mRNA decoding in human is kinetically and structurally distinct from bacteria

Abstract

In all species, ribosomes synthesize proteins by faithfully decoding messenger RNA (mRNA) nucleotide sequences using aminoacyl-tRNA substrates. Current knowledge of the decoding mechanism derives principally from studies on bacterial systems¹. Although key features are conserved across evolution², eukaryotes achieve higher-fidelity mRNA decoding than bacteria³. In human, changes in decoding fidelity are linked to ageing and disease and represent a potential point of therapeutic intervention in both viral and cancer treatment^4-6. Here we combine single-molecule imaging and cryogenic electron microscopy methods to examine the molecular basis of human ribosome fidelity to reveal that the decoding mechanism is both kinetically and structurally distinct from that of bacteria. Although decoding is globally analogous in both species, the reaction coordinate of aminoacyl-tRNA movement is altered on the human ribosome and the process is an order of magnitude slower. These distinctions arise from eukaryote-specific structural elements in the human ribosome and in the elongation factor eukaryotic elongation factor 1A (eEF1A) that together coordinate faithful tRNA incorporation at each mRNA codon. The distinct nature and timing of conformational changes within the ribosome and eEF1A rationalize how increased decoding fidelity is achieved and potentially regulated in eukaryotic species.

Fig. 1. smFRET and cryo-EM investigations of structural dynamics during mRNA decoding.

a, Schematic of FRET donor (P-site tRNA) and acceptor (aa-tRNA) fluorophores. b, Example smFRET data (10-ms time resolution) of a decoding reaction from the perspective shown in a showing progression from IC to CR and GA to AC for a single trace (left), and a population histogram of n traces (right). c, Transition density plot showing the FRET efficiency before and after each transition detected in this population of traces using hidden Markov model (HMM) idealization. d, Population histograms as in b of decoding reactions in the presence of an mRNA displaying a near-cognate (nc) A-site codon, GTPγS, PLT (10 µM), SR-A3 (10 µM), ANS (50 µM) or HHT (50 µM), showing stalling or rejection along the reaction coordinate. e, Overview of four cryo-EM reconstructions along the decoding reaction coordinate, filtered by local resolution and contoured at 3σ. Insets: details of eEF1A interacting with the GAC (top; 3σ) and the aa-tRNA (Phe-tRNA^Phe) ASL (bottom; 4σ) in the CR-to-GA transition. f,g, tRNA motions in the transition between the CR and GA complexes (f) and the GA and AC complexes (g) showing the positions of the FRET label attachment points and the distance between them, coloured as in a.

Fig. 2. Domain closure and initial ternary complex binding.

a, Cryo-EM density of the SSU from the GA complex coloured by backbone root-mean-squared deviation (r.m.s.d.) compared with the CR complex, contoured at 3σ. b, Overview of the SSU from the GA complex, showing the positions of the ternary complex, P-site tRNA and eIF5A and illustrating the size of the mobile shoulder domain (surface representation). Inset: solvent-exposed post-translationally modified C-terminal helix of eS6; known phosphorylation sites on the structured part of the C terminus are indicated in yellow. c, Magnification of eEF1A contacts with the SSU (surface representation) in the CR complex. The PLT-binding site is indicated. d, Overview of missing intersubunit bridges in the CR complex. Known phosphorylation and acetylation (Ac, orange) locations on eL24 are shown as spheres. SSU is shown in surface representation. Inset: atomic model and cryo-EM density illustrating the separated elements of bridge B8 and the α2 helix of eEF1A in the CR complex. Cryo-EM density is contoured at 3σ. Alignment is on the LSU core.

Fig. 3. Structural remodelling during initial selection.

a, Remodelling of the decoding centre to recognize the codon–anticodon helix in the CR (top) to GA (bottom) transition, highlighting the monitoring bases (red) and post-transcriptionally and post-translationally modified residues (yellow outline). b, Ternary complex contacts at the subunit interface near bridge B8 in the CR (top) and GA (bottom) complexes. Inset (left): coordination of a catalytic water in the eEF1A G domain in the GA complex; eEF1A-focused refinement. Insets (right): formation of the temporary bridge B8 through the α2 helix of eEF1A. c, Overlay of CR (grey) and GA (coloured) complexes showing SSU domain closure and ternary complex movements (top), combined movement of the SSU shoulder and ternary complex (middle; LSU alignment), and ternary complex movements in addition to those induced by SSU shoulder domain closure (bottom; SSU-shoulder alignment). Cryo-EM density is contoured at 3σ. Alignment is on the LSU core, unless otherwise noted.

Fig. 4. Subunit rolling and tRNA accommodation during proofreading selection.

a, Cryo-EM density of the SSU of the GA complex coloured by backbone r.m.s.d. compared with the AC complex, contoured at 3σ. b, Overview of the factor-binding site in the GA (left) and AC (right) complexes, showing closure of the eEF1A-binding site between the SSU and LSU due to SSU rolling. aa-tRNA has been omitted for clarity. The black bar shows the distance between the phosphates of LSU rRNA G4600 and SSU rRNA A464. c, Example smFRET trace (blue) and an HMM idealization (red) from the perspective of FRET between the two tRNAs of an uninhibited decoding reaction showing reversible excursions from the GA to the AC state preceding stable AC-state formation (left). Transition density plot showing the FRET efficiency before and after each transition detected in this population of traces by HMM idealization after the first visit to the AC state (right), showing persistent fluctuations back to the GA state. d, Intersubunit bridges formed and broken by SSU rolling in the GA-to-AC transition (bottom), contoured at 3σ.

Fig. 5. Binding sites for ribosome inhibitors.

a,b, Overview of the binding sites for PLT (red) (a) and SR-A3 (coral) (b) on eEF1A between domain III (DIII, cyan) and the G domain (blue) bound to a GA-ribosome complex. Focus refined on eEF1A. The black arrow indicates the hydroxyl moiety that differentiates SR-A3 from ternatin-4. c–e, Structures of ANS (light orange) (c) and HHT (orange) (d) in the PTC and LTM (dark purple) (e) and cycloheximide (CHX, purple) (f) in the E site from consensus LSU focused refinements. The cryo-EM density from structures stalled with either PLT, ANS, LTM and GTPγS (a–c) or SR-A3, HHT, CHX and GTPγS (d–f) is shown. Known resistance mutations (green), waters (red), Mg²⁺ (lime green) and K⁺ (steel blue) are indicated. Contour levels for cryo-EM density are indicated in σ units.

Extended Data Fig. 1. Overview of smFRET data showing stalling of decoding by small-molecule interventions and near-cognate aa-tRNA from two structural perspectives.

a, Schematic of the decoding reaction on human ribosomes as observed by smFRET. A reaction coordinate of four relatively long-lived ribosome states is inferred, (1) IC with an empty A site, (2) CR with ternary complex bound to the SSU A site but not the LSU, (3) GA with ternary complex bound to the SSU and docked at the LSU GTPase activating centre (GAC) and (4) AC without eEF1A and with A-site tRNA fully accommodated into the peptidyl transferase centre (PTC). Donor and acceptor fluorophore locations and FRET efficiencies for each state in the two FRET perspectives employed are indicated. Locations along the reaction coordinate inhibited by the drugs plitidepsin (PLT), SR-A3, anisomycin (ANS), homoharringtonine (HHT), cycloheximide (CHX) and lactimidomycin (LTM) are indicated. b, Example fluorescence (top) and FRET efficiency time traces (bottom) from the perspective of FRET between the two tRNAs at 10 ms time resolution in the absence of any inhibition. c, 1D FRET efficiency population histograms of pre-translocation complexes imaged at different concentrations of eIF5A (left) and fraction of classical-state (~0.7 FRET efficiency) ribosomes as a function of eIF5A concentration (right) estimated by fitting of a sum of gaussian functions to data such as on the left. The solid line represents a fit of an equilibrium binding equation (SI) to the data. Error bars represent SEM from duplicate experiments. d, 2D and 1D population FRET histograms (left) and transition density plot (right) for an uninhibited decoding reaction imaged at a time resolution of 100 ms at 25 °C. e, 2D and 1D population FRET histograms (left) and transition density plot (right) for an uninhibited decoding reaction imaged at a time resolution of 40 ms at 37 °C. On average, (d) the uninhibited reaction proceeds rapidly through the CR and GA states to reach the AC state. However, the transition density plots indicate that reversible transitions between states are common prior to forward progression along the reaction coordinate. f, 2D and 1D population FRET histograms (left) and transition density plot (right) for the cognate tRNA decoding reaction imaged at 10 ms time resolution from the tRNA–tRNA FRET perspective. g, 2D and 1D population FRET histograms (left) and transition density plot (right) for the near-cognate tRNA decoding reaction imaged at 10 ms time resolution from the tRNA–tRNA FRET perspective. h, Example traces of near-cognate decoding events imaged at 10 ms time resolution from the tRNA–tRNA structural perspective. i–m, 2D and 1D population FRET histograms (left) and transition density plots (right) for decoding reactions imaged at a time resolution of 100 ms from the tRNA–tRNA FRET perspective with inclusion of the indicated stalling agents. (i) The slowly hydrolysable GTP analogue GTPγS stalls the reaction in the GA state with fluctuations to the CR state. The eEF1A-dissociation inhibitors (j) PLT and (k) SR-A3 likewise stall the reaction predominantly in the GA state with rare excursions to the CR state. The peptidyl transferase inhibitors (l) ANS and (m) HHT stall the reaction in a GA-like state with rare excursions to the CR and AC states. Red and black lines in the 1D FRET histograms indicate fits of gaussian functions to the data. n, Schematic of the uL11-tRNA FRET perspective. o, 2D and 1D population FRET histograms (left) and transition density plots (right) for an uninhibited decoding reaction imaged at a time resolution of 100 ms from the uL11-tRNA FRET perspective. On average, (o) the uninhibited reaction proceeds rapidly through the CR/GA states to reach the AC state. However, the transition density plot indicates that reversible transitions between states are common prior to forward progression along the reaction coordinate. p, 2D and 1D population FRET histograms (left) and transition density plot (right) for the cognate tRNA decoding reaction imaged at 10 ms time resolution from the uL11-tRNA FRET perspective. q, 2D and 1D population FRET histograms (left) and transition density plot (right) for the near-cognate tRNA decoding reaction imaged at 10 ms time resolution from the uL11-tRNA FRET perspective. r–v, 2D and 1D population FRET histograms (left) and transition density plots (right) for decoding reactions imaged at a time resolution of 100 ms from the uL11-tRNA FRET perspective with inclusion of the indicated stalling agents. (r) The slowly hydrolysable GTP analogue GTPγS stalls the reaction in the GA state with fluctuations to the CR state. The eEF1A-dissociation inhibitors (s) PLT and (t) SR-A3 likewise stall the reaction predominantly in the GA state with rare excursions to the CR state. The peptidyl transferase inhibitors (u) ANS and (v) HHT stall the reaction in a GA-like state with rare excursions to the CR and AC states.

Extended Data Fig. 2. Cryo-EM data processing and refinement of the ribosome structures.

a, b, Flowcharts of cryo-EM data processing of the ribosome structures stalled with (a) plitidepsin, anisomycin, lactimidomycin and GTPγS (PLT, ANS, LTM and GTPγS) and (b) SR-A3, homoharringtonine, cycloheximide and GTPγS (SR-A3, HHT, CHX and GTPγS). For 3D classifications, the number of classes (K) and the regularization parameter (T) are indicted. For focused 3D classification and refinements, regions contained within the soft mask are indicated. To generate high-resolution consensus maps, the particles circled in light grey line were merged, pooled, filtered for duplicates and refined with the full pixel sizes. Focused refinements with signal subtraction and 3D classifications with signal subtraction were performed with ‘shiny’ particles reextracted from refined consensus LSU metadata. All processing was conducted in RELION 3.1, unless otherwise noted. Processing of consensus maps and final refinements/postprocessing of all maps was conducted in RELION 4.0. c–j, Cryo-EM maps filtered and coloured by local resolution (left) and Fourier shell correlation (FSC) curves (right) obtained by masking the two half maps and calculating the cross-correlation between the masked volumes in RELION 4.0 for (c) consensus LSU, (d) consensus SSU, (e) initiation (IC), (f) CR, (g) GA and (h) AC complexes stalled with PLT, ANS, LTM and GTPγS and (i) consensus LSU and GA complexes stalled by SR-A3, HHT, CHX and GTPγS. Resolution was estimated using the 0.143 cutoff criterion (black dotted line). Cross-validation was used to optimize the weight on the experimental density in REFMAC to prevent overfitting. Cryo-EM density contour levels are indicated in σ units. Refinement procedures are described in Methods. For more details on cryo-EM processing, see also Extended Data Table 1.

Extended Data Fig. 3. High-resolution structural features observed in the human ribosome consensus reconstruction.

a–c, Cryo-EM density of complexes stalled with plitidepsin, anisomycin, lactimidomycin and GTPγS (PLT, ANS, LTM and GTPγS) highlighting the locations of visualized post-transcriptional (dark blue) and post-translational (dark red) modifications on (a) the LSU and (b) the SSU from the high-resolution consensus reconstruction and (c) aa-tRNA (Phe-tRNA^Phe), P-site tRNA (Met-tRNA^fMet), eEF1A and eIF5A from the GTPase activated reconstruction. d–n, High-resolution structural features visible within the consensus cryo-EM map, including (d) the centre of the LSU core, (e) the start codon–anticodon nucleotides in the P site (f) polyamines, (g, k) hydroxylhistidines, (h) fully hydrated Mg²⁺ ions (lime green, left) and partially hydrated and fully coordinated and Mg²⁺ ions (top, middle and right), (i) 2′O-Me uridine (Um), (j) pseudouridine (Ψ), (l) methyl-histidine, (m) trimethyl-lysine and (n, o) methyl-lysine. Protein and nucleic acid modifications are indicated with black arrows. Cryo-EM density is from post-processed high-resolution consensus LSU map and is contoured at 3 σ for panel m and 5 σ for all remaining panels. See also Methods, Extended Data Fig. 2 and Extended Data Table 1.

Extended Data Fig. 4. Interactions between eIF5A, P-site tRNA and the human ribosome.

a, Overview of the E site from the indicated orientation (centre), showing interactions between uL1, uL5, eL42, eIF5A, the L1 stalk rRNA, H74 and P-site tRNA. Lactimidomycin (LTM) shown in ball-and-stick representation. Insets running left to right show zoom-ins of the position of eIF5A relative to LTM, H74 base G3922, and eL42 (left), the interactions between P-site tRNA base pair G20-C57 and uL5/eL42 and P-site tRNA D-loop residues 16-21 interaction with eIF5A (middle) and the CCA-end of P-site tRNA (right). b, Overview of the P-site tRNA acceptor stem from the indicated direction showing interactions with eS25 (N terminus), uS13 (C terminus), uS19 (C terminus), uS9 (C terminus) and SSU bases of h31 (m¹acp³Ψ1248, red) and h29 (1639-1642, PE loop). Insets running left to right show zoom-ins of interactions between the P-site tRNA and: eS25, uS13 and the PE loop (left); mRNA, uS9 and m¹acp³Ψ1248 (middle); and uS19 and uS13 (right). Cryo-EM density and atomic model are from the GTPase activated complex. All cryo-EM density is filtered by local resolution and is contoured in units of σ as indicated.

Extended Data Fig. 5. Remodelling of interactions between the aa-tRNA and the ribosome during mRNA decoding.

a, Interactions between the tRNA CCA-ends and the peptidyl transferase centre (PTC). b–d, Overview of aa-tRNA interactions with the L11 stalk (H42 and H44), H89 and the LSU A-site finger (ASF) in (b) the CR, (c) GA and (d) AC complexes stalled with plitidepsin, anisomycin, lactimidomycin and GTPγS. The relative position of aa-tRNA and P-site tRNA and the ASF, H89 and the GTPase activating centre (GAC, left). A zoom in on the region within the dotted line is provided with (right) and without (middle) experimental density, showing the interaction with aa-tRNA (Phe-tRNA^Phe) bases G19 and C56. The vertical green fields show the overall position of the tRNA bases G19 and C56 relative to the LSU. These show the stacking between G19 and C56 of the aa-tRNA elbow with G1981 of H44 and A2009 of H42, respectively, in the CR complex. In the GA complex the aa-tRNA has moved past H89 further towards the P site and the SSU, shifting the stacking interaction between the elbow and H42 and H44 such that C56 of the elbow now stacks on G1981 of H44 while G19 no longer makes any stacking interaction. In the AC complex, G19 and C56 interact weakly with the ASF. e, View of aa-tRNA accommodation showing the spatial relationship between the aa-tRNA, the P-site tRNA and the accommodation corridor. The inset shows the movement of the aa-tRNA CCA end due to subunit rolling, the white tRNA is a model of an accommodated tRNA with its anticodon stem loop (ASL) aligned onto that of an aa-tRNA on an unrolled ribosome in the GA complex. The dashed green line shows the likely path of the aa-tRNA CCA end. f, g, Zoom-ins of the accommodation corridor corresponding to the dashed rectangle in (e) showing (f) experimental cryo-EM density and (g) atomic model showing the additional crowding due to the eukaryote extension of uL3. All cryo-EM density is filtered by local resolution and is contoured at 3 σ.

