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. 2022 Jul 25;50(14):8240–8261. doi: 10.1093/nar/gkac631

Figure 5.

Figure 5.

Three-color smFRET to probe eIF4F- and eIF4A-mRNA interaction dynamics. (A) Schematic of the three-color smFRET experiment with two donors (on eIF4A and mRNA) and one acceptor (eIF4E). A FRET signal between Cy5-labeled eIF4E and the Cy3.5-labeled mRNA is tracked at the same time as a FRET signal between Cy3-eIF4A and Cy5-eIF4E. (B) Relative incidence of eIF4A-mRNA binding occurring with and without FRET to eIF4E. n is the number of molecules analyzed to enumerate the event types on each mRNA. (C) Reaction pathway and representative smFRET trace showing concomitant mRNA binding of eIF4E and eIF4A with eIF4E–eIF4A FRET, consistent with eIF4F–mRNA binding, on JJJ1. The eIF4A–mRNA lifetime measured in panel E is indicated. (D) Reaction pathway and representative single-molecule fluorescence trace for eIF4A–mRNA binding without eIF4E–eIF4A FRET on JJJ1. These events result both from ‘free’ eIF4A–mRNA interaction (‘eIF4A(–EG)’), and eIF4F–mRNA interaction where eIF4E is unlabeled (‘eIF4A(+EG)’. The Cy3 and Cy5 signals were manually corrected by linear subtraction to equalize their background values, for clarity of presentation. (E) eIF4A–mRNA dissociation rates following eIF4A–mRNA binding with eIF4E, i.e. with observable eIF4E–eIF4A FRET as shown in panel C. The Cy3 and Cy5 signals were manually corrected by linear subtraction to equalize their background values, for clarity of presentation. (F) eIF4A–mRNA dissociation rates following eIF4A–mRNA binding without FRET to eIF4E. (G) eIF4A–mRNA association rates across all binding event types.