Structural studies of nedicistrovirus IRES-driven, initiation factor-independent translation shed light on key steps of eukaryotic translation elongation.

Structural studies of nedicistrovirus IRES-driven, initiation factor-independent translation shed light on key steps of eukaryotic translation elongation - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Search Log in Primary site navigation Search Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Nucleic Acids Res . 2026 Jun 4;54(10):gkag510. doi: 10.1093/nar/gkag510 Structural studies of nedicistrovirus IRES-driven, initiation factor-independent translation shed light on key steps of eukaryotic translation elongation Swastik De 1, 4 , Clara G Altomare 2, 4 , Irina S Abaeva 3 , Prikshat Dadhwal 4, 5 , Priyanka Garg 6 , Francisco Acosta-Reyes 7 , Zuben P Brown 8 , Tatyana V Pestova 9, ✉ , Christopher U T Hellen 10, ✉ , Joachim Frank 11, 12, ✉ Author information Article notes Copyright and License information 1 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States 2 Department of Biological Sciences, Columbia University, New York, NY 10027, United States 3 Department of Cell Biology, SUNY Downstate Health Sciences University, Brooklyn, NY 11203, United States 4 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States 5 Department of Biological Sciences, Columbia University, New York, NY 10027, United States 6 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States 7 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States 8 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States 9 Department of Cell Biology, SUNY Downstate Health Sciences University, Brooklyn, NY 11203, United States 10 Department of Cell Biology, SUNY Downstate Health Sciences University, Brooklyn, NY 11203, United States 11 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States 12 Department of Biological Sciences, Columbia University, New York, NY 10027, United States ✉ To whom correspondence should be addressed. Email: tatyana.pestova@downstate.edu ✉ Correspondence may also be addressed to Christopher U.T. Hellen. Email: christopher.hellen@downstate.edu ✉ Correspondence may also be addressed to Joachim Frank. Email: jf2192@cumc.columbia.edu 4 Swastik De and Clara G Altomare should be regarded as Joint First Authors. Received 2025 Sep 30; Revised 2026 Apr 22; Accepted 2026 May 3; Collection date 2026 Jun 10. © The Author(s) 2026. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( https://creativecommons.org/licenses/by-nc/4.0/ ), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com . PMC Copyright notice PMCID: PMC13235965  PMID: 42240621 Previous version available: This article is based on a previously available preprint posted on bioRxiv on September 29, 2025: " Structural Studies of Nedicistrovirus IRES-Driven, Initiation Factor-independent Translation Shed Light on Key Steps of Eukaryotic Translation Elongation ". Abstract We utilized the nedicistrovirus (NediV) intergenic region (IGR) internal ribosomal entry site (IRES)-mediated, initiation factor-independent translation initiation system and determined high-resolution structures of 80S ribosome complexes with the NediV IRES in various functional states, including binary complexes, aminoacyl–transfer RNA (tRNA)-bound complexes, and complexes with elongation factor eEF2. In binary complexes, the NediV IRES primarily occupies the ribosomal P site, exhibiting conformational flexibility and engaging the ribosome at multiple interaction sites. Upon translocation, the IRES undergoes structural rearrangements, including destabilization of its PKI domain, facilitating the transition to canonical elongation. Crucially, we captured an eEF2-bound complex, along with an eEF1A-bound failed decoding complex featuring a mismatched tRNA, the latter representing the first instance of a canonical elongation complex visualized in the presence of a natural, hydrolysable nucleotide and without the addition of any trapping agents. These findings provide a comprehensive structural overview of IGR IRES-mediated translation initiation and its transition to elongation, revealing key mechanistic details of viral translation and proofreading. Graphical Abstract Graphical Abstract. Open in a new tab Introduction Translation is the process through which ribosomes decode the genetic instructions in messenger RNA (mRNA) to assemble amino acids into polypeptides. Initiation of translation requires the precise positioning of the initiator aminoacyl–transfer RNA (tRNA) and the start codon of the mRNA in the ribosomal P site. In bacteria, this process is relatively simple, requiring just three initiation factors and registration on the ribosome of the mRNA by a short Shine–Dalgarno sequence near its 5′ end. In contrast, eukaryotic translation initiation is far more complex, involving more than a dozen eukaryotic initiation factors (eIFs) to recruit the ribosome and initiate translation from an AUG start codon via a cap-dependent scanning mechanism [ 1 – 3 ]. The eukaryotic mRNA 5′ cap structure, 7-methyl-guanosine-(5′)-ppp-(5′)N, facilitates ribosome recruitment through the eIF4F cap-binding complex, enabling the 40S ribosomal subunit, associated with eIFs 1, 1A, and 3, as well as the ternary complex (TC) formed by eIF2, GTP, and Met–tRNA i Met , to scan for the start codon. However, many viruses circumvent host translational control by utilizing internal ribosomal entry sites (IRES), structured RNA elements that enable 5′ end-independent translation, particularly under conditions where cap-dependent translation is compromised, such as viral infection or cellular stress [ 4 , 5 ]. Different viral IRESs vary in their dependency on canonical initiation factors. Among them, the dicistronic RNA genomes of dicistroviruses contain an IRES in the intergenic region (IGR) that mediates initiation of translation of the downstream open reading frame (ORF) 2 by an exceptionally streamlined mechanism. IGR IRESs (recently termed type 6 IRESs) [ 6 ] bind directly to ribosomal 40S subunits and/or 80S ribosomes, and translation proceeds from a defined location without the involvement of initiator tRNA or eIFs [ 7 , 8 ]. Instead, ribosomes enter directly into the elongation phase of translation [ 9 , 10 ]. Translation mediated by IGR IRESs is therefore resistant to, or even stimulated by, regulatory mechanisms and pharmacological compounds that inhibit the canonical initiation process [ 7 , 11 , 12 ]. The ability of IGR IRESs to bypass the conventional initiation process renders them a useful tool both for the synthesis of biologics in eukaryotic cell-free translation systems (e.g. [ 13 – 16 ]) and to facilitate characterization of the mechanism and inhibitors of downstream steps in eukaryotic translation, including elongation [ 9 , 17 – 19 ] and termination [ 20 – 22 ]. Type 6 IRESs form several subgroups, each with shared structural elements and sequence motifs that underlie differences in mechanism of action. Type 6a IGR IRESs, the first to be characterized, are exemplified by cricket paralysis virus (CrPV). They are ∼190 nt long and consist of three pseudoknot (PK) domains. The nested domain 1 (PKII) and domain 2 (PKIII) are principally responsible for the affinity of ribosomal binding: the conserved internal L1.1a and L1.1b loops in domain 1 bind to the L1 stalk of the ribosomal 60S subunit, and the SL2.1 and SL2.3 stem-loops in domain 2 bind to ribosomal proteins eS25 and uS7, close to the ribosomal E site and the mRNA exit channel [ 23 , 24 ]. Domain 3 (PKI) binds in the decoding center of the 40S ribosomal A site, placing the adjacent region of ORF2 into the mRNA-binding channel [ 24 ]. PKI consists of an element that mimics a tRNA anticodon stem loop (ASL) engaged in codon–anticodon interaction with an mRNA triplet, and a conformationally dynamic loop (L3.2) known as the variable region loop (VRL) [ 25 ] that influences subsequent translocation steps [ 26 ]. In a process termed pseudo-translocation, eEF2 brings PKI from the ribosomal A to the P site and the first codon of ORF2 into the A site; however, this state is meta-stable and the ribosome can undergo spontaneous back-translocation if the codon in the A site is not decoded [ 9 , 20 , 27 , 28 ]. Pseudo-translocation