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. 2021 Oct 20;40(23):e108299. doi: 10.15252/embj.2021108299

Figure EV4. Effect of the exit tunnel mutations on the IRD counteraction.

Figure EV4

  • A
    Inserted amino acid length‐dependent IRD‐counteracting effects in the uL22∆loop‐ribosome. Messenger RNA coding 5‐aa (panel 1) or 30‐aa (panel 2) insertion and the EPDP used in Fig 1G were individually translated by customized PURE systems, including ribosome variants as indicated. Individual translation continuation indices of wild type (black line), ∆bL31 (blue line), or uL22∆loop ribosomes (orange line) are shown.
  • B
    Structure of the uL23 signaling loop (PDB: 4V5H) and schematic drawing of the tunnel structure. Deleted residues in the uL23∆loop mutation are indicated in red.
  • C
    Translation continuity of ORFs with the IRD‐inducing motif located in the middle of the ORFs. Messenger RNAs encoding rpoD (panel 1), yihI (panel 2), or GFP‐10E (panel 3) carrying 10 consecutive D/E residues were translated in vitro and the translation continuities were evaluated as described above. The mean values ± SE estimated from three independent technical replicates are shown.
  • D
    Translation continuity of ORFs with an IRD sequence without a preceding nascent polypeptide occupying the tunnel. Messenger RNAs encoding mgtL (panel 1), FLAG tag (panel 2) or a mixture of 5 aa‐ EPDP constructs in Fig 1F and G (panel 3) were translated in vitro and analyzed as described above. The mean values ± SE estimated from three independent technical replicates are shown.