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Cryo-EM of elongating ribosome with EF-Tu•GTP elucidates tRNA proofreading

Abstract

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 challenge2,3,4. 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.

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Fig. 1: Cryo-EM of an elongation event reveals structural intermediates.
Fig. 2: EF-Tu and ribosome rearrangements during mRNA decoding.
Fig. 3: Differences between cognate and near-cognate tRNA accommodation.
Fig. 4: Schematic of mRNA decoding.

Data availability

The models generated and analysed during the current study are available from the RCSB PDB: 6WD0 (structure I-A), 6WD1 (structure I-B), 6WD2 (structure II-A), 6WD3 (structure II-B1), 6WD4 (structure II-B2), 6WD5 (structure II-C1), 6WD6 (structure II-C2), 6WD7 (structure II-D), 6WD8 (structure III-A), 6WD9 (structure III-B), 6WDA (structure III-C), 6WDB (structure IV-A), 6WDC (structure IV-B), 6WDD (structure V-A), 6WDE (structure V-B), 6WDF (structure VI-A), 6WDG (structure VI-B), 6WDH (structure IV-B1-nc), 6WDI (structure IV-B2-nc), 6WDJ (structure V-A1-nc), 6WDK (structure V-A2-nc), 6WDL (structure V-B1-nc) and 6WDM (structure V-B2-nc). The cryo-EM maps used to generate models are available from the EMDB: EMD-21619 (structure I-A), EMD-21620 (structure I-B), EMD-21621 (structure II-A), EMD-21622 (structure II-B1), EMD-21623 (structure II-B2), EMD-21624 (structure II-C1), EMD-21625 (structure II-C2), EMD-21626 (structure II-D), EMD-21627 (structure III-A), EMD-21628 (structure III-B), EMD-21629 (structure III-C), EMD-21630 (structure IV-A), EMD-21631 (structure IV-B), EMD-21632 (structure V-A), EMD-21633 (structure V-B), EMD-21634 (structure VI-A), EMD-21635 (structure VI-B), EMD-21636 (structure IV-B1-nc), EMD-21637 (structure IV-B2-nc), EMD-21638 (structure V-A1-nc), EMD-21639 (structure V-A2-nc), EMD-21640 (structure V-B1-nc) and EMD-21641 (structure V-B2-nc).

References

  1. Hopfield, J. J. Kinetic proofreading: a new mechanism for reducing errors in biosynthetic processes requiring high specificity. Proc. Natl Acad. Sci. USA 71, 4135–4139 (1974).

    ADS  PubMed  CAS  Article  PubMed Central  Google Scholar 

  2. Voorhees, R. M. & Ramakrishnan, V. Structural basis of the translational elongation cycle. Annu. Rev. Biochem. 82, 203–236 (2013).

    PubMed  CAS  Article  Google Scholar 

  3. Pavlov, M. Y. & Ehrenberg, M. Substrate-induced formation of ribosomal decoding center for accurate and rapid genetic code translation. Annu. Rev. Biophys. 47, 525–548 (2018).

    PubMed  CAS  Article  Google Scholar 

  4. Rodnina, M. V., Fischer, N., Maracci, C. & Stark, H. Ribosome dynamics during decoding. Phil. Trans. R. Soc. Lond. B 372, 20160182 (2017).

    Article  CAS  Google Scholar 

  5. Loveland, A. B., Demo, G., Grigorieff, N. & Korostelev, A. A. Ensemble cryo-EM elucidates the mechanism of translation fidelity. Nature 546, 113–117 (2017).

    ADS  PubMed  PubMed Central  CAS  Article  Google Scholar 

  6. 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 (2018).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  7. Moazed, D. & Noller, H. F. Intermediate states in the movement of transfer RNA in the ribosome. Nature 342, 142–148 (1989).

    ADS  PubMed  CAS  Article  Google Scholar 

  8. Yusupov, M. M. et al. Crystal structure of the ribosome at 5.5 Å resolution. Science 292, 883–896 (2001).

    ADS  PubMed  CAS  Article  Google Scholar 

  9. Stark, H. et al. Visualization of elongation factor Tu on the Escherichia coli ribosome. Nature 389, 403–406 (1997).

    ADS  PubMed  CAS  Article  Google Scholar 

  10. Ehrenberg, M. & Blomberg, C. Thermodynamic constraints on kinetic proofreading in biosynthetic pathways. Biophys. J. 31, 333–358 (1980).

