Extended Data Fig. 1: Characterization of SRI-41315 in an in vitro translation system.
From: The eRF1 degrader SRI-41315 acts as a molecular glue at the ribosomal decoding center
a, Effect of SRI-41315 on protein synthesis. Representative titrations of SRI-41315 into in vitro translation reactions of a radiolabeled 3xFlag-tagged model protein ending with the indicated stop codon or a nonstop (NS) control, analyzed by SDS-PAGE and autoradiography on film (top) or by phosphorimaging (bottom). NC, expected nascent protein chain; NC-tRNA, nonhydrolyzed peptidyl-tRNA adduct. Orange dots, smaller products specifically observed with SRI-41315, representative of 5 replicates with similar results. b, SRI-41315 does not trap nascent proteins on ribosomes. In vitro translation reactions as in panel a without or with 0.5 µM eRF1(AAQ) and/or 100 µM SRI-41315 added after 5 min were size fractionated on 10-50% sucrose gradients. The total (T) and eleven fractions collected from the top of each gradient were analyzed by SDS-PAGE and autoradiography. Note: eRF1(AAQ) but not SRI-41315 retains NC-tRNAs and NCs hydrolyzed from tRNAs during SDS-PAGE in ribosomal fractions, representative of 3 replicates with similar results.