Extended Data Fig. 6. tRNA positions and ternary complex interactions in decoding intermediate complexes in human and bacteria.

a, b, Superposition of early (CR) (a) and late (GA) (b) ternary complex-bound decoding intermediates from human (coloured) and bacteria from (grey; ‘Structure II’, PDB-ID: 5UYL; ‘Structure III’, PDB-ID: 5UYM). c, d, tRNA motions in the transition between the CR and GA complexes (c) and the GA and AC complexes (d) in human. e, f, tRNA motions in the transition between ‘Structure II’ in reference and ‘Structure III’ in reference (e) and ‘Structure III’ in reference and PRE-C in bacteria (f). Alignment on the P-site tRNA. g, h, Overview of the SSU in human (AC complex) (g) and bacteria (PRE-C, PDB-ID: 7N1P) (h) showing the size of the mobile shoulder domain (surface representation). The decoding centre is shown in red. i, j, Overview of the absence (human, GAcomplex) (i) and presence (bacteria, ‘Structure III’, PDB-ID: 5UYM) (j) of intersubunit bridge B8 between h14, uL14 and bL19 (bacteria only). The shoulder domain is shown in surface representation. Insets, comparison of switch-I (SW-I) in eEF1A (left) and EF-Tu (right). k, l, Close-up view of bridge B8, same structures as in (i, j) for human (k) and bacteria (l). Alignment on the LSU core.

Extended Data Fig. 7. Changes in key decoding centre interactions along the decoding reaction coordinate.

a–c, Views of the decoding centre of IC, CR, GA and AC reconstructions, from left to right. Overview of aa-tRNA (Phe-tRNA^Phe) in the decoding centre as seen from the leading edge of the SSU (a). The monitoring bases (red) G626, A1824 and A1825 (530, 1492 and 1493 in E. coli, respectively) are disengaged in the IC and CR complexes. A1824 resides inside h44, hydrogen bonding between the amino group of Am3760 (A1913 in E. coli) of H69 and N3 of A1824 replaces the stacking interaction between these two bases observed in bacteria where Am3760 resides inside h44. A1825 is disordered rather than, as observed in bacteria, flipped out close to its ‘monitoring’ position, possibly due to the empty space available to it inside h44 due to the distal position of Am3760 in human compared to bacteria. G626 is in an anti-conformation, rather than in a syn conformation as observed in bacteria, it is stacked with C614 and positioned away from the decoding centre. Ribosomal proteins uS12 and eS30 are disengaged. In the GA and AC complexes the decoding centre is fully structured around the codon–anticodon minihelix and the monitoring bases occupy positions like those observed in bacteria. A1824 and A1825 reside outside h44 forming A-minor interactions with the codon–anticodon pair. Am3760 has moved away from its CR position and is hydrogen bonded to the aa-tRNA base 3′ of the anticodon, as in bacteria. Domain closure has brought G626, C614 and uS12 roughly 3 Å further into the decoding centre. G626 now hydrogen bonds with A1824 and the first and second bases of the anticodon. C614 coordinates a Mg²⁺ molecule with the third base of the mRNA codon and uS12 hydroxy-pro62. Additionally, uS12 Gln61 forms hydrogen bonds with the second base of the mRNA codon and A1824. C1331 (C1054 in E. coli) forms a Pi-stacking interaction with the third base of the anticodon while C1698 (C1397 in E. coli) intercalates into the mRNA one base 3′ of the codon. The N-terminal tail of eS30 has become structured and Met1 hydrogen bonds with A1825 while His3 hydrogen bonds to the aa-tRNA residue 3′ of the anticodon and forms a salt bridge to C615. View of the same process from the SSU side highlighting intercalation of C1698 into the mRNA (b). Close-up view of the same sequence of events focused on the h44 side of the decoding centre shown with cryo-EM density (c). Density for A1824 is strong in all four reconstructions, indicating stable localization and a switch-like behaviour while density for A1825 is absent in IC, weak in CR and strong in GA and AC, indicating a stepwise transition from disordered to ordered positioning of this base. Strong density places G626 in an anti-conformation in all four reconstructions (inset), unlike the syn to anti flip observed in bacteria in the CR to GA transition, due to the stacking between G626, C614 and G625 these bases appear to move as a rigid body with the rest of the shoulder domain. d, Equivalent cryo-EM structures of decoding intermediates from E. coli from the same view as (c). From left to right, POST complex (PDB-ID: 7N31), ‘Structure I’ (PDB-ID: 5UYK), ‘Structure III’ (PDB-ID: 5UYM), PRE-C complex (PDB-ID: 7N1P). All cryo-EM density is filtered by local resolution and is contoured at 5 σ.