is followed by delivery of a cognate aminoacyl–tRNA to the ribosome in complex with eEF1A and GTP (the elongation factor 1∙GTP∙aa–tRNA TC), release of the aminoacyl–tRNA into the A site, its accommodation, and its eEF2-mediated translocation to the ribosomal P site. This final stage is accompanied by partial unwinding of PKI, such that the ASL-like element relocates to a position equivalent to that of the acceptor stem of E-site tRNA [ 29 ]. There are several other distinct structural subclasses of type 6 IRES, the best-characterized of which [type 6b, exemplified by Taura syndrome virus (TSV) and Israeli acute paralysis virus (IAPV)] contain divergent motifs in L1.1a and L1.1b loops and an additional, functionally important stem-loop in domain 3 [ 30 , 31 ]. Type 6b IRESs are thought to promote adoption by the 40S-IRES complex of a conformation that facilitates ribosomal subunit joining [ 32 ] and influences reading frame selection [ 33 ]. Specific motifs and structures therefore determine specific aspects of function, such as attachment to the ribosomal A site, mimicry of the ASL/codon interaction, engagement with the L1 stalk, and facilitation of subunit joining. Recently identified subclasses of type 6 IRESs lack one or more structural elements that are typical of type 6a and type 6b IRESs and utilize even simpler mechanisms for initiation [ 34 – 36 ]. For example, type 6d IRESs, epitomized by nedicistrovirus (NediV), are only ∼165 nt long, and while they also consist of three PK domains, they lack a SL2.3 stem-loop and canonical motifs in the L1.1a/L1.1b loops [ 35 ]. They bypass the entire canonical initiation process, like type 6a and 6b IRESs, but also bypass the subsequent pseudo-translocation step, and instead bind directly to the P site, placing the first codon of the downstream ORF in the A site, where it is accessible for decoding by the TC. This mode of direct P-site engagement was structurally demonstrated by cryo-electron microscopy (cryo-EM) in the ribosome-bound HalV IRES [ 34 ]. Once initiation is complete, the structured IRES domains dissociate from the ribosome, leaving only the ORF mRNA for the ribosome to transition into the canonical elongation cycle. In each elongation cycle, eEF1A within the TC delivers the incoming aa–tRNA to the A site of the ribosome. Noncognate codon–anticodon mismatches result in the immediate rejection of the TC, while near-cognate and cognate pairings trigger GTP hydrolysis on eEF1A, leading to its release and the accommodation of the aa–tRNA into the A site [ 37 ]. Following this step, eEF2, bound to GTP, triggers intersubunit rotation, a rotation of the small subunit relative to the large subunit [ 38 , 39 ], and thereby translocates the tRNAs together with their bound mRNA (“tRNA2–mRNA”) from the P and A sites into the hybrid P/E and A/P sites, and eventually into the E and P sites. The spatial precision of this process is essential for maintaining high translational fidelity. Although spontaneous intersubunit rotation does occur, leading to translocation of the tRNA2–mRNA moiety, eEF2·GTP accelerates this process [ 19 ]. By binding to the A site, eEF2 induces specific ribosomal conformational changes, thereby ensuring maintenance of the reading frame and enforcing translational fidelity [ 40 , 41 ]. The ribosome is prone to frameshifting during this phase, but the diphthamide modification of eEF2 helps prevent these errors by stabilizing the codon in its correct position [ 38 , 40 , 42 ]. Published structures of eEF2-bound ribosomes span a range of species, from yeast ( Saccharomyces cerevisiae ) (e.g. PDB ID 7OSM) to mammals like rabbit ( Oryctolagus cuniculus , PDB IDs 6MTD and 6MTE) and humans (PDB ID 6Z6M), with resolutions ranging from 11.7Å to 3.0 Å [ 42 – 44 ]. These structures capture ribosomes in different eEF2-bound conformations, including rotated and nonrotated states (such as in PDB IDs 6MTE and 6MTD, bound to SERBP1 and GDP) as well as those with ligands like GDPNP (PDB ID 2P8W) and GDPCP (PDB ID 7OSM) [ 38 , 42 ]. Additional structures of ribosome-eEF2 complexes include Nelfinavir-treated neuronal ribosomes (PDB ID 7LS2), Puromycin-treated ribosomes with EBP1, SERBP1, and eEF2 (PDB ID 6Z6M), and viral IRES-bound ribosomes (PDB IDs 5IT7 and 6D9J) [ 29 , 44 – 46 ]. Crucially, only one of these structures of eEF2-bound ribosomes [ 29 ] formed under natural reaction conditions, and all other structures correspond to complexes assembled in the presence of non-native nucleotides, antibiotics, or biochemical inhibitors. Only a limited number of published structures have captured complexes with eEF1A, including those stabilized by translation inhibitors such as Didemnin B (PDB ID 5LZS), Angiogenin (PDB ID 9BDP), or in the presence of plitidepsin, GTPγS, and anisomycin (PDB ID 8G60) [ 47 – 49 ], as well as one obtained through in situ imaging of yeast lamellae (PDB ID 8Z71) [ 50 ]. Current knowledge of elongation cycle dynamics relies heavily on these static structures, which represent ribosome complexes stabilized in specific states using GTP analogs or other chemical means. However, they leave considerable gaps in knowledge as they do not include short-lived intermediate states instrumental for ensuring fidelity in eukaryotic translation. This study used cryo-EM to determine the structures of rabbit ribosomal initiation and early elongation complexes assembled on the NediV IRES. Included is the visualization of one previously unreported intermediate state in the mammalian translocation cycle: the eEF1A-bound ribosome with A*/T–tRNA stalled at a codon–anticodon mismatch. Its structure reveals a distortion in the tRNA anticodon loop and unusual eEF1A positioning, as well as offering insights into the structure of the TC in this state. Notably, this study characterizes biologically relevant complexes on pathway bearing GTP, validating previous conclusions drawn from kinetically trapped complexes. The structures we present form a detailed molecular timeline of IGR IRES-mediated translation initiation and its seamless handoff to canonical elongation. It is seen in intriguing detail how the NediV IRES directly binds to the 80S ribosome’s P site, bypassing pseudo-translocation, and thereby establishes a stable platform for accurate initiation. Our structural snapshots illuminate how IRES dynamics go hand in hand with ribosomal conformational changes and the binding of elongation factors in enabling efficient translation. Most importantly, we observe a novel eEF1A-bound failed decoding complex, captured without chemical trapping, that highlights a mechanistic basis for fidelity control. These insights not only clarify the NediV IRES’s unique strategy but also shed light on the nature of conserved translational checkpoints, underscoring the utility of IGR IRESs in dissecting translation mechanisms. Materials and methods Plasmids The transcription vector for rabbit cytoplasmic tRNA Ala (GenBank accession no. Y17041 ), and expression vectors for eRF1 and a truncated form of eRF3 lacking a.a. 1–138 have been described [ 9 , 51 ]. Transcription vectors for NediV IGR IRES-containing mRNAs were made by GeneWiz (South Plainfield, NJ). pUC57-NediV (wt) [ 35 ] contains a stable hairpin (5′-GGCTCGAGGCCCGGTGACGGGCCTCGGGCC-3′ [ΔG = −32.70 kcal/mol]) and NediV nt 1161–1606, inserted downstream from a T7 promoter between BamH1 and XbaI sites of pUC57. The second codon [ACA (Thr)] of the ORF was substituted by a UAA stop codon in the NediV-[Ala.STOP] variant ( Supplementary Fig. S3D ) Purification of factors, ribosomal subunits, and aminoacylation of tRNA Native eEF1H, eEF2, total aa–tRNA synthetases and 40S and 60S ribosomal subunits were purified from rabbit reticulocyte lysate (Green Hectares, Oregon, WI) [ 52 , 53 ]. eRF1 and eRF3 were purified after expression in Escherichia coli [ 51 ]. mRNAs and tRNA were transcribed using T7 RNA polymerase (Thermo Fisher). In vitro transcribed tRNA Ala was refolded and then aminoacylated using total native aminoacyl–tRNA synthetases [ 9 ]. Assembly and analysis of ribosomal complexes Binary IRES/80S ribosome complexes were assembled by incubating 7 pmol NediV (wt) mRNA for 5 min at 37°C in 40ul reaction volumes that contained buffer A [2 mM