The mechanism of ribosomal recruitment during translation initiation on the Type 2 encephalomyocarditis virus IRES

Figures

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  Figure EV1. Secondary structure of the EMCV IRES. The EMCV IRES secondary structure model featuring individual domains, the region corresponding to cryo-EM density (red), and specific binding of eIF4A/4G to the JK domain.

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  Figure EV2. 48S complex formation on the wt EMCV IRES and the IRES with mutated AUG826. (A) Purified initiation factors, nPTB and 40S subunits, analyzed by SDS-PAGE followed by SimplyBlue staining. (B) Toe-printing analysis of 48S complex formation on wt EMCV IRES mRNA and IRES mRNA with mutated AUG826 in the presence of 40S subunits, initiator Met-tRNAiMet, nPTB and indicated initiation factors. Lanes C, T, A, and G depict wt EMCV sequence generated using the same primer. The positions of initiation codons are indicated on the left and of assembled 48S complexes on the right. Source data are available online for this figure.

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  Figure EV3. Cryo-EM data processing workflow and structural characterization of EMCV IRES 48S initiation complexes. (A) Data processing pipeline for structure determination of EMCV IRES 48S initiation complexes. (B) Class Ia (left): 48S initiation complex showing Met-tRNAiMet, eIF2, eIF1A, eIF3 (eIF3a/c/d subunits are indicated), and EMCV IRES density bound at the 40S subunit head. Classes II–IV: A series of reconstructions capturing progressive 40S head rotation, accompanied by repositioning of the EMCV IRES. Fragmented extra density attributed to the IRES is highlighted (circled), and a sky-blue model of the EMCV IRES is overlaid to illustrate its positional shift. Gaussian filtering reveals that this density remains connected to the IRES across all states. The horizontal arrow from left to right denotes the direction of progressive rotational movement of the 40S head, representing a continuum of structural intermediates.

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  Figure 1. Translation initiation complex on the EMCV IRES. (A–C) Cryo-EM map of the 48S initiation complex assembled on the EMCV IRES, shown in different orientations. The 40S ribosomal subunit, ribosomal proteins uS13 and uS19, associated initiation factors, Met-tRNAiMet and EMCV IRES are shown in distinct colors and are labeled accordingly. (D–G) Zoomed-in views of the EMCV IRES density map (rendered at 70% surface transparency) superimposed with the atomic model, displayed in different orientations. The individual domains of the IRES are indicated in these views. (H–K) Zoomed-in views of the cryo-EM density map, with superimposed atomic models, showing the key interactions between the EMCV IRES (red) and initiator tRNA (magenta), uS13 (blue) and uS19 (green). The densities of the different components are color-coded using the ChimeraX surface color tool (Meng et al, 2023). Panel (I) also shows interaction between the start codon (AUG) of the mRNA and the anti-codon of the initiator tRNA at the P site of the 40S subunit.

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  Figure EV4. Local resolution estimation and assignment of nucleotides of EMCV mRNA. (A) The local resolution estimation of the 40S head part along with the EMCV IRES and Met-tRNAiMet. (B, C) The zoomed view of the Id subdomain of the EMCV IRES (in mesh) showing the loop formation through base pairing among RNA bases. The models fitted within densities of the EMCV IRES (red), uS19 (green), and uS13 (blue) are showing the relative position of Id on the 40S head.

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  Figure 2. Specific interactions of EMCV IRES domain I with the head of 40S ribosomal subunit. (A–C) Zoomed-in views of the interaction between protein uS13 (blue) of the 40S subunit and the EMCV IRES, as shown by atomic models in three orientations, with interacting residues labeled. (D–F) Close-up views of the Id subdomain of the EMCV IRES interacting with uS13, presented in different orientations, with map density rendered at 70% transparency to show the fitting of the atomic model. (G–I) Interaction between protein uS19 (green) of the 40S subunit and the EMCV IRES (red) shown by atomic models presented in different orientations, with interacting residues labeled. (J–L) Close-up views of the Ib and Id subdomains of the EMCV IRES interacting with uS19, presented in different orientations, with map density rendered at 70% transparency to show the fitting of the atomic model.

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  Figure 3. The conformation of the EMCV IRES domain I tetraloop and its interactions with initiator tRNA. (A) Close-up view of the atomic model of the 40S subunit head region, showing interactions between the EMCV IRES (red), initiator tRNA (magenta), and 40S subunit proteins uS19 (green) and uS13 (blue). (B) Structural comparison reveals that the acceptor arm of the P-site-bound tRNA undergoes a ~ 9.7 Å displacement from its position associated with eIF2 in canonical 48S complexes (PDB id: 8OZ0) (tRNA is in light pink) to a new position where it interacts directly with the EMCV IRES (tRNA is in magenta). (C) Interaction between the tetraloop of Ic domain of the EMCV IRES and initiator tRNA, and codon-anticodon recognition between initiator tRNA at the P site and the EMCV initiation codon AUG834. (D–I) structural details of the EMCV IRES tetraloop regions interacting with initiator tRNA. (D, E) Interaction is shown by atomic models and by atomic models fitted into Coulomb density (rendered at 70% transparency, and highlighting the front TψC arm), respectively. (F, G) Same interaction is shown but with a focus on the rear TψC arm. (H, I) Interaction between the variable loop of initiator…

