The mechanism of ribosomal recruitment during translation initiation on Type 2 IRESs

Figures

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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 tool67. 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 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 and 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 and G) Same interaction is shown but with a focus on the rear TψC arm. (H and I) Interaction between the variable loop of…

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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 anaysis of 48S complex formation on wt and mutant EMCV IRES mRNAs in the presence of individual 40S subunits, Met-tRNAiMet, nPTB and indicated eIFs. 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.

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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) On the right, formation of the 80S complex is prevented by a steric clash between the 40S-bound EMCV IRES and the 60S subunit. Removal of the EMCV IRES (red) is required to prevent a significant steric clash. (B) 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. (C) Close-up view on a steric clash between the EMCV IRES and the 60S subunit. (D) The EMCV IRES appears to compete for the same binding site with uS19 that H38 occupies during 80S formation. (E) Interactions among the 40S subunit, the EMCV IRES, initiator tRNA (magenta), and eIF2α. (F) Interactions between the 40S subunit, initiator tRNA (light pink) and eIF5B after dissociation of eIF2 during subunit joining (pdb id: 7SYV). (G) Superimposition of 40S complexes shown in (E) and (F). (H) The ribosomal positioning of initiator tRNA (magenta) bound to the EMCV IRES and eIF2α in 48S initiation complexes. (I) The ribosomal positioning of initiator tRNA…

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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. 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.

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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. 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.