Extended Data Fig. 6: eIF5 transiently binds initiation complexes.
From: eIF1 and eIF5 dynamically control translation start site fidelity
(a) Schematic of predicted single-molecule data that monitor eIF5-Cy5.5. Initial appearance of tRNAi-Cy3 (green) fluorescence indicates loading of the 43S initiation complex onto the mRNA. Either direct appearance of eIF5-Cy5.5 (purple) fluorescence or appearance of tRNAi-to-eIF5 FRET indicates the presence of eIF5 on initiation complexes. Appearance of eIF5B-Cy3.5 (orange) indicates binding of eIF5B. Appearance of 60S-Cy5 (red) fluorescence indicates the 60S subunit joined to form the 80S ribosome; the relative proximity of tRNAi-Cy3 and 60S-Cy5 labeling sites in the 80S initiation complex yields low FRET. The kinetic parameters were defined as follows. ‘eIF5 first binding time’ as the time elapsed from initial appearance of the tRNAi-Cy3 signal to the first appearance of eIF5-Cy5.5 signal. ‘eIF5 first lifetime’ as the duration of the first eIF5-Cy5.5 binding event. ‘eIF5 rebinding time’ as the time elapsed from departure of the previous eIF5 protein until the start of the next binding event. ‘eIF5 subsequent lifetimes’ as the duration of the subsequent eIF5 binding events. ‘eIF5 final binding time’ as the time elapsed from appearance of the tRNAi-Cy3 signal until appearance of the final eIF5-Cy5.5 signal prior to eIF5B binding. ‘eIF5 final lifetime’ was the duration of the final eIF5 binding event. Since most complexes contained a single eIF5 binding event, the ‘first’ and ‘final’ eIF5 binding events were identical on most complexes. ‘eIF5B binding time’ was defined as the time elapsed from disappearance of the final eIF5 signal until appearance of the eIF5B-Cy3.5 signal. ‘60S joining time’ was defined as the time elapsed from appearance of eIF5B-Cy3.5 signal until appearance of 60S-Cy5 signal. ‘eIF5B-80S lifetime’ was defined as the duration of the eIF5B-Cy3.5 signal on the 80S initiation complex. (b) Cumulative probability plots of the indicated kinetic parameters on β-globin mRNA at the indicated concentrations of eIF5-Cy5.5. (c) Example single-molecule data of an initiation complex that was bound multiple times by eIF5-Cy5.5. (d) Plot of the percent of initiation complexes that contained either a single or multiple eIF5-Cy5.5 binding events at the indicated eIF5-Cy5.5 concentrations. (e) Plot of the percent of initiation complexes where eIF5B bound either during (overlapped), within 100 ms of ( < 0.1 s), or after 100 ms ( > 0.1 s) the final eIF5 binding event at the indicated concentrations of eIF5-Cy5.5. In all experiments, unlabeled eIF1 was present at a final concentration of 290 nM and eIF5-Cy5.5 was present at the indicated final concentrations (by Cy5.5 dye). See Supplementary Table 3 for the number of complexes and binding events analyzed in each experiment.
