Abstract
One of the most critical steps of protein synthesis is coupled translocation of messenger RNA (mRNA) and transfer RNAs (tRNAs) required to advance the mRNA reading frame by one codon. In eukaryotes, translocation is accelerated and its fidelity is maintained by elongation factor 2 (eEF2)1,2. At present, only a few snapshots of eukaryotic ribosome translocation have been reported3,4,5. Here we report ten high-resolution cryogenic-electron microscopy (cryo-EM) structures of the elongating eukaryotic ribosome bound to the full translocation module consisting of mRNA, peptidyl-tRNA and deacylated tRNA, seven of which also contained ribosome-bound, naturally modified eEF2. This study recapitulates mRNA–tRNA2-growing peptide module progression through the ribosome, from the earliest states of eEF2 translocase accommodation until the very late stages of the process, and shows an intricate network of interactions preventing the slippage of the translational reading frame. We demonstrate how the accuracy of eukaryotic translocation relies on eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs. Our findings shed light on the mechanism of translation arrest by the anti-fungal eEF2-binding inhibitor, sordarin. We also propose that the sterically constrained environment imposed by diphthamide, a conserved eukaryotic posttranslational modification in eEF2, not only stabilizes correct Watson–Crick codon–anticodon interactions but may also uncover erroneous peptidyl-tRNA, and therefore contribute to higher accuracy of protein synthesis in eukaryotes.
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Data availability
Atomic coordinates and cryo-EM maps generated during this study are available through the PDB and the Electron Microscopy Data bank (EMDB). The complexes have been deposited under following accession codes: PRE-H1 (8CCS, EMD-16563), PRE-H2 (8CDL, EMD-16591), TI-1 (8CF5, EMD-16616), TI-2 (8CDR, EMD-16594), TI-3 (8CG8, EMD-16634), TI-4 (8CEH, EMD-16609), TI-5 (8CIV, EMD-16684), NR (8CGN, EMD-16648), TI-1* (8CKU, EMD-16702) and TI-4* (8CMJ, EMD-16729).
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Acknowledgements
We are grateful to the staff of the Dubochet Center for Imaging (DCI-Lausanne) for cryo-imaging and preliminary data analysis. We thank Y. Polikanov from the University of Illinois at Chicago for providing the plasmid for the over-expression of the CCA-adding enzyme. We also thank C. Crucifix from the IGBMC cryo-EM platform for her expertise in grid preparation. This work was supported by La Fondation pour la Recherche Médicale grant no. DEQ20181039600 (to M.Y.).
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M.Y. and G.Y. conceived the project, N.M. conducted all biochemical experiments, and N.M and A.M. collected cryo-EM data. Main data analysis was performed by N.M. L.J. and A.M. also contributed to the data analysis. N.M. prepared the manuscript. G.Y. reviewed and edited the manuscript. G.Y. supervised the project. All authors gave their input in the preparation of the final manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Large-scale conformational changes of the small ribosomal subunit.
Intersubunit view of the full SSU (left grid) and a zoomed top view of the SSU head domain (right grid) for each solved translocation complex. SSU is coloured on the basis of root-mean-square deviation values of atomic positions relative to the non-rotated (NR) state by 25 S alignment. White arrows indicate shoulder domain closure (upwards) or opening (downwards). Dark grey arrows indicate small subunit rotation (clockwise) or back-rotation (anti-clockwise) around the rotation axes shown in black. Light grey arrows indicate forward tilting or swiveling motions around the axes shown in light gray. All angles are calculated relative to the NR state.
Extended Data Fig. 2 Progressive anticodon advancement in chimeric states of translocation.
Positions of peptidyl- (green) and deacyl-tRNA (yellow) anticodons in chimeric states of translocation TI-2 to TI-5 relative to final positions observed in the NR state (white) and the early chimeric ap0/P-pe0/E state (PDB ID 7OSM). Individually displayed anticodon nucleotides (top to bottom in the 3′ – 5′ direction) reveal distinct SSU sub-chimeric states: ap1-pe1 (in TI-2 and TI-3) and ap2-pe2 (in TI-4 and TI-5).