    ADS  PubMed  PubMed Central  CAS  Article  Google Scholar 

  11. 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  PubMed Central  CAS  Article  Google Scholar 

  12. Fischer, N. et al. Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM. Nature 520, 567–570 (2015).

    ADS  PubMed  Article  CAS  Google Scholar 

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

    ADS  PubMed  PubMed Central  CAS  Article  Google Scholar 

  14. Noel, J. K. & Whitford, P. C. How EF-Tu can contribute to efficient proofreading of aa-tRNA by the ribosome. Nat. Commun. 7, 13314 (2016).

    ADS  PubMed  PubMed Central  CAS  Article  Google Scholar 

  15. Sanbonmatsu, K. Y., Joseph, S. & Tung, C. S. Simulating movement of tRNA into the ribosome during decoding. Proc. Natl Acad. Sci. USA 102, 15854–15859 (2005).

    ADS  PubMed  CAS  Article  PubMed Central  Google Scholar 

  16. Abeyrathne, P. D., Koh, C. S., Grant, T., Grigorieff, N. & Korostelev, A. A. Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome. eLife 5, e14874 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  17. Thompson, R. C., Dix, D. B., Gerson, R. B. & Karim, A. M. A GTPase reaction accompanying the rejection of Leu-tRNA2 by UUU-programmed ribosomes. J. Biol. Chem. 256, 81–86 (1981).

    PubMed  CAS  Article  Google Scholar 

  18. Dunkle, J. A. et al. Structures of the bacterial ribosome in classical and hybrid states of tRNA binding. Science 332, 981–984 (2011).

    ADS  PubMed  PubMed Central  CAS  Article  Google Scholar 

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

    PubMed  CAS  Article  Google Scholar 

  20. Ieong, K. W., Uzun, Ü., Selmer, M. & Ehrenberg, M. Two proofreading steps amplify the accuracy of genetic code translation. Proc. Natl Acad. Sci. USA 113, 13744–13749 (2016).

    PubMed  CAS  Article  PubMed Central  Google Scholar 

  21. Yang, H., Perrier, J. & Whitford, P. C. Disorder guides domain rearrangement in elongation factor Tu. Proteins 86, 1037–1046 (2018).

    PubMed  CAS  Article  Google Scholar 

  22. Kothe, U. & Rodnina, M. V. Delayed release of inorganic phosphate from elongation factor Tu following GTP hydrolysis on the ribosome. Biochemistry 45, 12767–12774 (2006).

    PubMed  CAS  Article  Google Scholar 

  23. Kavaliauskas, D. et al. Structural dynamics of translation elongation factor Tu during aa-tRNA delivery to the ribosome. Nucleic Acids Res. 46, 8651–8661 (2018).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  24. Berchtold, H. et al. Crystal structure of active elongation factor Tu reveals major domain rearrangements. Nature 365, 126–132 (1993).

    ADS  PubMed  CAS  Article  Google Scholar 

  25. Polekhina, G. et al. Helix unwinding in the effector region of elongation factor EF-Tu-GDP. Structure 4, 1141–1151 (1996).

    PubMed  CAS  Article  Google Scholar 

  26. 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  CAS  Article  Google Scholar 

  27. Voorhees, R. M., Schmeing, T. M., Kelley, A. C. & Ramakrishnan, V. The mechanism for activation of GTP hydrolysis on the ribosome. Science 330, 835–838 (2010).

    ADS  PubMed  PubMed Central  CAS  Article  Google Scholar 

  28. Kothe, U., Wieden, H. J., Mohr, D. & Rodnina, M. V. 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 (2004).

    PubMed  CAS  Article  Google Scholar 

  29. Schuette, J. C. et al. GTPase activation of elongation factor EF-Tu by the ribosome during decoding. EMBO J. 28, 755–765 (2009).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  30. Villa, E. et al. Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis. Proc. Natl Acad. Sci. USA 106, 1063–1068 (2009).

