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

Nature volume 584, pages 640–645 (2020)Cite this article

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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.

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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).

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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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  1. Gabriel Demo

    Present address: Central European Institute of Technology, Masaryk University, Brno, Czech Republic

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

    Anna B. Loveland, Gabriel Demo & Andrei A. Korostelev

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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.

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Correspondence to Andrei A. Korostelev.

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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. c–f, 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.

a–r, Density for EF-Tu in 30S-open (II) and 30S-closed (III) conformations is shown relative to the sarcin-ricin loop (SRL) (a–c, g–i and m–o) or at the tRNA interacting face (d–f, j–l and p–r; 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. d–g, 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. e–h, 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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