Extended Data Fig. 3 Nucleotide-binding pockets of eEF2 in translocation intermediates from TI-1 to TI-5.
Unfiltered, unsharpened maps (σ = 3) of the nucleotide-binding pocket found in domain I of eEF2 (red). TI-1 and TI-3 show density for the non-hydrolysable analogue of GTP (GMPPCP) and switch loop 1 (sw1). GDP-containing TI-2, TI-4 and TI-5 show no density for sw1. Additional density corresponding to inorganic phosphate was identified in TI-4.
Extended Data Fig. 4 eEF2 interactions with the eukaryotic 80 S ribosome.
a, Contacts of eEF2 domains (I in green, II in magenta, III in pink, IV in red, and V in blue) with ribosomal elements of large 60 S (RNA in light grey and proteins in dark grey) and small 40 S subunit (RNA in light blue and proteins in dark blue) in translocation intermediate complexes TI-1 to TI-5. Sordarin is depicted in orange. b-d, Quantification of contact areas of eEF2 domains I to V with 80 S, in b, large 60 S subunit, in c, and small 40 S subunit, in d. Eukaryote-specific interactions between eEF2 and SSU proteins eS6 and eS24 during factor recruitment in early translocation (TI-1), and eS30 once eEF2 is fully accommodated on 80 S (TI-2 to TI-5). Black arrows indicate areas of eukaryote-specific contacts.
Extended Data Fig. 5 Conformational adaptations of eEF2 and contact areas with the tRNA2-mRNA translocating module.
a, Alignment on 25 S (left) and global eEF2 alignment (right) on eEF2 in TI-5 for GMPPCP bound intermediates TI-1 and TI-3, and GDP-containing intermediates TI-2, TI-4 and TI-5. Atoms are colored on the basis of root-mean-square deviation values of atomic positions relative to TI-5. b, Buried surface measurements corresponding to contacts of the tRNA2-mRNA module with 80 S ribosome (pink) and domain IV of eEF2 (red) in the course of elongation from PRE-H1 to TI-5. c, Unfiltered, unsharpened density of S. cerevisiae eEF2 (e.g. TI-4) contoured at σ = 3.
Extended Data Fig. 6 mRNA path on the small subunit of the eukaryotic 80 S ribosome.
a, Lateral view of the full 40 S small subunit (SSU) of the eukaryotic 80 S ribosome trapped in the PRE-hybrid-1 reconstruction (PRE-H1) reveals the path of messenger RNA (magenta) at the SSU head-body domain interface. Unsharpened, unfiltered high-resolution cryo-EM maps are shown at σ = 3.5. 18 S ribosomal RNA and SSU proteins are colored in light and dark blue, respectively. b, Zoom on the thirteen nucleotide-long messenger RNA and interacting residues of 18 S rRNA and the conserved SSU protein uS12. +1 indicates the first nucleotide of the start codon. c, Close-up view of the interactions between messenger RNA and ribosomal SSU elements in the PRE-H1 complex. π-π stacking is found between −1 and G1150, while the p-π effect is observed in the following interacting pairs: −3 and G904, and +3 and C1637.
Extended Data Fig. 7 Effect of sordarin binding to eEF2 in the context of eukaryotic translocation.
a-c, Global alignments reflecting general conservation of overall eEF2 conformation during early, in a, and late states of translocation, in b and c. d-f, Domain III alignment shows displacement of eEF2 domain V towards the interface with domain III. g-I, Zoom on the sordardin binding site for respective structural superimpositions on eEF2 domain III, as shown in d-f. The arrow indicates the displacement of domain V relative to domain III. Remodelling of the switch loop 2 (sw2) has not been observed. Structural alignments are performed for late translocation states before, in b, e and h, and after GTP hydrolysis, in c, f and i. eEF2 domains are colored as follows: domain I in green, domain II in magenta, domain III in pink, domain IV in red, and domain V in blue. Unfiltered, unsharpened of sordarin is shown at σ = 4 and depicted in orange. Lighter nuances correspond to structures without sordardin, TI-1* and TI-4*. j, Chemical structure of sordarin.