    ADS  PubMed  CAS  Article  PubMed Central  Google Scholar 

  31. Pape, T., Wintermeyer, W. & Rodnina, M. V. 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  PubMed Central  CAS  Article  Google Scholar 

  32. Hausner, T. P., Atmadja, J. & Nierhaus, K. H. 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  CAS  Article  Google Scholar 

  33. Moazed, D., Robertson, J. M. & Noller, H. F. Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA. Nature 334, 362–364 (1988).

    ADS  PubMed  CAS  Article  Google Scholar 

  34. Daviter, T., Wieden, H. J. & Rodnina, M. V. 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  CAS  Article  Google Scholar 

  35. Maracci, C., Peske, F., Dannies, E., Pohl, C. & Rodnina, M. V. Ribosome-induced tuning of GTP hydrolysis by a translational GTPase. Proc. Natl Acad. Sci. USA 111, 14418–14423 (2014).

    ADS  PubMed  CAS  Article  PubMed Central  Google Scholar 

  36. Koripella, R. K. et al. A conserved histidine in switch-II of EF-G moderates release of inorganic phosphate. Sci. Rep. 5, 12970 (2015).

    ADS  PubMed  Article  CAS  Google Scholar 

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

    ADS  PubMed  CAS  Article  Google Scholar 

  38. Ogle, J. M. & Ramakrishnan, V. Structural insights into translational fidelity. Annu. Rev. Biochem. 74, 129–177 (2005).

    PubMed  CAS  Article  Google Scholar 

  39. Demeshkina, N., Jenner, L., Westhof, E., Yusupov, M. & Yusupova, G. A new understanding of the decoding principle on the ribosome. Nature 484, 256–259 (2012).

    ADS  PubMed  CAS  Article  Google Scholar 

  40. Whitford, P. C. et al. Accommodation of aminoacyl-tRNA into the ribosome involves reversible excursions along multiple pathways. RNA 16, 1196–1204 (2010).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  41. 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  CAS  Article  Google Scholar 

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

    ADS  PubMed  CAS  Article  Google Scholar 

  43. Noller, H. F., Lancaster, L., Zhou, J. & Mohan, S. The ribosome moves: RNA mechanics and translocation. Nat. Struct. Mol. Biol. 24, 1021–1027 (2017).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  44. Zhang, J., Pavlov, M. Y. & Ehrenberg, M. Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions. Nucleic Acids Res. 46, 1362–1374 (2018).

    PubMed  CAS  Article  Google Scholar 

  45. Zhang, J., Ieong, K. W., Johansson, M. & Ehrenberg, M. Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome. Proc. Natl Acad. Sci. USA 112, 9602–9607 (2015).

    ADS  PubMed  CAS  Article  PubMed Central  Google Scholar 

  46. Gromadski, K. B. & Rodnina, M. V. Kinetic determinants of high-fidelity tRNA discrimination on the ribosome. Mol. Cell 13, 191–200 (2004).

    PubMed  CAS  Article  Google Scholar 

  47. Johansson, M., Bouakaz, E., Lovmar, M. & Ehrenberg, M. The kinetics of ribosomal peptidyl transfer revisited. Mol. Cell 30, 589–598 (2008).

    PubMed  CAS  Article  Google Scholar 

  48. Fu, Z. et al. Key intermediates in ribosome recycling visualized by time-resolved cryoelectron microscopy. Structure 24, 2092–2101 (2016).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  49. Kaledhonkar, S. et al. Late steps in bacterial translation initiation visualized using time-resolved cryo-EM. Nature 570, 400–404 (2019).

    ADS  PubMed  PubMed Central  CAS  Article  Google Scholar 

  50. 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.e3 (2018).

    PubMed  CAS  Article  Google Scholar 

  51. 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 (2018).

    ADS  PubMed  PubMed Central  Article  CAS  Google Scholar 

  52. Svidritskiy, E. & Korostelev, A. A. Conformational control of translation termination on the 70S ribosome. Structure 26, 821–828.e3 (2018).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  53. Lancaster, L. & Noller, H. F. Involvement of 16S rRNA nucleotides G1338 and A1339 in discrimination of initiator tRNA. Mol. Cell 20, 623–632 (2005).