dithiothreitol, 100 mM potassium chloride, 20 mM Tris (pH 7.5), 2.5 mM magnesium chloride, and 0.25 mM spermidine] supplemented with 3.2 pmol 40S subunits and 5 pmol 60S subunits. Ribosomal elongation complexes were assembled by incubating 7 pmol NediV (wt) or NediV-[Ala.STOP] mRNA for 5 min at 37°C in 40 μl reaction volumes that contained buffer A supplemented with 1 mM GTP and 3.2 pmol 40S subunits, 5 pmol 60S subunits, 7pmol Ala–tRNA Ala , 10 pmol eEF1H, 15 pmol eEF2, 15 pmol eRF1, and 20 pmol eRF3. Assembly and translocation of complexes was confirmed by toe-printing using avian myeloblastosis virus reverse transcriptase (Promega) and a cognate primer as described [ 35 ]. Sample preparation and data collection For (A) the NediV-IRES:80S complex (binary setup), (B) the NediV-IRES∙80S∙tRNA∙eEF2 complex (elongation setup) and (C) the NediV-IRES∙80S∙tRNA∙eRF1 complex (termination setup), 3 μl of ribosomal complexes at concentrations of (A) 500 nM and (B, C) 80 nM were applied to plasma-treated, thin carbon-coated holey carbon grids (QUANTIFOIL R1.2/1.3) [ 54 ], blotted for (A) 5 s and (B, C) 9 s, and rapidly frozen in liquid ethane using an FEI Vitrobot. Following freezing, the grids were transferred to a Polara-G2 microscope (FEI, now part of Thermo Fisher Scientific, OR), operating at 300 kV and equipped with a Gatan K2 Summit direct detector. For the NediV-IRES∙80S complex, 10 690 frame stacks were captured in counting mode with a dose rate of 8e − /pix/s at a magnification of 31 000×, corresponding to a calibrated pixel size of 1.24 Å. For the NediV-IRES∙80S∙tRNA∙eEF2 complex, 4193 frame stacks were recorded in counting mode with a dose rate of 8e − /pix/s at a magnification of 23 000×, yielding a calibrated pixel size of 1.66 Å. For the NediV-IRES∙80S∙tRNA∙eRF1 complex, 3273 frame stacks were collected in counting mode at a dose rate of 16e − /pix/s at a magnification of 39 000×, with a calibrated pixel size of 0.95 Å. Frame stacks for all complexes were recorded in automatic mode using Leginon software [ 55 ], which determined that defocus values ranged (A) from 2.5 to 3.5 μm and (B, C) from 1.5 to 3.5 μm. Frame alignment was performed using Motioncor2 [ 56 ], and data collection was monitored in real time using APPION [ 57 ]. Data processing and classification For all ribosomal complexes, motion-corrected micrographs were manually screened for quality. CTF parameters were estimated using GCTF [ 58 ]. Particle picking was performed using Gaussian picking in GAUTOMATCH, with a particle diameter set to 350 Å. Subsequent 2D and 3D classifications and refinements were carried out using RELION 4 [ 59 , 60 ] and Cryosparc [ 61 ]. The picked particles were binned four times and subjected to 2D classification to separate 80S ribosomes from nonribosomal particles and noise. Initial consensus models for the 80S particle sets were generated using the 3D Refine procedure. A mask enclosing ∼1/3 of the 40S, IRES, and 60S was created to prioritize IRES density and applied in RELION for separate 3D classification of each dataset. The resulting classes were divided into IRES-containing complexes and E-site tRNA-containing complexes. The latter, as they are no longer bound to the IRES but retain the linked ORF portion of the viral mRNA, can be considered elongation complexes on the canonical pathway. High-resolution reconstructions of IRES-containing complexes without tRNA or additional factors were also obtained. Masked refinements were performed on the unbinned data for selected classes using RELION, followed by nonuniform refinement in cryoSPARC [ 61 ] to improve resolution in flexible regions. These analyses of the NediV-IRES:80S dataset yielded clean density maps, primarily consisting of 80S ribosomes with P-site-loaded IRES, 80S ribosomes with A-site-loaded IRES, and empty 80S ribosomes. For the NediV-IRES∙80S∙tRNA∙eEF2 dataset, similar masked classifications identified high-resolution classes, including 80S with a nontranslocated IRES and A-site tRNA, 80S with a singly translocated IRES and P-site tRNA, 80S with a singly translocated IRES, P-site tRNA, and eEF2, and 80S in a canonical elongation complex with eEF1A and A*/T tRNA. Finally, the NediV-IRES∙80S:tRNA∙eRF1 dataset yielded multiple termination complexes (to be presented elsewhere) as well as a canonical elongation complex containing eEF2 and P-site tRNA. Despite these refinements, some factor densities remained poorly resolved. To better address dataset heterogeneity, cryoSPARC variability analysis and CryoDRGN [ 62 ] were employed. Model building Atomic models were built for the major complexes: (A) the NediV-IRES·80S binary complexes, (B) the NediV-IRES·80S·tRNA complex, (C) the NediV-IRES·80S·tRNA·eEF2 complexes, and (D) the NediV-IRES·80S·tRNA·eEF1A complex. For the binary complexes(A), the 80S ribosome from PDB 5LZS was used as the initial model. For (B), (C), and (D), the starting models were 5LZS (ribosome and eEF1A) and 6GZ5 (eEF2 and tRNAs) respectively. The ribosomal RNA (rRNA) sequences were changed for rabbit rRNA based on the study by De et al . (2025) [ 63 ]. The NediV IRES was modeled de novo in Coot [ 64 ], guided by its predicted secondary structure [ 35 ]. All atomic models were further adjusted manually in Coot and ChimeraX [ 65 ] to optimize fit into the experimental density maps. Final refinements were performed using Phenix real-space refine [ 66 ], with appropriate secondary structure restraints and validation metrics monitored throughout. Results Cryo-EM analysis of ribosomes bound to the NediV IRES Cryo-EM analysis of binary NediV IRES∙80S ribosome complexes revealed two distinct classes displaying a canonical nonrotated ribosomal state (i.e. without intersubunit rotation) with domain 3 (PKI) bound to either the ribosomal P site or to the A site (Fig. 1A and B , and Supplementary Figs S1 , and S2A and B ). The resolution of these classes was 3.1 and 3.5 Å, respectively (Table 1 , and Supplementary Tables S1 and S2 ). As with other type 6 IRESs, NediV IRES∙80S complexes are conformationally dynamic and sample different rotational states [ 27 , 34 , 67 – 69 ]. Another IRES-containing class with a rotated ribosome was obtained, but the IRES density was too noisy for model building or further analysis ( Supplementary Fig. S3A–C ). Figure 1. Open in a new tab Structural Features of the NediV IRES in the Initiation Complex. ( A, B ) Cryo-EM density maps of the NediV IRES (dark orange) bound to the mammalian 80S ribosome (gray). The maps reveal two distinct conformational classes: the more abundant P-site-loaded state ( A ) and the less-stable A-site-loaded state ( B ). ( C , D ) Zoomed-in view of the NediV IRES within the ribosomal mRNA cleft, with the model shown as a cartoon and the map displayed as a transparent surface. Panel (C) highlights the well-ordered IRES occupying the P site in a canonical, nonrotated ribosomal state. Panel (D) shows the less-ordered IRES in the A site, which appears to block access to both the A and P sites. ( E ) Zoomed-in view of the P-site-bound IRES and 18S rRNA residues, demonstrating the high-quality resolution of the map in this region, which enabled detailed structural analysis of the IRES-ribosome interactions. ( F ) Predicted secondary structure of the full-length NediV IRES [ 35 ], color-coded to delineate its characteristic domains, including the nested PKs (PKII and PKIII) and the independently folded PKI. ( G ) A structural comparison of the NediV IRES (type 6d) and CrPV IRES (type 6a) (PDB ID 6D90) showing the length difference between the IRESs after aligning the PKI for both the IRESs. Table 1. Cryo-EM data collection, refinement, and validation statistics Ribosomal complex P-site IRES(PDB 9q1s; EMDB 72137) A-site IRES(PDB 9q1q; EMDB 72136) P-site IRES A-site tRNA(PDB 9q2o; EMDB 72170) eEF2 IRES(PDB 9q2t; EMDB 72175) Canonical eEF1A(PDB 9q2p; EMDB 72171) Canonical eEF2(PDB 9q2m; EMDB 72168) Data collection and processing Electron microscope Polara F30 Polara F30 Polara F30 Polara F30 Polara F30 Polara F30 Detector K2 Summit K2 Summit K2 Summit K2 Summit K2 Summit K3 Summit Magnification 31 000 31 000 31 000 23 000 23 000 39 000 Voltage (kV) 300 300 300 300 300 300 Electron exposure (e–/Å 2 ) 42 42 23 23 23 71 