Figure EV5. Supplemental data for the Discussion section.

• A, B
  Dissociation of short peptidyl‐tRNA from the ribosome after IRD. N‐terminally His₆‐tagged GFP(5aa)‐EPDP sequence (A) was translated by PURE system and mixed with an equal volume of HKM buffer (25 mM HEPES‐KOH pH 7.4, 100 mM KCl, 5 mM MgCl₂) or HKM buffer supplemented with 100 mM MgCl₂. Then the mixture was fractionated by SDG in the presence of 10 mM or 50 mM magnesium ion. Recorded A₂₅₄ values (upper) and the distribution of aborted peptidyl‐tRNA detected by anti‐His₆ Western blotting (bottom) are shown (B).
• C–E
  Influence of N‐terminal amino acid motifs that determine the initiation rate on IRD. Some of the 3 amino acid motifs reported by Verma et al, 2019 (KYY, KIH, IGK and TVG) were fused to N‐terminal EPDP motif (C) and in vivo translated to evaluate the TC index. Representative image of ³⁵S‐Methionine‐labeled products separated by SDS–PAGE (D) and two‐dimensional plot of the TC indices of three independent technical replicates and averaged van der Waals radii of 3 aa motif (E) are shown with the result of Spearman's correlation test.