Figures and data
(A) Different views of EMCV IRES-48S PIC (Map B1) by 45⁰ rotation along one axis. The map shows densities assigned to 40S ribosome, RNA (in channel), ternary complex and IRES density at the inter-subunit region of head. (B) View of Map B and zoomed view of densities corresponding to IRES and tRNAi.
Cryo-EM data and model statistics
(A) Fitting of tRNAi-base paired to start codon AUG (left). Zoomed view of codon-anticodon interaction (middle), and B-factor for codon-anticodon interaction (right). (B) The tRNAi in EMCV IRES-48S PIC is in PIN state similar to that in human 48S PIC PIN state (PDB Id-7QP7) and its anticodon shows ∼7 Å shift as compared to human 48S PIC open state (PDB Id-7QP6). (C) Comparison of ribosomal conformation (18S rRNA) of EMCV IRES 48S PIC with Human open and closed state (PDB Id-7QP6 and 7QP7, respectively). Focusing on the entry site and the helices governing latch conformation-Helix 34 moves toward helix 18 by 9 Å. (D) Movement of eS17 in open and closed states of ribosome. Zoomed view of comparison between eS17 in EMCV IRES 48S PIC and Human 48S PIC open state-showing an upward shift of the N terminal domain by 10Å.
(A) Extra density connecting the head of 40S to tRNAi elbow region is contributed by EMCV IRES RNA in Map B1. Rotated views of RNA density showing its connection to the 40S head via uS13 and uS19 and to tRNAi via its elbow region. (B) Organization of EMCV IRES domains from D-L, where H-L makes the functional IRES moiety (Hellen and Wimmer et al 1995). (C) Deciphering the architecture of the obtained IRES density. The density could be interpreted as a long main stem (S1) extending away from the ribosome where the base is anchored to the ribosome by two branches (B1 and B2), and to tRNAi by one branch (B3), further divided into two sub-branches (B3a and B3b). (D) Secondary structure of apical region of domain I (made using RNAfold) marking the Stem and branches, along with imported loops. (E) Fitting of I domain apex in the density (Rotated views). The sub-domains are coloured as proposed in Fig 3C-D.
(A) Model showing connections of I domain apex with uS13, uS19, and tRNAi. (B) uS13 interacts with B3 branch or sub-domain of IRES via its alpha helix (100-117 residues). (C) Multiple point of contacts between uS19 and I domain motifs-RAAA and AAG. The electrostatic potential map of uS19 suggests that EMCV IRES interacts via ionic interactions with its phosphate backbone. (D) Sequence alignment of h38 with I domain apex of EMCV IRES showing sequence identity. Overlapping of uS19 from 80S (PDB Id-4UG0) and EMCV IRES-48S PIC show that the interaction of uS19 with EMCV IRES is similar to interaction with that of h38. (E) The fit of GNRA or GCGA stem and its contact with tRNAi at the elbow region and acceptor stem.
(A) Inter-subunit view of EMCV IRES 48S PIC showing position for ternary complex on map. Fitting of eIF2α and eIF2γ in its corresponding density in Map B1. (B) Overlapping EMCV IRES 48S PIC on mammalian late-stage 48S PIC (PDB Id-6YAN), indicates a shift in position of eIF2γ towards 40S head. (C) Zoomed view showing position of eIF2γ, tRNAi, eIF2α from EMCV IRES 48S PIC with respect to position on mammalian late-stage 48S PIC (PDB Id-6YAN). The black arrow indicates the shift in position.
(A) EMCV IRES secondary structure depicting the role or binding partners for each domain in context to translation initiation. The known tertiary structure of EMCV IRES and its binding partners are depicted. The domains responsible for binding PTB1 are boxed. (B) Conservation of I domain apex sequence and secondary structure across type 2 IRES family. (C) Comparison of secondary structure of EMCV and FMDV IRES to that of Polioviral IRES (type 1).
(A) Isolation of EMCV IRES-48S PIC from Talon Affinity Chromatography-Talon Affinity chromatography profile and analysis of Elution fractions-pelleted by using 30% cushion on 1.5% Agarose gel, followed by Uranyl acetate staining of elution pellet and observation under transmission electron microscope at 57k X. (B) Processing of Cryo-EM data and features of obtained maps using CryoSPARCv4.3.0 and GSFSC resolution estimates for different maps obtained.
(A) Local resolution estimation of Map B. (right) Surface view; (right-top) zoomed view on IRES density; (left-bottom) cross-section of Map B to show resolution at the core of the ribosome. (B) Local resolution estimation of Map B1. (right) Surface view; (left-bottom) cross-section of Map B1 to show resolution at the core of ribosome. (C) Extra density at the mRNA entry site of Map B/B1. Ribosomal proteins and RNA in contact with the unassigned extra density. (D) Superimposition of EMCV IRES-48S PIC on human 48S PIC (PDB Id-8OZ0) shows the extra density coincides with the location of eIF3g-RRM and downstream mRNA. (E) Extra density at the mRNA entry site of Map A. Ribosomal proteins and RNA in contact with the unassigned extra density.
(A) From Map B to model (EMCV IRES-48S PIC). Obtained Model showing 40S ribosome, RNA in channel, initiator tRNA, IRES domain. (right) Fourier Shell Correlation (FSC) of Map to Model fit. (B) From Map B1 to model (EMCV IRES-48S PIC). Obtained Model showing 40S ribosome, RNA in channel, initiator tRNA, IRES domain, eIF2αγ. (right) FSC of Map to Model fit. (C) From Map A to model by fitting of 40S ribosome without any factors. (right) FSC of Map to Model fit. (D) The sequence of EMCV IRES mRNA in the channel is provided. Residues in orange colour has no density in the obtained map B. (E) Comparing the state of 18S rRNA in Map A and Map B. (F) eS17-NTD position in 40S ribosome. (right) Fitting of eS17 in EMCV IRES-48S PIC map.
(A) Process of fitting I domain apex from Alphafold3 generated model. (B) Correlation coefficient per residue in I domain apex for fit in the density map. (C) B-factor for each residue in the final model. The range is depicted in the colour key where blue is low, and red is high. (D) Possibility of fitting H domain in the IRES density. H domain model was generated by Alphafold3 (ptm= 0.31). (E) Possibility of fitting J-K domain (PDB-8HUJ) in density. The J-K domain is way too long to fit in map and does not account for certain region. The extending loops would clash with 40S ribosome and tRNAi.
(A) Interaction of uS19 and uS13 in 40S subunit with 60S subunit-Helix 38 and uL5 to form inter-subunit bridges-B1a and B1b/c, respectively in 80S (PDB Id-4UG0). (B) Estimation of clash with IRES domain-I during 60S joining to form 80S-elongation competent complex (PDB Id-4UG0). uL5 and helix 38 of 28S rRNA would clash with the IRES I domain. (left) Superimposition of 80S ribosome on EMCV IRES-48S PIC. (C) Reported GNRA (GUGA) interaction with the minor groove formed by C-G stem in bacterial RNaseP (PDB Id-3Q1Q).