    PubMed  CAS  Article  Google Scholar 

  54. Walker, S. E. & Fredrick, K. Preparation and evaluation of acylated tRNAs. Methods 44, 81–86 (2008).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  55. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    PubMed  Article  Google Scholar 

  56. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    PubMed  CAS  Article  Google Scholar 

  57. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    PubMed  PubMed Central  Article  Google Scholar 

  58. Chen, J. Z. & Grigorieff, N. SIGNATURE: a single-particle selection system for molecular electron microscopy. J. Struct. Biol. 157, 168–173 (2007).

    PubMed  CAS  Article  Google Scholar 

  59. Gabashvili, I. S. et al. Solution structure of the E. coli 70S ribosome at 11.5 A resolution. Cell 100, 537–549 (2000).

    PubMed  CAS  Article  Google Scholar 

  60. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    PubMed  CAS  Article  Google Scholar 

  61. Lyumkis, D., Brilot, A. F., Theobald, D. L. & Grigorieff, N. Likelihood-based classification of cryo-EM images using FREALIGN. J. Struct. Biol. 183, 377–388 (2013).

    PubMed  CAS  Article  Google Scholar 

  62. Wieden, H. J., Wintermeyer, W. & Rodnina, M. V. 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).

    ADS  PubMed  CAS  Article  Google Scholar 

  63. Cornish, P. V., Ermolenko, D. N., Noller, H. F. & Ha, T. Spontaneous intersubunit rotation in single ribosomes. Mol. Cell 30, 578–588 (2008).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  64. Jenner, L. B., 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  CAS  Article  Google Scholar 

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

    PubMed  CAS  Article  Google Scholar 

  66. Dinos, G., Kalpaxis, D. L., Wilson, D. N. & Nierhaus, K. H. 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 (2005).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  67. Semenkov, Y. P., Rodnina, M. V. & 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. USA 93, 12183–12188 (1996).

    ADS  PubMed  CAS  Article  PubMed Central  Google Scholar 

  68. Petropoulos, A. D. & Green, R. Further in vitro exploration fails to support the allosteric three-site model. J. Biol. Chem. 287, 11642–11648 (2012).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

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

    ADS  PubMed  PubMed Central  CAS  Article  Google Scholar 

  70. Grant, T., Rohou, A. & Grigorieff, N. cisTEM, user-friendly software for single-particle image processing. eLife 7, e35383 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  71. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  72. Passos, D. O. & Lyumkis, D. Single-particle cryoEM analysis at near-atomic resolution from several thousand asymmetric subunits. J. Struct. Biol. 192, 235–244 (2015).

    PubMed  CAS  Article  Google Scholar 

  73. Cardone, G., Heymann, J. B. & Steven, A. C. One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions. J. Struct. Biol. 184, 226–236 (2013).

    PubMed  Article  Google Scholar 

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

    PubMed  CAS  Article  Google Scholar 

  75. Polikanov, Y. S., Steitz, T. A. & Innis, C. A. A proton wire to couple aminoacyl-tRNA accommodation and peptide-bond formation on the ribosome. Nat. Struct. Mol. Biol. 21, 787–793 (2014).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  76. Jin, H., Kelley, A. C. & Ramakrishnan, V. Crystal structure of the hybrid state of ribosome in complex with the guanosine triphosphatase release factor 3. Proc. Natl Acad. Sci. USA 108, 15798–15803 (2011).

    ADS  PubMed  CAS  Article  PubMed Central  Google Scholar 

  77. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    PubMed  CAS  Google Scholar 

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

    PubMed  Article  CAS  Google Scholar 

  79. Chapman, M. S. Restrained real-space macromolecular atomic refinement using a new resolution-dependent electron-density function. Acta Crystallogr. A 51, 69–80 (1995).

    Article  Google Scholar 

  80. DeLano, W. L. The PyMOL Molecular Graphics System. (DeLano Scientific, 2002).

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

    PubMed  Article  CAS  Google Scholar 

  82. Zhou, G., Wang, J., Blanc, E. & Chapman, M. S. Determination of the relative precision of atoms in a macromolecular structure. Acta Crystallogr. D 54, 391–399 (1998).