Defocus range (μm) −3.5 to −1.5 −3.5 to −1.5 −3.5 to −1.5 −3.5 to −1.5 −3.5 to −1.5 −3.5 to −1.5 Pixel size (Å) 1.24 1.24 1.66 1.66 1.66 0.95 Symmetry imposed C1 C1 C1 C1 C1 C1 Initial particle images (no.) 499 723 499 723 546 318 546 318 546 318 347 454 Final particle images (no.) 264 326 27 739 23 922 17 367 107 135 34 773 FSC threshold 0.143 0.143 0.143 0.143 0.143 0.143 Map resolution (Å) 3.1 3.5 4.1 4.1 3.4 3.3 Refinement Initial model used (PDB code) 5LZS 5LZS 5LZS 5LZS 5LZS 5LZS Model composition Atoms 208 596 218 573 219 082 231433 225 300 225 300 Protein residues 10 853 11 894 11 583 12 565 12 137 12 234 Nucleotide residues 5645 5734 5752 5739 5740 5612 Ligands Zn:8 Mg:206 Zn:8 Mg:206 Zn:8 Mg:206 Zn:5 Mg:205 Zn:8 Mg:276 Zn:8 Mg:206 RMS deviations Bond lengths (Å) 0.002 0.004 0.002 0.002 0.004 0.013 Bond angles (°) 0.477 0.793 0.583 0.525 0.555 0.608 Validation MolProbity score 2.14 2.14 2.62 2.46 1.77 2.56 Clashscore 8.13 2.38 26.6 21.8 6.79 20.9 Poor rotamers (%) 4.84 8.55 4.92 3.72 1.47 4.62 Ramachandran plot Favored (%) 96.99 93.68 96.91 96.86 96.10 96.41 Allowed (%) 3.01 6.22 3.06 3.10 3.86 3.53 Disallowed (%) 0.00 0.10 0.03 0.04 0.04 0.07 Open in a new tab The ribosome-bound NediV IRES has the characteristic architecture of type 6 IRESs, comprising the nested PKII and PKIII and the independently folded PKI (Fig. 1F and Supplementary Fig. S1 ). The NediV PKI domain closely resembles PKI of the type 6a CrPV IRES, which comprises helix P3.1 [11 base-pairs (bp) and one unpaired nucleotide], helix P3.2 (5 bp), the 9 nt-long VRL that connects them, and one unpaired nucleotide. The equivalent elements in the NediV IRES are helix P3.1 (comprising 9 bp and two unpaired nucleotides), helix P3.2 (5 bp), the 7 nt-long VRL, and one unpaired nucleotide [ 35 ]. The principal differences between type 6d NediV-like IRESs and type 6a IRESs are that domain 1 in the former has conserved motifs in loops L1.1a and L1.1b that are unlike those in type 6a IRESs, and domain 2 has a simple loop in place of the SL2.3 stem-loop (Fig. 1G ). These differences likely account for the different positions of type 6a and 6d IRESs in binary ribosomal complexes, as described below. Type 6a IRESs bind in the A site [ 24 ], whereas PKI is in the P site in the overwhelming majority of binary NediV IRES:80S complexes (Fig. 1A and C ). This location is consistent with toe-printing and other biochemical data, which showed that in elongation-competent complexes, the NediV IRES occupies the P site and the first codon of ORF2 is placed in the A site [ 35 ]. Binding of the NediV IRES to the P site is stabilized by three, and possibly four sets of interactions with the ribosome ( Supplementary Fig. S2 ). First, the L1.1 region in domain 1 binds to ribosomal protein uL1 of the 60S subunit and to nucleotides of the L1 stalk ( Supplementary Fig. S2B–D ). These interactions anchor domain 2 securely to facilitate the insertion of the remainder of the IRES into the mRNA cleft and intersubunit space. PKI binds to the ribosome via residue G1466, which interacts with nt. 1701 of the 18S rRNA at a distance of 7.7 Å, while G1467 is positioned near 18S rRNA nt.1248, with a distance of 5.8 Å (Fig. 1E and Supplementary Fig. S4A ). G1468 interacts with Arg140 of uS19, maintaining a distance of 9.0 Å, and IRES residues A1498 and A1500 are in close proximity to Gln113 and Phe124 of ribosomal protein uS5 and A1485 to Arg66 of uS11, creating stabilizing interactions on both sides of PKI and place PKI firmly into the ribosomal P site ( Supplementary Fig. S4A–C ). The third set of interactions involves the VRL, which interacts with ribosomal protein uS7 ( Supplementary Fig. S2B and C ). Finally, C1423, in the highly conserved L2.2 loop in domain 2, is in close proximity to eS25 at the head of the 40S subunit ( Supplementary Fig. S2E and F ), but details of this interaction could not be established because of the lower resolution of the map in this area. The inability of the NediV IRES to establish a stable interaction with eS25 may be a contributing factor to the weak binding of this IRES to the 40S subunit alone, and to its preferred binding to the P site in 80S ribosomes. The functional importance of each of these binding sites has been confirmed by mutational analysis of the NediV IRES and of the related type 6d Antarctic picorna-like virus (APLV) IRES [ 35 ]. Notably, identical or similar interactions have been observed in the binary 80S∙HalV IRES complex [ 34 ], and in the post-translocation type 6a (CrPV) and type 6b (TSV and IAPV) IRESs [ 20 , 32 , 69 ]. The NediV IRES forms a complex, by binding directly to the 80S ribosome, that is directly analogous to complexes formed on type 6a and 6b IRESs after they have bound to 80S ribosomes and undergone pseudo-translocation. A substantially smaller proportion of binary complexes contain the NediV IRES bound to the A site: our cryo-EM analysis revealed this conformation in only 5.5% of particles (27 739 out of 499 723), indicative of a transient and unstable nature. Consistently, domain 3 (PKI) in these complexes appears disordered, allowing only a portion of the IRES entering the A site to be atomically modeled (Fig. 1B and D ). The P site-loaded state thus appears to be more stable than the A site-loaded state, and is well-resolved, ordered and (as described above) forms multiple contacts with the ribosome. While the A site is accessible for binding of aminoacyl–tRNA in the P site-bound state, the NediV IRES binds to the A site of nonrotated ribosomes in a perpendicular position, extending through the P site and effectively blocking access of aminoacylated tRNA to both A and P sites. This A site-loaded complex is therefore structurally distinct from pre-pseudotranslocation A site complexes assembled on type 6a and 6b IRESs [ 27 , 32 , 67 ]. Binary NediV IRES/80S ribosomal complexes yielded strong toeprints that correspond to the P-site-loaded ribosomal complex and a second weak set [ 35 ] that may reflect the noncanonical positioning of the IRES in the A site observed here. As binding of the IRES to the ribosome is reversible, the A site-bound state can dissociate, allowing the IRES to bind to the P site, but it could also potentially relax into the more stable P site-bound state through the intrinsic conformational dynamics of the IRES-ribosome complex, thereby committing the ribosome to elongation, as subsequent A site binding of aa–tRNA would preclude re-occupation of the A site by the IRES. However, we need to emphasize that this A site-loaded state represents a minor population, and there is currently no direct evidence that it lies on the productive translation pathway. The structure of 80S∙NediV IRES∙aa–tRNA complexes Decoding of the A-site codon in binary 80S∙NediV IRES complexes yields a transient ternary [80S∙IRES∙aa–tRNA] complex, and although the latter corresponds to a key stage in translation mediated by type 6 IRESs, its structure has not previously been determined. To capture this complex, the NediV wt IRES was incubated with Ala–tRNA Ala , eEF1H (a complex of eIF1A and its guanine-nucleotide-exchange factor eEF1B), eEF2, GTP, and mammalian 80S ribosomes pre-assembled from purified 40S and 60S subunits (also defined as ‘elongation setup’ in the ‘Materials and methods’ section). This preparation yielded high-quality cryo-EM data, with 4139 micrographs collected at a resolution of 1.66 Å per pixel. Following particle selection and 2D classification, 251 173 particles corresponding to 80S ribosomes bound to the IRES and Ala–tRNA Ala , together with heterogeneous densities from elongation factors or additional tRNAs, were refined into ten distinct ribosomal classes. Of these, only one class lacked bound factors and displayed sufficiently well-resolved tRNA density to permit model building at 4.1 Å resolution ( Supplementary Tables 1 and 2 ). Since Ala–tRNA Ala binding is mediated by eEF1H·GTP, the absence of this factor together with full accommodation of the tRNA in the A site (Fig. 2A and C ) indicates successful direct recognition of the A-site codon by cognate tRNA and consequent triggering of GTP hydrolysis, leading to release of Ala–tRNA Ala and dissociation of eEF1H∙GDP. Figure 2. Open in a new tab Translation Elongation