Extended Data Fig. 7: eIF5 competes with eIF1 to bind initiation complexes.
From: eIF1 and eIF5 dynamically control translation start site fidelity
(a) Plot of the percentage of initiation complexes bound by eIF5 in the presence of 290 nM or 10 nM eIF1 on the AUG model RNA. (b) Cumulative probability plot of the eIF5 first binding time on initiation complexes in the presence of 290 nM or 10 nM eIF1 on the AUG model RNA. (c) Schematic of the single-molecule translation initiation assay to monitor eIF1-Cy5 and eIF5-Cy5.5 binding simultaneously using the AUG model RNA. In this experiment, only the 532 nm excitation laser was used. This experimental scheme directly detected loading of the 43S initiation complex (via tRNAi-Cy3, green) and eIF5B-Cy3.5 (orange), as the conjugated dyes are directly excited by the 532 nm laser. By contrast, eIF1-Cy5 (red) and eIF5-Cy5.5 (purple) are not excited directly by the 532 nm laser and thus their binding is detected solely via FRET with the tRNAi-Cy3 donor. (d) Representative single-molecule data of initiation complexes that progressed to eIF5B binding using the experimental scheme outlined in panel c. In total, 77 initiation complexes were analyzed, and all eIF1 and eIF5 binding events were mutually exclusive. Furthermore, eIF5 was the final binding event prior to eIF5B binding on 95% of the complexes. We suspect that the remaining 5% where eIF1 was last were likely followed by binding of an unlabeled eIF5 protein immediately prior to eIF5B binding. (e) Schematic of an alternative single-molecule translation initiation assay to monitor eIF1-Cy5 and eIF5-Cy5.5 binding simultaneously using the AUG model RNA. In this experiment, 532 nm and 640 nm excitation lasers were used simultaneously. This experimental scheme directly detected binding of all fluorescently-labeled components in the assays, independent of FRET. (f) Representative single-molecule data of initiation complexes that progressed to eIF5B binding using the experimental scheme outlined in panel e. As in panels c and d, eIF1 and eIF5 binding was mutually exclusive on initiation complexes that progressed to eIF5B binding. (g) On about 5% of complexes, we observed eIF1 and eIF5 binding events that overlapped; however, these complexes never progressed to eIF5B binding (that is, were unsuccessful) and very likely represent aberrant complexes or non-specific binding events.