    PubMed  CAS  Article  Google Scholar 

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

    ADS  PubMed  CAS  Article  Google Scholar 

  84. Adams, P. D. et al. The Phenix software for automated determination of macromolecular structures. Methods 55, 94–106 (2011).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

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

    ADS  PubMed  PubMed Central  CAS  Article  Google Scholar 

  86. 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 (2005).

    PubMed  CAS  Article  Google Scholar 

  87. 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  CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank M. Rigney for help with grid preparation and screening at the cryo-EM facility at Brandeis University; C. Xu and K. Song for data collection at the cryo-EM facility at UMass Medical School; D. Conte Jr., D. Ermolenko, A. Korennykh and members of the Korostelev laboratory for comments on the manuscript; and D. Grunwald and D. Susorov for help with the video. This study was supported by NIH grants R01 GM106105, R01 GM107465 and R35 GM127094 (to A.A.K.). A.B.L. performed part of this work as a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation.

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Authors and Affiliations

Authors

Contributions

A.B.L. and A.A.K. developed the concept of the work. A.B.L., G.D. and A.A.K. designed the methodology. A.B.L. and A.A.K. validated the methodology. A.B.L. and G.D. carried out the experiments. A.A.K. provided resources. A.B.L. and A.A.K. wrote the original draft of the manuscript; A.B.L., G.D. and A.A.K. reviewed and edited the manuscript. A.B.L. generated graphics and figures for the manuscript. A.A.K. acquired funding and supervised the project.

Corresponding author

Correspondence to Andrei A. Korostelev.

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The authors declare no competing interests.

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Peer review information Nature thanks Yves Mechulam and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Classification procedure and FSC curves for maps of cognate ternary complex decoding.

a, Scheme of the maximum-likelihood classification strategy to obtain the final maps and state occupancies for the cognate 29-s dataset. Transparency for EF-Tu and line-thickness for tRNA depict full/partial occupancy or strong/weak features. Gray shading in 30S subunit indicates the closed 30S subunit conformation. Brackets with red multiplication signs (e.g. x3), indicate the number of states that recur, and in such cases the lowest and highest resolution is listed along with a summed occupancy for all states. b, EF-Tu-bound particles from 17 s, 29 s and 120 s were processed together to obtain final maps for states II-A to III-C. cf, Fourier shell correlations (FSC) between even- and odd-particle half maps show that average resolutions range from 3.0 to 4.0 Å for modelled maps. c, Substrate ribosome states. d, EF-Tu-bound ribosome states with an open 30S subunit. e, EF-Tu-bound ribosome states with a closed 30S subunit. f, Accommodation and product ribosome states.

Extended Data Fig. 2 Classification procedure and FSC curves for maps of near-cognate ternary complex decoding.

a, Scheme depicts maximum-likelihood classification strategy to obtain the final maps and state occupancies for the near-cognate 30-s dataset. b, Fourier shell correlation (FSC) between even- and odd-particle half maps for near-cognate ribosome states that were modelled. c, Cryo-EM maps for 16 states of the elongation reaction with near-cognate tRNA, and their assignment as substrates, EF-Tu-bound intermediates, or products of the reaction. The maps are coloured to show the 50S ribosomal subunit (light blue), 30S ribosomal subunit (yellow), E-tRNA (orange), P-tRNA (dark blue), near-cognate A-tRNA (red), and EF-Tu (magenta).

Extended Data Fig. 3 Cryo-EM density and interactions of EF-Tu.