Extended Data Fig. 8 Interactions of eEF2 with small subunit proteins uS12 and eS30.
a, Inter-domain remodeling of eEF2 and conformational changes of SSU proteins uS12 and eS30 accompanying SSU back-rotation during respective transitions between translocation intermediates TI-1 to TI-5. b, Curves depicting contacts between SSU proteins uS12 with eEF2 domains II (dark yellow) and III (orange), and eS30 with domain IV (blue).
Supplementary information
Supplementary Information
Supplementary Table 1 and Figs 1 and 2.
Supplementary Video 1
Conformational changes of the eukaryotic ribosome during tRNA translocation. The video recapitulates mRNA–tRNA progression through the ribosome starting from pretranslocation hybrid intermediates (PRE-H1 to PRE-H2), eEF2 recruitment (TI-1) and accommodation (TI-2). The complex then undergoes sequential transitions from TI-2 to TI-5, resulting in the POST state. Peptidyl-tRNA in shown in green, deacyl-tRNA in violet, eEF2 in red, LSU (60S) is depicted in light blue, SSU (40S) head and body domains in light brown and orange, respectively.
Supplementary Video 2
eEF2-assisted advancement of tRNAs during translocation on the eukaryotic ribosome. Morphing of unfiltered, unsharpened cryo-EM maps reveals structural dynamics of the mRNA–tRNA2 module advancement during translocation. The sequence reflects progressive transitions between hybrid (PRE-H1, PRE-H2 and TI-1) and chimeric states (TI-2 to TI-5), yielding the classical POST state. Peptidyl-tRNA is shown in green, deacyl-tRNA in yellow, eEF2 in red and mRNA in magenta.
Supplementary Video 3
Accuracy of ribosomal translocation in eukaryotes: opening of the decoding centre and role of diphthamide. The video shows an intricated network of interactions on the atomic level, which maintains the mRNA translational reading frame during translocation. It also shows the locked conformation of the eukaryotic decoding centre, which undergoes unlocking as domain IV protrudes into the A-site following the accommodation of eEF2 on the ribosome. Peptidyl-tRNAPhe (green), deacyl-tRNAfMet (violet), mRNA (magenta), SSU (light brown), uS12 (orange), potassium ion (dark violet) and eEF2 (red). PDB ID 7OSM corresponds to the crystal structure of the early intermediate translocation complex.
Supplementary Video 4
Role of elongation factor 2 (eEF2) during eukaryotic ribosome translocation. Interface view of the SSU (40S) illustrates SSU rotation and head domain swivelling during mRNA–tRNA2 translocation. Progressive advancement of ASLs of peptidyl-tRNA (green) and deacyl-tRNA (violet) is shown from early hybrid to late chimeric states of translocation as the ligands proceed to their classical positions in the POST state. eEF2 contributes to translocation most probably by preventing reverse rotation of the SSU head domain, acting as a molecular doorstop. The head of SSU (40S) is shown in orange, SSU body in light brown and eEF2 in red.
Supplementary Video 5
Role of the 1-methyl-3-α-amino-α-carboxyl-propyl pseudouridine 1,191 (m1acp3Ψ1191) hypermodification in eukaryotic ribosome translocation. The heavily modified uridine, found in the hairpin loop of the universally conserved helix 31 of 18S rRNA lies at the interface of SSU (40S) head and body domains. During translocation, m1acp3Ψ1191 acts by repetitively guiding tRNA from P to E site.
Supplementary Video 6
Maintenance of the translational reading frame during eukaryotic translocation. TI-4 is given as an example. Residues of SSU (40S) head and body domains, assisted by residues of eEF2 domain IV including diphthamide, form an extensive network of interactions with codon–anticodon duplexes and maintain the translational reading frame during translocation in eukaryotes. Peptidyl-tRNA is shown in green, deacyl-tRNA in violet, eEF2 in red, mRNA in magenta, residues of SSU head and body domains are depicted in light brown and orange, respectively, and uS9 is shown in dark blue.
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Milicevic, N., Jenner, L., Myasnikov, A. et al. mRNA reading frame maintenance during eukaryotic ribosome translocation. Nature 625, 393–400 (2024). https://doi.org/10.1038/s41586-023-06780-4
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DOI: https://doi.org/10.1038/s41586-023-06780-4
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