Complexes on the NediV IRES. ( A, B ) Cryo-EM reconstructions illustrating the transition from initiation to the first elongation step. The NediV IRES is shown in dark orange, with the ribosome in gray. ( A ) Shows the initial TC with Ala–tRNA (cyan) bound in the A site. ( B ) Shows the post-translocation complex, featuring eEF2 (crimson) and a translocated P-site tRNA (teal). ( C , D ) Focused views of the complexes shown in panels (A) and (C), respectively, providing a detailed look at the positioning of the IRES and its interactions with the ribosome, tRNA, and eEF2. ( E ) Structural alignment of the binary (80S + IRES) and quaternary (80S + IRES + tRNA + eEF2) complexes, showing the conformational changes of the IRES itself. Panels ( E ) illustrates how Domains 2 and 3 of the IRES move by ∼13–14 Å relative to their position in the binary complex as the ribosome transitions to the elongation state. ( F ) A close-up view of IRES Domain 1 models, showing the conformational rearrangements that occur to accommodate the full elongation machinery. The NediV IRES remains stably bound to the ribosomal P site (Fig. 2B and D ) in presence of the tRNA, showing minimal movement relative to the 18S rRNA. C1243 in the L2.2 loop remains in close proximity to eS25 as in the binary complex ( Supplementary Fig. S2E ), and there is no significant compression or expansion of domains 1 and 2 in this complex relative to the binary complex (Fig. 2C – F ). Indeed, the initial binding of Ala–tRNA Ala to the ribosomal A site appears to stabilize the overall ribosome-IRES interaction, particularly enhancing the rigidity of domains 1 and 2 (Fig. 2D ). These and other interactions observed in the binary complex anchor domains 1 and 2 of the IRES, allowing the ribosome to adopt both canonical and hybrid states, which are required for elongation. This dynamic behavior is consistent with that of type 6a IRES elements, such as CrPV [ 27 ], but differs significantly from IAPV and TSV type 6b IRESs, which stabilize the ribosome in the nonrotated state [ 32 , 69 ]. The IRES in the Ala–tRNA Ala -bound complex is rotated 2–3 degrees away from the L1 stalk relative to its position in the binary 80S∙NediV IRES complex. The dynamic behavior of the 40S subunit in these complexes mirrors the dynamics observed in canonical elongation, where tRNA delivery to the ribosomal A site induces conformational changes in the ribosome, including reorientation of G626 and flipping-out of the ‘monitoring’ nucleotides A1824 and A1825 from helix 44 (corresponding to G530, A1492, and A1493 in E. coli ), as well as domain closure [ 47 , 70 – 72 ] ( Supplementary Fig. S5 ). The structure of the post-translocation complex with eEF2 The 80S∙IRES∙aa–tRNA complex resembles neither the rotated-1 state [ 70 ] nor the rotated-2-Pre state [ 72 ], with a meager 1.1-degree rotation observed in our structure. Instead, it has formed during the canonical elongation process, except that the P site in the 80S∙IRES∙aa–tRNA complex is occupied by PKI rather than by aminoacylated tRNA. This complex is thus primed for the binding of eEF2 and subsequent translocation. To investigate the binding of eEF2 and the ensuing translocation step, we assembled the 80S∙IRES∙aa–tRNA∙eEF2 complex by incubating the NediV IRES with Ala–tRNA Ala , eEF1H, eEF2, GTP, and mammalian 80S ribosomes pre-assembled from purified 40S and 60S subunits (defined as the ‘elongation setup’ in the ‘Materials and methods’ section). A total of 4139 micrographs were collected at 1.66Å/pixel resolution. 3D classification identified one class with 18 360 particles ( Supplementary Table 1 ), capturing the ribosome in the nonrotated state. In this state, eEF2 occupies the A site, tRNA has been translocated to the P site, and the IRES has also been singly translocated (Fig. 2C – F ). The position of the 40S subunit head remains largely unchanged in this eEF2/P-site tRNA state compared to the 80S∙NediV IRES complex with aa–tRNA in the A site. Detailed structural comparisons show that these complexes closely resemble the POST-3 state of the elongation complex (PDB ID 6GZ5) [ 40 ]. The present eEF2-bound structure maintains a near-zero-degree rotation for both the head and body, suggesting that it corresponds to a complex at the end of the translocation cycle, immediately prior to the release of eEF2. Two key structural differences are apparent between the IRES in this and the preceding two complexes. Domains 2 and 3 have moved ∼13–14 Å relative to their position in the binary complex, to a position close to the 40S head (Fig. 2E and F ). While the P site-bound PKI was stabilized in the presence of A site tRNA (Fig. 2D ), the translocated PKI in complexes containing P-site tRNA was destabilized, and is visible only up to A1473, 14.8Å from C40 of the tRNA (Fig. 3A and B ). The destabilization of NediV PKI in the E site, which is reminiscent of the disassembly of the analogous domain in the CrPV IRES upon translocation from the P to the E site [ 29 ], may support the threading of the viral ORF2 coding region into the mRNA channel, preparing the ribosome for elongation. The IRES mRNA is well resolved all the way along its path through the ribosome. Past the A site, it follows the mRNA cleft while interacting closely with the 18S rRNA (sandwiched between residues 1825 and 1701) and ribosomal proteins uS3, and uS5 ( Supplementary Figs S2B , and S6A and B ). Figure 3. Open in a new tab Elongation Complex in the Presence of eEF2. ( A ) Cryo-EM structure of a fully translocated elongation complex, captured from the termination setup. This state contains a translocated partial P-site tRNA (dodger blue, labeled as P–tRNA*), an E-site tRNA (teal), and eEF2 (crimson). This class represents the transition from IRES-mediated initiation to canonical elongation. ( B, C ) Focused views of the ligands in the complex in panel (A). Panel (B) shows the cryo-EM map/densities, and panel (C) shows the atomic model, highlighting the arrangement of eEF2 and the two tRNAs. Notably, the P-site tRNA density is very noisy and only can be visualized at its lower half (labeled as P–tRNA*), while the off-pathway E-site tRNA is exceptionally stable, reflecting the strength of the stable occupation of the E site. ( D , E ) Comparative analysis of the tRNAs in the E and P sites with the corresponding ones from PDB ID 6GZ5. These panels highlight the unusually close proximity between the P-site and E-site tRNAs, with their backbones nearing a steric clash, a novel observation in eukaryotic elongation complexes. ( F ) A comparative analysis of the eEF2 conformation in our canonical complex versus the POST-3 state (PDB ID 6GZ5). We also isolated a distinct group of 34 773 particles from our termination setup experiment (see the ‘Materials and methods’ section) in which we could identify eEF2 on the ribosome (Fig. 3A ). In this class we captured an eEF2-containing 80S ribosome with two tRNAs present and only the ORF mRNA visible (Fig. 3B and C ), marking the transition into a canonical elongation state. This class displays a unique arrangement of eEF2 and the P- and E- tRNAs, with a slight (1.4°) intersubunit rotation, engaging in previously characterized tRNA-mRNA and tRNA–ribosomal interactions [ 41 ] (Fig. 3A ). The lower half of the P-site tRNA site is significantly better resolved than the rest of the molecule (Fig. 3B – E ), reflecting the strength of the codon–anticodon interaction established after the first translocation step. Although the appearance of deacylated tRNA in the E site may reflect binding due to its presence in molar excess over ribosomes during sample preparation (e.g. [ 70 ]), stable occupation of the E site by deacylated tRNA is a characteristic of eukaryotic elongation complexes [ 72 , 73 ]. The structural map strongly resembles the classical POST-3 state of elongation, with tRNAs in the P and E sites and the L1 stalk rotated into a stabilized conformation, consistent with observations in bacterial and rabbit elongation complexes [ 40 , 41 , 74 ]. The subtle tilt of the 40S subunit head (1.4°) could either be a remnant of the prior translocation event or an inherent feature of mammalian ribosomes at rest. A particularly striking feature of this class is the