ar, Density for EF-Tu in 30S-open (II) and 30S-closed (III) conformations is shown relative to the sarcin-ricin loop (SRL) (ac, gi and mo) or at the tRNA interacting face (df, jl and pr; tRNA is omitted for clarity). Maps were filtered according to local resolution, using blocfilt (Methods). Numbers 1–3 indicate domains of EF-Tu. EF-Tu is shown in magenta with switch I region (Sw I) and switch II region (Sw II) shown in blue, tRNA is shown in green, 30S subunit is shown in gold, 50S subunit is shown in cyan. a, Map II-A (shown at 3σ). b, Map II-B1 (3.25σ). c, Map II-B2 (2.5σ). d, Map II-A has strong density for both switch I and switch II regions (2σ). e, Map II-B1 has weaker density for switch I (2σ). f, In map II-B2, density for switch I is missing even at lower contour levels (shown at 1.5σ). g, Map II-C1 (1σ). h, Map II-C2 (1σ). i, Map II-D has only domain 3 density at low contour levels (0.5σ). j, Map II-C1 has only weak density for switch I and switch II (0.25σ). k, Map II-C2 is missing ordered density for most of switch I (0.5σ). l, Map II-D has weak density only for domain 3 (0.5σ). m, Map III-A shows EF-Tu away from the SRL when the 30S closure is intermediate (2σ). n, Map III-B shows EF-Tu next to the SRL, when 30S subunit is in the closed conformation (2σ). o, Map III-C shows GTPase domain away from the SRL and rotated by approximately 90° relative to domain 2 (1.5σ). p, Map III-A has density for both switch I and switch II (1.5σ). q, In map III-B, density for both switch I and switch II is missing (1.5σ). r, Map III-C has weak density for switch I (1.25σ). s, Classification with a larger mask around EF-Tu reveals a map with weak density at helix D of domain 1, which corresponds to the binding site for the C terminus of L7/L12. Map was low-pass filtered to 6 Å and is shown at 1σ. t, The putative interaction of L7/L12 with domain 1 of EF-Tu (left) differs from that of L7/L12 with domain 1 of EF-G (right). Rigid-body fitted structure of EF-Tu and L7/L12 as in a was aligned to EF-G from PDB: 4V5F85 via GTPase domains. u, v, An independent classification strategy yielded a map (shown with 4× binning and at 0.75σ) with density sufficient to fit a dimer of the L7/L12 C-terminal domain. The density bridges domain 3 of EF-Tu with L11. Model for L7/L12 N terminus (green cyan) from PDB:1ZAX86 is shown on both panels. u, Tentative fit utilizes the L7/L12 dimer interface observed in the X-ray crystallographic structure (PDB: 1CTF87). v, Alternatively, two monomers may be docked independently (bottom based on PDB: 1CTF) and top based on PDB: 4V5F). w, Cryo-EM map II-C1 shows ordered density for A*/T tRNA when EF-Tu domain 1 undocks from the tRNA and SRL, while domain 2 (magenta) remains bound. Map is shown at 3σ for A*/T tRNA and 2σ for EF-Tu. x, Comparison between II-A and II-D shows that density is missing for A76 and Phe of Phe-tRNAPhe upon release of EF-Tu domain 2 in II-D. Right, map II-A, filtered via blocfilt and B-factor sharpened (−75 Å2), with ordered EF-Tu shows strong density for A76 and Phe (2.75σ). Left, map II-D was filtered via blocfilt and B-factor sharpened (−50 Å2) and is shown at 0.75σ to accentuate weak density. A*/T tRNA from II-A is shown in grey for reference after structural superposition of structures via 23S rRNA. y, Comparison of EF-Tu in structure III-A (resembling a pre-GTP-hydrolysis state, grey, only EF-Tu is shown) and III-B (post-GTP hydrolysis, coloured as in Fig. 1) reveals roles of the 30S shoulder in bringing EF-Tu towards the SRL and of L11 in optimally positioning EF-Tu for GTP hydrolysis. Alignment of structures was achieved by superposition of 23S rRNA. z, Superposition shows similarity of structure III-B (coloured) with 70S•Phe-tRNAPhe •EF-Tu stalled with GDPCP in previous work (PDB: 5UYM5).

Extended Data Fig. 4 Cryo-EM density of the decoding centre of the open and closed 30S conformations.