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  Figure 4. Influence of mutations in the apex of domain I on 48S complex formation on the EMCV IRES. (A) Secondary structure model of the apex of domain I showing introduced mutations. (B, C) Toe-printing analysis of 48S complex formation on wt and mutant EMCV IRES mRNAs in the presence of individual 40S subunits, Met-tRNAiMet, nPTB and indicated eIFs. The efficiency of 48S complex formation on AUG834 (red numbers) was quantified taking the efficiency of complex formation on the wt EMCV IRES mRNA as 100%. Standard deviations (omitted for clarity) did not exceed 6%. Lanes C, T, A, and G depict the wt EMCV sequence generated using the same primer. The positions of initiation codons are indicated on the left and of assembled 48S complexes on the right. The division between sequence lanes and lanes 1–12 in (A) indicates that these two sets of lanes were derived from the same gel, exposed for different lengths of time. Source data are available online for this figure.

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  Figure EV5. Influence of the nature of initiator tRNA and substitution of eIF2 by eIF5B or eIF2D on 48S complex formation on the EMCV IRES. (A) Toe-printing analysis of 48S complex formation on wt EMCV IRES mRNA in the presence of either native or in vitro transcribed Met-tRNAiMet, 40S subunits, nPTB and indicated initiation factors. The efficiency of 48S complex formation on AUG834 (red numbers) was quantified on the basis of three experiments with the efficiency of complex formation in the presence of eIF1, eIF1A and native Met-tRNAiMet defined as 100%. Standard deviations (omitted for clarity) did not exceed 8%. Black numbers represent relative efficiencies of 48S complex formation on AUG834 and AUG826 in each condition. (B) Toe-printing analysis of 48S complex formation on wt EMCV IRES mRNA and mRNA with mutated AUG826 in the presence of 40S subunits, initiator tRNA, nPTB and indicated initiation factors. The efficiency of 48S complex formation on AUG834 (red numbers) was quantified taking the efficiency of complex formation in the presence of eIF2 defined as 100%. Standard deviations (omitted for clarity) did not exceed 15%. (A, B) Lanes C, T, A, and G depict wt EMCV sequence generated…

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  Figure 5. Release of the EMCV IRES from the 40S ribosomal head is necessary for joining of the 60S subunit and formation of the 80S ribosome. (A) Close-up view of the 80S complex, showing that its formation is stabilized by the inter-subunit bridge B1a, which is established between H38 of the 60S subunit and uS19 of the 40S subunit. (B) Close-up view on a steric clash between the EMCV IRES and the 60S subunit. (C) The EMCV IRES appears to compete for the same binding site on uS19 that H38 occupies during 80S formation. (D) On the right, formation of the 80S complex is prevented by a steric clash between the 40S-bound EMCV IRES (red) and the 60S subunit. Release of the EMCV IRES is required to prevent this clash. (E) Interactions among the 40S subunit, the EMCV IRES (red), initiator tRNA (magenta) and eIF2α (yellow) (this study). (F) Interactions between the 40S subunit, initiator tRNA and eIF5B after dissociation of eIF2 during subunit joining (pdb id: 7SYV). (G) Superimposition of 40S complexes shown in (E) and (F). (H) Zoomed-in view of the ribosomal positioning of initiator tRNA bound to the EMCV IRES and eIF2α in 48S initiation complexes. (I) Close-up of the ribosomal positioning of…

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  Figure 6. Influence of mutations in the stem of domain I on 48S complex formation on the EMCV IRES. (A) Secondary structure model of the EMCV IRES showing introduced mutations. (B, C) Toe-printing analysis of 48S complex formation on wt and mutant EMCV IRES mRNAs in the presence of individual 40S subunits, Met-tRNAiMet, nPTB and indicated eIFs. The efficiency of 48S complex formation on AUG834 (red numbers) was quantified taking the efficiency of complex formation on the wt EMCV IRES mRNA as 100%. Standard deviations (omitted for clarity) did not exceed 8%. Lanes C, T, A, and G depict the wt EMCV sequence generated using the same primer. The positions of initiation codons are indicated on the left and of assembled 48S complexes on the right. Source data are available online for this figure.

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  Figure 7. Influence of mutations in the region upstream of domain I and in domain L downstream of the JK domain on 48S complex formation on the EMCV IRES. (A) Secondary structure model of the EMCV IRES showing introduced mutations. (B) Toe-printing analysis of 48S complex formation on wt and mutant EMCV IRES mRNAs in the presence of individual 40S subunits, Met-tRNAiMet, nPTB and indicated eIFs. The efficiency of 48S complex formation on AUG834 (red numbers) was quantified taking the efficiency of complex formation on the wt EMCV IRES mRNA as 100%. Standard deviations (omitted for clarity) did not exceed 6%. Lanes C, T, A, and G depict wt EMCV sequence generated using the same primer. The positions of initiation codons are indicated on the left and of assembled 48S complexes on the right. Source data are available online for this figure.