Extended Data Fig. 8. eEF1A interactions with the ribosome in the codon recognition and GTPase activated complexes.

a, Zoomed-out view of Fig. 3b, showing interactions between eEF1A, the SSU and the LSU in the CR complex (top) and the GA complex (bottom). In the GA complex the eEF1A G domain packs against the SRL and its α2 expansion segment forms a temporary bridge B8 between h14 and uL14. b, View of same process from the head domain of the SSU with aa-tRNA hidden for clarity, showing the interaction between DII of eEF1A with the C-terminus of uS12. c, View of the interaction between the eEF1A G domain and the ribosome as seen from the GAC in the CR complex (top) and the GA complex (bottom). The GTPγS molecule is tightly coordinated in the G-domain by switch I (SW-I), switch II (SW-II), the P loop and other G-domain elements. In the GA complex, the G domain of eEF1A docks onto the GAC, packing the α2 expansion segment tighter against SW-I. The SRL coordinates Arg69 of the SW-I element and the ‘catalytic’ His95 of the SW-II element, respectively, priming eEF1A for GTP hydrolysis. Cryo-EM density from a focused refinement on eEF1A is shown for the GA complex. d, e, The geometry of the catalytic His95 in the eEF1A G domain in the G interaction between the eEF1A G domain and the ribosome as seen from the GAC in the CR complex (top) and the GA complex. Contour levels for cryo-EM density are indicated in σ units.

Extended Data Fig. 9. Remodelling of intersubunit bridges during mRNA decoding.

a, Overview of all intersubunit bridges on the human ribosome. b, Overviews of bridges formed (cyan) and not formed (yellow) in the four decoding complexes. c, Overview of the changes in bridging interactions during the transitions between the four decoding complexes. All cryo-EM density is filtered by local resolution and is contoured at 3 σ. See Methods for more details.

Extended Data Fig. 10. Allosteric interaction between the leading and lagging edges of the ribosome affect mRNA decoding.

a, Overlay of the interaction between eIF5A and P-site tRNA for the rolled AC complex (solid) and the unrolled GA complex (transparent), showing movements of eIF5A towards the P site and the P-site tRNA towards the E site as a response to SSU rolling. LSU rRNA base G4385 is shown in red. b, Overlay of the E site for the rolled AC complex (solid) and the unrolled GA complex (transparent), showing conformational changes in the E site as a consequence of SSU rolling. c, Cryo-EM density for the N-terminal tail of eIF5A in the GA (left) and AC (right) complexes, showing its restructuring as a response to SSU rolling. Cryo-EM density is filtered by local resolution and contoured at 2 σ with a 1 σ gaussian filter. d, Catalytic efficiency of decoding on fast ribosomes as a function of the ligand bound in the ribosomal E site, slow ribosomes carried out the decoding reaction with 10-20× lower speed. e, Fraction of ribosomes that carried out decoding fast as a function of the ligand bound in the ribosomal E site. Approximately 50% of ribosomes with an empty E site carry out the decoding reaction slowly, whereas with any E site ligand only about 15% do so. This implies that natural as well as small-molecule ligands that bind the E site are able to affect the conformation of the ribosome in a way that accelerates binding of ternary complex to the A site. Each dot represents one experimental replicate, the horizontal bar represents the average.