16S rRNA is shown in yellow, 23S rRNA is shown in cyan, codon is shown in magenta, cognate tRNA is shown in green and near-cognate tRNA is shown in red. All maps have been local-resolution-filtered using blocfilt and B-factor-sharpened (−75 Å2). a, Structure I-A with the decoding centre in an open conformation; 16S residues of helix 44 including residues 1492–1493 are shown at 2.5σ, G530 of the 16S is shown at 4σ, 23S rRNA residue A1913 at 2.5σ, and the weaker codon density is shown at 1.0σ. b, Structure II-A with the decoding centre in an open conformation in the presence of A*/T tRNA; the codon and A*/T tRNA are shown at 2.5σ, h44 is shown at 2.5σ, G530 is shown at 4.5σ, A1913 is shown at 3σ. c, Structure II-C1 with the decoding centre in an open conformation in the presence of A*/T tRNA and with EF-Tu in an extended, post-GTP hydrolysis conformation; the codon and A*/T tRNA are shown at 3.75σ, h44 is shown at 2.5σ, G530 is shown at 3σ, A1913 is shown at 2.5σ. d, Structure III-A with the decoding centre in an intermediate conformation in the presence of A*/T tRNA; the codon and A*/T tRNA are shown at 3.5σ, h44 is shown at 3.5σ, G530 is shown at 5σ and A1913 at 2.5σ. e, Structure III-B with a closed conformation of the decoding centre in the presence of A/T tRNA; the codon and A/T tRNA are shown at 4.5σ, h44 is shown at 4.5σ, G530 is shown at 5σ and A1913 is shown at 5σ. f, Structure IV-B with the decoding centre in a closed conformation during accommodation; the codon, EA-1 tRNA, A1913 and 16S h44 are shown at 5σ, and G530 is shown at 6σ. g, Structure V-B with the decoding centre in a closed conformation in the presence of the accommodated A/A tRNA; the codon and A/A tRNA, 23S rRNA and G530 are shown at 5σ, and h44 at 4.5σ. h, Density for the modified nucleotide at position 37, 2-methylthio-N6-(2-isopentenyl)-adenosine, of the anticodon in V-B (cryo-EM map was filtered with blocfilt and B-factor-sharpened (−100 Å2) and is shown at 4σ). i, Structure VI-B with the decoding centre in a closed conformation in the presence of A/P* tRNA; the codon, A/T tRNA, h44 and G530 are shown at 3.5σ, and A1913 at 3σ. j, Near-cognate structure V-A1-nc with the decoding centre in an open conformation in the presence of A*/A tRNA with the accommodated CCA end; the codon and A*/A tRNA are shown at 3.75σ, h44 and A1913 are shown at 3.5σ and G530 is shown at 5σ. k, Near-cognate structure V-A2-nc with the decoding centre in a closed conformation in the presence of A/A tRNA; the codon and A/A tRNA are shown at 6σ, h44 is shown at 5σ, G530 at 7σ and A1913 at 3σ.

Extended Data Fig. 5 Cryo-EM density for tRNAs during cognate and near-cognate tRNA decoding.

Cognate tRNA is shown in green, near-cognate tRNA is shown in red, 23S rRNA including ASF (residues 860:915), H89 (residues 2450–2500), and H90–H92 (residues 2513:2571) are shown in cyan, and the mRNA codon is shown in magenta. Cryo-EM maps were local-resolution-filtered using blocfilt. a, b, Structure II-D and cryo-EM map shown at 2σ. c, d, Structure IV-A and cryo-EM map shown at 3σ. e, f, Structure IV-B and cryo-EM map shown at 3σ. g, h Structure V-A and cryo-EM map shown at 3σ. i, j, Structure IV-B1-nc and cryo-EM map shown at 2σ. k, l, Structure IV-B2-nc and cryo-EM map shown at 2σ. m, n, Structure V-A1-nc and cryo-EM map shown at 3σ. o, p, Structure V-A2-nc and cryo-EM map shown at 3σ. q, Charging of tRNAPhe with phenylalanine, as assessed by 6.5% acid-PAGE and methylene-blue staining (see uncropped gel image in Supplementary Information). r, Charging of tRNAfMet by formyl-methionine (as in q).

