nature 464, 1012–1017 (2010). PubMed PMC

Grant T, Rohou A & Grigorieff N cisTEM, user-friendly software for single-particle image processing. Elife 7, doi:10.7554/eLife.35383 (2018). PubMed DOI PMC

Zivanov J et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7, doi:10.7554/eLife.42166 (2018). PubMed DOI PMC

Passos DO & Lyumkis D Single-particle cryoEM analysis at near-atomic resolution from several thousand asymmetric subunits. J Struct Biol 192, 235–244, doi:10.1016/j.jsb.2015.10.002 (2015). PubMed DOI

Cardone G, Heymann JB & Steven AC One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions. J Struct Biol 184, 226–236, doi:S1047–8477(13)00208–6 [pii] 10.1016/j.jsb.2013.08.002 (2013). PubMed DOI PMC

Abel K, Yoder MD, Hilgenfeld R & Jurnak F An alpha to beta conformational switch in EF-Tu. Structure 4, 1153–1159 (1996). PubMed

Polikanov YS, Steitz TA & Innis CA A proton wire to couple aminoacyl-tRNA accommodation and peptide-bond formation on the ribosome. Nat Struct Mol Biol 21, 787–793, doi:nsmb.2871 [pii] 10.1038/nsmb.2871 (2014). PubMed DOI PMC

Jin H, Kelley AC & Ramakrishnan V Crystal structure of the hybrid state of ribosome in complex with the guanosine triphosphatase release factor 3. Proc Natl Acad Sci U S A 108, 15798–15803, doi:10.1073/pnas.1112185108 (2011). PubMed DOI PMC

Pettersen EF et al. UCSF Chimera—A visualization system for exploratory research and analysis. Journal of Computational Chemistry 25, 1605–1612, doi:10.1002/jcc.20084 (2004). PubMed DOI

Korostelev A, Bertram R & Chapman MS Simulated-annealing real-space refinement as a tool in model building. Acta Crystallogr D Biol Crystallogr 58, 761–767 (2002). PubMed

Chapman MS Restrained real-space macromolecular atomic refinement using a new resolution-dependent electron-density function. Acta Crystallographica Section A 51, 69–80, doi:10.1107/S0108767394007130 (1995). DOI

DeLano WL The PyMOL Molecular Graphics System. (DeLano Scientific, 2002).

Emsley P & Cowtan K Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126–2132 (2004). PubMed

Zhou G, Wang J, Blanc E & Chapman MS Determination of the relative precision of atoms in a macromolecular structure. Acta Crystallographica. Section D, Biological Crystallography 54, 391–399 (1998). PubMed

Laurberg M et al. Structural basis for translation termination on the 70S ribosome. Nature 454, 852–857 (2008). PubMed

Adams PD et al. The Phenix software for automated determination of macromolecular structures. Methods (San Diego, Calif.) 55, 94–106, doi:10.1016/j.ymeth.2011.07.005 (2011). PubMed DOI PMC

Gao YG et al. The structure of the ribosome with elongation factor G trapped in the posttranslocational state. Science 326, 694–699 (2009). PubMed PMC

Diaconu M et al. Structural basis for the function of the ribosomal L7/12 stalk in factor binding and GTPase activation. Cell 121, 991–1004, doi:10.1016/j.cell.2005.04.015 (2005). PubMed DOI

Leijonmarck M & Liljas A Structure of the C-terminal domain of the ribosomal protein L7/L12 from Escherichia coli at 1.7 A. J Mol Biol 195, 555–579 (1987). PubMed

Ribosome inhibition by C9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP

Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation