Extended Data Figure 1. eRF1^AAQ stalls ribosomes at stop codons.

a, Line diagrams of mRNA encoding nascent chain (NC) substrates used in this study. The cytosolic portion of human Sec61β (residues 1-68, orange) was modified to contain an N-terminal 3× Flag tag (green) for affinity purification and the autonomously-folding villin headpiece (VHP, blue) domain. The three stop codons were individually inserted after Val68 of Sec61β to generate substrates for eRF1^AAQ-mediated stalling, or the mRNA was truncated after the same residue to generate an independently-stalling substrate. b, In vitro translation reactions of NC-stop substrates containing the indicated stop codon (see panel a) in the presence of ³⁵S-methionine without or with excess eRF1^WT or eRF1^AAQ. Reactions were for 25 min at 32°C and directly analyzed by SDS-PAGE and auto-radiography. The terminated (NC) and tRNA-associated (NC-tRNA) nascent chain products are indicated. Addition of eRF1^AAQ selectively prevents peptide hydrolysis when the stop codon is reached. c, Anti-Flag affinity purifications of ribosome-nascent chains (RNCs) stalled either by mRNA truncation or at the UAA stop codon with eRF1^AAQ (see panel a) were immunoblotted for the splitting factors Hbs1 and ABCE1. The different amounts of Hbs1 and ABCE1 co-purified despite identical nascent chain sequences in each RNC complex suggest that eRF1^AAQ selectively traps ABCE1 on pre-termination complexes. d, SDS-PAGE and Coomassie staining of affinity-purified eRF1^AAQ-stalled ribosome-nascent chains containing the UGA stop codon utilised for cryo-EM analysis. Bands corresponding to ribosomal proteins, ABCE1, and eRF1^AAQ, which were verified by immunoblotting and mass spectrometry (data not shown), are indicated.