unprecedentedly close proximity of the P-site and E-site tRNAs. Across all classes, the E-site tRNA displays exceptional stability and high occupancy, potentially having entered the ribosome via the open L1 stalk. Meanwhile, the P-site tRNA part of the map exhibits lower resolution, with the anticodon arm, T-arm, D-arm, and acceptor stem largely unresolved, indicating weaker stabilization. Notably, the P-site tRNA appears to invade the E site, bringing its backbone into close contact with the backbone of the nearest E-site tRNA residues, nearing steric clash ( Supplementary Fig. S7 ). Both structural classes exhibit nearly identical positioning of eEF2, which interacts with the ribosomal GTPase-associated center (GAC) of the large ribosomal subunit. The ribosome-eEF2 complex undergoes significant conformational rearrangements upon GTP hydrolysis, as observed in 80S∙ADPR-eEF2∙GDPNP and 80S∙ADPR-eEF2∙GDP∙sordarin structures [ 38 , 41 ]. These rearrangements shift domains G′, I, and II of eEF2 toward the GAC while displacing domains III, IV, and V further away. The Switch 1 loop, a key regulatory element, adopts an ordered conformation in the GTP-bound state, similar to EF-G [ 75 , 76 ], engaging the terminal phosphate groups of GTP via Mg²⁺ coordination. Following GTP hydrolysis, the Switch 1 loop becomes disordered, as confirmed by 80S∙ADPR-eEF2∙GDP∙sordarin structures (PDB ID 5JUU) [ 69 ]. This disordering occurs in proximity to the sarcin-ricin loop, a crucial ribosomal site for triggering GTP hydrolysis. Post-hydrolysis, domains III and V shift relative to Switch 2 in domain I, with domain III rotating by ∼5 degrees and domains IV and V rotating together by ∼9 degrees. This movement repositions the diphthamide-containing tip of domain IV by ∼6 Å toward the top of helix 44 in the small ribosomal subunit (2P8X [ 38 ], 5JUU [ 69 ]). In our structures, eEF2 exhibits hallmark features of the post-GTP hydrolysis state: The Switch I loop is partially disordered; although the backbone could be modeled in both classes, the His108 side chain is only faintly visible in the IRES-containing eEF2 complex and better visible in the canonical eEF2 complex. Switch II is completely unresolvable in both complexes, which is consistent with a GDP-bound or nucleotide-release state. Additionally, domain II is rotated relative to the GTP-bound conformation, and the diphthamide-containing loop of domain IV is retracted by ∼6 Å (Fig. 3F ). While no nucleotide density is observed in the IRES-bound eEF2 class, the canonical eEF2 class contains interpretable density for the nucleoside and two phosphates. We attempted to model both GTP and GDP into the density, and found that GDP provides a better fit ( Supplementary Fig. S8A–C ). These features clearly distinguish our structures from previously published GTP-bound eEF2 conformations such as 6GZ5 [ 40 ], where both Switch loops were resolved. Together, the data support the assignment of our eEF2 conformations to the post-GTP hydrolysis state. Domain IV of eEF2 contains three loops—loop 1, loop 2, and loop 3, all critical for its function. Consistent with the results of Flis et al . (2018) [ 40 ] and Djumagulov et al . (2021) [ 42 ], our analysis shows that the diphthamide residue of domain IV stacks against the ribose of A36 in peptidyl–tRNA ( Supplementary Fig. S8D ). Loop 2 interacts with helix 32, anchoring domain IV near the A site, while loop 3 engages with 18S rRNA h44, likely to stabilize the reading frame ( Supplementary Fig. S8D ). Additional stabilizing interactions, previously underexplored, occur between eEF2 domain IV and 28S rRNA of the 60S subunit, involving water- or metal-ion-mediated contacts: Trp 685 with C1’ of C3761 (7.22 Å), Ile 722 with C1’ of A3760 (5.58 Å), and Gly 719 with C1’ of A3760 (5.1 Å) ( Supplementary Fig. S8E and F ). Overall, the comparison of these two structural classes (one with IRES-bound 80S in complex with eEF2 and the other with canonical mRNA-bound 80S in complex with eEF2) highlights a clear transition from an IRES-dependent initiation complex to a canonical elongation complex ( Supplementary Fig. S9 ), with eEF2 playing a central role in coordinating tRNA translocation and stabilizing the ribosomal complex. While the first class still retains a partially visible IRES, the second class represents a fully canonical elongation state with only the ORF mRNA present along with tRNAs bound to the 40S subunit. Both classes feature a slightly rotated ribosome. The structure of an eEF1A-bound, failed decoding complex One of the classes (107 135 particles; 3.4 Å resolution) from our elongation setup, which includes eEF1H, eEF2, and Ala–tRNA Ala , shows clear map coverage for eEF1A, A*/T-Ala–tRNA Ala , P–tRNA Ala , E–tRNA Ala , and the translocated mRNA encoding Ala–Thr–His (Fig. 4A – C and Supplementary Fig. S3D ). Determining the exact codon register is complicated by blurred mRNA density arising from residual heterogeneity within the class. Nevertheless, the exclusive presence of Ala–tRNA Ala allowed placement of the Ala codon (GCU) in the P site and modeling of the mRNA as a singly translocated complex. In this structure, the A site contains a mismatched codon–anticodon pair, resulting in a disordered anticodon stem but a well-resolved tRNA body, consistent with an Ala–tRNA Ala undergoing rejection from the A site (Fig. 4A – D ). Another class from the same elongation setup shows density consistent with eEF1A, A*/T-Ala–tRNA Ala , P-site tRNA Ala , and E-site tRNA Ala ; however, it was not included in further analysis due to its lower resolution and insufficient map quality. Figure 4. Open in a new tab eEF1A-Mediated tRNA Delivery and Failed Decoding. ( A ) Cryo-EM map of a canonical A*/T complex, featuring an E-site tRNA (teal), a P-site tRNA (dodger blue), and an A*/T–tRNA (cyan) bound to eEF1A (crimson). This structure captures a key intermediate state during the proofreading process, where a mismatched tRNA is being rejected from the A site. ( B , C ) Focused views of the ligands in the complex ( A ), showing the cryo-EM map ( B ) and the atomic model ( C ). These panels highlight the positioning of eEF1A and the A*/T–tRNA, demonstrating the unique failed decoding conformation where eEF1A is shifted along its axis away from the A site. ( D ) Structural comparison of the failed decoding complex with the Didemnin B-trapped cognate complex (PDB ID 5LZS) and with the complex in the presence of plitidepsin, GTPγS, and anisomycin (PDB ID 8G60).The structures were aligned using the 60S subunit, and the relative positions of the tRNAs and eEF1As were compared by measuring RMSDs both with and without alignment. Comparison with the ribosome-bound eEF1A structure from PDB ID 5LZS [ 47 ] shows substantial differences when aligned based on the 60S subunit: the root-mean-square deviation (RMSD) of the peptide backbone is 9.1 Å for Domain 1, 5.96 Å for Domain 2, and 9.1 Å for Domain 3, with an overall protein RMSD of 8.5 Å. However, when the individual eEF1As are superimposed, the overall RMSD drops to 1.28 Å, with our eEF1A shifted by ∼10 Å away from the A site (Fig. 4D , right panel, and Supplementary Fig. S10A ). In contrast, comparison with the codon-recognition state from PDB ID 8G60 shows much closer agreement: the RMSD of the peptide backbone is 1.7 Å for Domain 1, 1.77 Å for Domain 2, and 2.23 Å for Domain 3, with an overall protein RMSD of 1.86 Å, and an RMSD of 1.21 Å after superimposing the individual eEF1As. The Cα atom of residue Lys165 in eEF1A differs by only ∼0.9 Å from 8G60, compared to a ∼10.1 Å shift relative to 5LZS, indicating that the position of eEF1A in our structure closely resembles the codon-recognition intermediate. The A*/T–tRNA adopts a conformation highly similar to the A/T–tRNA in PDB 5LZS, with an RMSD of 4.14 Å for the phosphate backbone, except for the flexible residues 31–38, which could not be modeled (Fig. 4D , left panel). In contrast, comparison with PDB 8G60 shows a much larger deviation, with an RMSD of 9.05 Å between untranslated raw coordinates. For example, the backbone of residue 57 in our tRNA aligns closely with 5LZS (∼2 Å separation) but is displaced by ∼8.4 Å relative to 8G60. Overall, these comparisons indicate that our complex combines