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.
(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 ∼10 Å.
(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 domain I apex in the density (Rotated views). The sub-domains are coloured as proposed in Fig 3C-D.
(A) Model showing connections of domain I 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 domain I 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 domain I 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 the positions of eIF2γ, tRNAi, and eIF2α in the EMCV IRES 48S PIC relative to those in the mammalian late-stage 48S PIC (PDB ID 6YAN). The black arrow indicates the shift in position. (D) Superimposition of the EMCV IRES-48S PIC on the human late-stage 48S PIC (before 60S joining; PDB Id-8PJ5), showing the conformation of tRNAi in association with eIF2 and IRES and tRNAi with eIF5B in the canonical context, respectively. (right) Zoomed view of tRNAi conformation in both complexes.
(A) EMCV IRES secondary structure depicting the role of 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 domain I 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).
