Extended Data Figure 8. eIF1 binding kinetics depend on the identity of the translation start site.

(a) Cumulative probability plots of the indicated eIF1 kinetic parameters on eIF5B-bound initiation complexes (successful) on the model AUG mRNAs in ideal (ACCAUGGA) or poor (CCCAUGCA) Kozak context. Lines represent fits to exponential functions. (b) Cumulative probability plots of the indicated eIF1 parameters observed on any loaded initiation complexes (both unsuccessful and successful) on the indicated model RNAs. Lines represent fits to exponential functions. (c) Plot of the population-weighted mean elapsed times for the indicated eIF1 parameters on the indicated mRNAs. All values plotted here were derived from events that occurred on any loaded initiation complex (both unsuccessful and successful). (d, e) Cumulative probability plots of the indicated kinetic parameters on eIF5B-bound initiation complexes on the indicated model mRNAs in the presence of 290 nM (panel d) or 5 nM (panel e) unlabeled eIF5. Lines represent fits to exponential functions. (f) Plot of the population-weighted mean eIF1 binding time on the indicated model RNAs in the presence of molar excess eIF5 (290 nM) or a limiting eIF5 concentration of 5 nM. (g) Plot of the percent of initiation complexes that classified into the three broad categories of eIF1-Cy5 binding outlined in Fig. 4b on β-globin mRNA. ‘Indeterminant’ captures complexes that were too ambiguous to classify. In all experiments, eIF1-Cy5 was present at a final concentration of 40 nM (by Cy5) and unlabeled eIF5 was present at either 290 or, when indicated, at 5 nM. See Supplementary Table 1 for the number of complexes and binding events analyzed in each experiment.