a tRNA conformation resembling the classical A/T state, albeit with a variably positioned anticodon loop, with an eEF1A conformation characteristic of a codon-recognition intermediate (Fig. 4D ). Together, these features suggest that this complex represents a failed decoding intermediate, in which eEF1A has progressed toward a recognition-like state, but the tRNA has been unable to adopt a conformation productive for decoding. The acceptor arm of the A*/T–tRNA positions G1 near eEF1A residues Y254 and R322 ( Supplementary Fig. S10C ), while A76 inserts into a hydrophobic pocket formed by I259, G258, G257, I256, and Y254 ( Supplementary Fig. S10D ), resulting in tight binding of the tRNA to eEF1A. Notably, the PDB 5LZS complex was trapped with Didemnin B and a fully paired codon–anticodon interaction, whereas PDB 8G60 was captured in the presence of plitidepsin, GTPγS, and anisomycin. In contrast, our structure was obtained in the presence of GTP and contains a mismatched codon–anticodon pair (Fig. 4D ). In our structure, the A*/T–tRNA interacts with uL11 (C57 with Thr25) and eS30 (backbone contacts between residues 25–26 and amino acids 76–80), stabilizing its position in the A site ( Supplementary Fig. S11A and B ). eEF1A remains tightly associated with the tRNA through multiple contacts involving domain III α-helices and the tRNA backbone ( Supplementar y Fig. S10C and D ), while also engaging 28S rRNA via domain I and 18S rRNA via domain II. Additionally, domain II interacts with uS12 Arg142, potentially facilitating positioning of the eEF1A·tRNA complex within the A-site cleft ( Supplementary Fig. S10B ). The P- and E-site tRNAs adopt canonical conformations, and the ribosome remains largely nonrotated, with only a ∼1.4° rotation of the 40S body. Discussion Initiation of translation mediated by the NediV IRES involves its direct binding to the P site of the 80S ribosome, and this initiation process is therefore even simpler than on type 6a and type 6b IRESs, which involves binding of the IRES to the A site followed by pseudo-translocation to the P site. Here, we have determined the structures of ribosomal complexes at key stages during initiation and early elongation on this IRES. Initiation on the NediV IRES The multiple stabilizing interactions of the IRES with the ribosome in binary complexes (Fig. 1A and Supplementary Fig. S2 ) likely account for its ability to bind directly to the P site, bypassing the pseudo-translocation step, and for the strong preference of this class of IRES for binding to 80S ribosomes rather than to 40S subunits. The ability of the IRES to maintain a stable interaction with the ribosomal P site in the 80S∙NediV IRES∙aa–tRNA complex, even across varying rotational states (Fig. 2A ), is instrumental in facilitating initiation. This complex stabilizes the bound IRES and its interactions with the ribosome, places the IRES-linked ORF into the mRNA-binding channel and positions the tRNA into the A site for subsequent elongation steps. It therefore represents a crucial intermediate in this translation mechanism. Destabilization of PKI may begin during or immediately after the initial translocation step (Fig. 2H and Supplementary Fig. S9 ), similar to the disassembly of the analogous domain in the CrPV IRES upon its transition from the P to the E site [ 29 ]. The presence of the destabilized PKI in the E site of the 80S∙IRES E ∙aa–tRNA P ∙eEF2 complex and the absence of E-site tRNA (Fig. 2C and E ) might account for two characteristics of IGR IRES-mediated translation. Failure to establish an interaction between the 3′-terminal adenosine (A76) of E-site tRNA and 23S rRNA nucleotide C2394 in bacterial ribosomes decreases the rate of translocation [ 77 , 78 ]. A similar phenomenon could account for the slow rate of early cycles of translocation observed after initiation on IGR IRESs [ 19 , 79 ], when the E site is either vacant or contains the destabilized PKI, and has not yet been replaced by deacylated tRNA. Premature release of E-site tRNA from bacterial ribosomes is associated with high-level frameshifting [ 80 ] and analogously, the absence of base-pairing with deacylated tRNA in the E site at this early stage might contribute to the frameshifting observed during initiation on NediV and other IGR IRESs [ 34 – 36 , 81 ]. The movement of the A-site tRNA to the P site during the first translocation cycle (Fig. 4B and C , and Supplementary Fig. S9 ) has the potential to introduce steric clashes with IRES PKI, initiating its melting, and thereby facilitating its clearance from the E site, triggering a cascade that ultimately leads to dissociation of IRES domains 2 and 3 from the ribosome. Destabilization of PKI may thus facilitate the clearance of the E site by the IRES and may also aid in threading the viral ORF2 mRNA into the mRNA channel, ensuring a smooth transition to elongation. Given the observed rigidity of IRES interactions with the ribosome before translocation, this transition may represent a tightly regulated step that balances IRES-mediated control with the canonical elongation mechanism. The presence of eEF2 in a post-GTP hydrolysis state (Fig. 3F ) further suggests that this process is coordinated with the conformational dynamics of elongation factors, reinforcing the idea that once translocation occurs, the IRES relinquishes its structured hold on the ribosome, enabling productive translation of the viral message. Early steps in elongation The capture of the singly translocated 80S∙IRES E ∙aa–tRNA P ∙eEF2 complex, featuring eEF2 bound to the ribosome with tRNA occupying the P site, offers a crucial snapshot of this transition from IRES-mediated initiation to canonical elongation. The observed near-zero-degree rotation of the ribosome, coupled with the precise positioning of eEF2, suggests a highly orchestrated handoff. This state likely represents a late-stage translocation intermediate, poised for eEF2 release in which the ribosome is primed for the commencement of canonical elongation. It is plausible that eEF2 stabilizes this specific conformation, ensuring an efficient and seamless transition. A key structural feature of this complex is the movement in IRES domains 2 and 3 toward the 40S head (Fig. 2G – I ), accompanied by the destabilization of the PKI domain in the E site. The close proximity of the P-site and E-site tRNAs within our canonical 80S∙tRNA E ∙aa–tRNA P ∙eEF2 complex ( Supplementary Fig. S7 ) raises intriguing questions about the dynamics of tRNA release. The electrostatic repulsion between the closely packed tRNAs in P and E sites may be utilized for promoting E-site tRNA release. It could serve to ensure efficient ribosome clearance before the next elongation cycle, preventing potential clashes and maintaining translational fidelity. While the precise chemical state of the nucleotide on eEF2 remains unresolved due to resolution limitations, the observed density for eEF2 aligns with expected post-GTP hydrolysis conformations. This highlights the conserved role of the Switch 1 loop in transmitting conformational signals within eEF2 and coordinating ribosome-elongation factor interactions [ 38 , 41 ]. The presence of eEF2 in this state further implies that these interactions stabilize a translocation-ready conformation, setting the stage for elongation factor release and continued mRNA–tRNA movement. Finally, the critical role of the diphthamide modification in eEF2 in maintaining reading-frame integrity, as suggested by previous studies [ 40 – 42 , 82 ], is supported by our structural data. The observed interactions ( Supplementary Fig. S8B ) suggest that in this complex, diphthamide may play a crucial role in preventing mRNA backsliding during P-gate disruption as tRNAs and mRNA transition. Collectively, these findings underscore the central role of eEF2 in ribosomal translocation, by pointing to the existence of a highly conserved mechanism across IRES-mediated and canonical elongation pathways. A post-codon-sampling state of the eEF1A-bound ribosome The error rate of translation in eukaryotes is ∼10 −3 to 10 −5 per codon [ 83 , 84 ] and it cannot be accounted for solely by the difference in binding energy of cognate and near-cognate tRNAs to the A site codon [ 85 , 86 ]. Instead, accuracy results from two-step kinetic proof-reading [ 87 ]. During initial selection, the [EF-Tu/eEF1A∙GTP∙aa–tRNA] TC samples the A site codon of mRNA, and TCs that encounter a noncognate or near-cognate codon were thought to be rejected and to dissociate rapidly [ 88 ]. If the aa–tRNA encounters a cognate codon, establishment of codon–anticodon base-pairing induces conformational changes in the ribosome that trigger GTP hydrolysis, aa–tRNA is released from eEF1A∙GDP and accommodated in the PTC [ 89 ]. However, a recent Förster Resonance Energy Transfer (FRET) study that employed fluorescently tagged mRNA and tRNA suggested that after release of near-cognate TC, ribosomes adopt a structure that binds TCs faster than the original state, enabling further cycles of unsuccessful sampling [ 19 ]. In this study, release of TCs from the A site was assayed by loss of FRET signal, and it was not established whether TCs had dissociated from the ribosome or remained bound, out of FRET range. Levels of noncognate and near-cognate aa–tRNAs exceed those of cognate aa–tRNAs for any specific codon, leading to the suggestion [ 19 ] that this post-codon-sampling state will predominate. The presence of only one type of tRNA (tRNA Ala ) in the reaction mixture and a mismatched codon at the A site have enabled us to capture a complex that likely corresponds to this key step in the tRNA rejection process. Notably, this complex differs significantly from previously captured complexes with cognate tRNA in codon-recognition, GTPase-activated and accommodated post-GTP hydrolysis states [ 47 , 72 , 73 , 90 ]. The accumulation of a significant number of ribosomes in this novel state is likely a consequence both of multiple rounds of rejection with the same mismatched tRNA and of the altered ribosomal state discussed above. Notably, the eEF1A-containing structure described here was visualized without any trapping agents. This result highlights the advantage of using IGR IRESs for the structural characterization of specific steps in eukaryotic translation in defined conditions. The substantial shift of eEF1A and the A/T–tRNA away from the A site, coupled with the distorted anticodon loop, strongly suggests an active rejection mechanism. The observed 1.4-degree rotation of the 40S body could also play a role in this process, potentially contributing to the destabilization of the mismatched codon–anticodon interaction. The extensive interactions between A*/T–tRNA and the ribosome, including uL11, rRNA, and eS30 ( Supplementary Fig. S6A and B ), might serve to maintain the rejected tRNA in a holding pattern, prior to its eventual release. It is possible that the interactions with uS12 Arg142 ( Supplementary Fig. S10B ) is a key component of the sampling mechanism, as the same interaction is not seen in previous structures. Future studies employing time-resolved cryo-EM or biochemical assays could further elucidate the precise sequence of events leading to tRNA rejection and provide a more complete understanding of eEF1A’s role in maintaining translational fidelity. This study has provided key structural insights into the mechanism of translation initiation and early elongation mediated by the NediV IRES. We have shown how the NediV IRES directly binds to the ribosomal P site and how its stable interactions facilitate the positioning of the viral ORF for translation. Furthermore, our findings illuminate the dynamic transitions during early elongation, particularly the role of eEF2 and eEF1A in coordinating the handoff from IRES-mediated initiation to canonical elongation and the process of tRNA rejection during proofreading. These structures, particularly the unprecedented failed decoding complex captured without trapping agents, offer a deeper understanding of fundamental eukaryotic translation mechanisms and highlight the utility of IGR IRESs in dissecting these complex processes. Depositions All cryo-EM maps and corresponding atomic models from this study have been deposited in the EMDB and PDB. The P-site complex is available under accession numbers PDB ID 9Q1S and EMDB 72137, and the A-site complex under PDB ID 9Q1Q and EMDB 72136. The A–tRNA complex is deposited as PDB ID 9Q2O and EMDB 72170, the canonical eEF1A complex as PDB ID 9Q2P and EMDB 72171, the canonical eEF2 complex as PDB ID 9Q2M and EMDB 72168, and the IRES–eEF2 complex as PDB ID 9Q2T and EMDB 72175. Supplementary Material gkag510_Supplemental_Files gkag510_supplemental_files.zip (37.5MB, zip) Acknowledgements We want to thank Thea Hu (University of Bristol) for her help in PDB curation, proofreading, and preparing the graphical abstract. Authors contributions : S.D., C.A., P.D., F.A.R., P.G., and Z.B. analyzed the data. I.A. and C.A. were responsible for sample preparation and data collection. The manuscript was written by S.D., T.V.P., C.H., and J.F. T.V.P., C.H., and J.F. designed and supervised the project. Notes Present address: Department of Biological and Physical Sciences, Grayson College, Denison TX 75020, United States Present address: Hummingbird Scientific Pvt Ltd, Hyderabad 500016, Telangana, India Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, United States Present address: Thermo Fisher Scientific, Hillsboro, OR 97124, United States Contributor Information Swastik De, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States. Clara G Altomare, Department of Biological Sciences, Columbia University, New York, NY 10027, United States. Irina S Abaeva, Department of Cell Biology, SUNY Downstate Health Sciences University, Brooklyn, NY 11203, United States. Prikshat Dadhwal, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States; Department of Biological Sciences, Columbia University, New York, NY 10027, United States. Priyanka Garg, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States. Francisco Acosta-Reyes, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States. Zuben P Brown, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States. Tatyana V Pestova, Department of Cell Biology, SUNY Downstate Health Sciences University, Brooklyn, NY 11203, United States. Christopher U T Hellen, Department of Cell Biology, SUNY Downstate Health Sciences University, Brooklyn, NY 11203, United States. Joachim Frank, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States; Department of Biological Sciences, Columbia University, New York, NY 10027, United States. Supplementary data Supplementary data is available at NAR online. Conflict of interest None declared. Funding U.S. National Institutes of Health (R01 GM097014 to C.H., R01 GM29169to J.F., R21 AI188505 to C.H., R35 GM122602 to T.V.P, R35 GM139453 to J.F.). National Science Foundation (NSF Award 2432512 to J.F). Funding to pay the Open Access publication charges for this article was provided by the National Institutes of Health and the National Science Foundation. Data availability All cryo-EM maps and corresponding atomic models from this study have been deposited in the EMDB and PDB. The P-site complex is available under accession numbers PDB ID 9Q1S and EMDB 72137, and the A-site complex under PDB ID 9Q1Q and EMDB 72136. The A–tRNA complex is deposited as PDB ID 9Q2O and EMDB 72170, the canonical eEF1A complex as PDB ID 9Q2P and EMDB 72171, the canonical eEF2 complex as PDB ID 9Q2M and EMDB 72168, and the IRES–eEF2 complex as PDB ID 9Q2T and EMDB 72175. References 1. Jackson  RJ, Hellen  CUT. Pestova TV. 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The A–tRNA complex is deposited as PDB ID 9Q2O and EMDB 72170, the canonical eEF1A complex as PDB ID 9Q2P and EMDB 72171, the canonical eEF2 complex as PDB ID 9Q2M and EMDB 72168, and the IRES–eEF2 complex as PDB ID 9Q2T and EMDB 72175. Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press ACTIONS View on publisher site PDF (2.8 MB) Cite Collections Permalink RESOURCES Similar articles Cited by other articles Links to NCBI Databases Follow NCBI NCBI on X (formerly known as Twitter) NCBI on Facebook NCBI on LinkedIn NCBI on GitHub NCBI RSS feed Connect with NLM NLM on X (formerly known as Twitter) NLM on Facebook NLM on YouTube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov Back to Top

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