Extended Data Fig. 6 30S rotation in accommodation states and tRNA conformations in peptidyl-transfer states.

a, In state V-A, with a partially rotated 30S subunit, cryo-EM density is consistent with substrate aminoacyl-tRNAs. Cryo-EM map was filtered with blocfilt and B-factor-sharpened (−50 Å2) and is shown at 2.5σ. b, In state V-B, which is less rotated, cryo-EM density is consistent with product dipeptidyl-tRNA. Cryo-EM map was filtered with blocfilt and B-factor-sharpened (−50 Å2) and is shown at 3.5σ. c, Change of the 70S conformation in accommodation intermediates over time. The 30S subunit rotation angle decreases with time in ribosome states with A/A-like tRNA, consistent with accommodation completion in a non-rotated state. Rigid-body docking of 50S subunit, 30S head, 30S shoulder, and 30S body was performed into the cryo-EM maps obtained for the 17- and 120-s time points. Superposition with the 70S•tRNA•EF-Tu•GDPCP structure (PDB: 5UYM5,) was achieved by structural alignment of 23S rRNA. Rotation of the 30S body rRNA versus that of non-rotated (PDB: 5UYM) was determined in Chimera. dg, Superpositions of the aminoacyl moieties in V-A and V-B with crystallographic structures of the T. th. 70S ribosome captured with substrate analogues (PDB: 1VY4) and product analogue (PDB: 1VY5)75. d, e, Substrate Phe-tRNAPhe in A site (green) and fMet-tRNAfMet (blue) in V-A are shown compared to two 70S structures in the asymmetric unit of PDB: 1VY4 (grey). Superposition was achieved by structural alignment of 23S rRNA. f, g, Product dipeptidyl fMet-Phe-tRNAPhe in the A site (green) and deacylated P-tRNAfMet (blue) in V-B are shown compared to two 70S structures in the asymmetric unit of PDB: 1VY5 (grey). In V-B, formyl group was not resolved. h, P*/P tRNA elbow (blue) in a partially rotated 30S conformation (V-A) is displaced by 7-11 Å towards the E site, relative to its position in the classical-state P/P tRNA (grey; V-B). Superposition of Structures V-A and V-B was achieved by structural alignment of 23S rRNA. i, P*/P tRNA (blue) is shown relative to the E-site tRNA (orange) in structure V-A. j, k, Particle classification yields E-site tRNA-bound (j) and vacant (k) particles with similar conformations of 30S-domain-closed complexes. Cryo-EM maps with or without E-tRNA (see Methods) were low-pass filtered to 4 Å and B-factor-softened (50 Å2) and are shown at 3.2σ. l, m, Particle classification yields E-site tRNA-bound (l) and vacant (m) particles with similar conformations of 30S-domain-open complexes. Cryo-EM maps with or without E-tRNA (see Methods) prepared as in j, k are shown at 3σ.

Extended Data Fig. 7 Differences between near-cognate and cognate structural ensembles.

a, Comparison of particle populations in cognate and near-cognate samples (at 30 s) reveals more substrate and less intermediate (EF-Tu) and product states for the near-cognate reaction. b, Near-cognate tRNA-bound EF-Tu is less abundant in GDP-bound states (after GTP hydrolysis) than the cognate complex. c, d, Elbow-accommodated EA tRNAs sample open (c) and closed (d) 30S conformations. eh, CCA-accommodated tRNAs sample open and closed 30S conformations. Notably, state V-A1-nc, with an open 30S, shows a destabilized amino acid in the PTC. i, 30S-subunit partial rotation moves the ASF (cyan) in the cognate EA-1 and EA-2 states from their position in the EF-Tu release or accommodated A/A states (grey) allowing tRNA accommodation. j, Near-cognate tRNA (red) accommodation and interactions with the ASF differ from those of cognate tRNA (green).

Extended Data Fig. 8 Local resolutions of each cognate modelled class assessed by blocres.

See Methods for details.

Extended Data Fig. 9 Local resolutions of each near-cognate modelled class assessed by blocres.

See Methods for details.

Supplementary information

Supplementary Information

This file contains detailed descriptions of all states, a Supplementary Discussion, Supplementary Tables 1-3, Supplementary Figure 1 and Supplementary References.

Reporting Summary

Video 1

Animation showing an elongation event, with near-cognate tRNA rejected at three stages of decoding (initial selection, EF-Tu-dependent proofreading and EF-Tu-independent proofreading), followed by delivery and acceptance of the cognate tRNA•EF-Tu•GTP ternary complex.

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Loveland, A.B., Demo, G. & Korostelev, A.A. Cryo-EM of elongating ribosome with EF-Tu•GTP elucidates tRNA proofreading. Nature 584, 640–645 (2020). https://doi.org/10.1038/s41586-020-2447-x

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