Extended Data Fig. 2: eIF1 stably binds and dynamically samples individual complexes.
From: eIF1 and eIF5 dynamically control translation start site fidelity
(a) Schematic of predicted single molecule data for eIF1-Cy5 experiments. Initial appearance of tRNAi-Cy3 (green) to eIF1-Cy5 (red) FRET signifies that a 43S initiation complex loaded onto the tethered mRNA. This initial FRET event is defined as the ‘initial eIF1 binding event’ and the duration of this event defined as the ‘initial eIF1 binding lifetime’. The complex then contains multiple subsequent tRNAi-Cy3 to eIF1-Cy5 FRET events, which are defined as subsequent eIF1 binding events. The dwell time between loss of the previous eIF1 signal and appearance of the next eIF1 signal is defined as the ‘eIF1 rebinding time’. The duration of all subsequent eIF1 binding events is defined as the ‘subsequent eIF1 binding lifetime’. Appearance of eIF5B-Cy3.5 (orange) signal signifies successful entry to the final initiation steps that culminate with joining of the 60S ribosomal subunit. The dwell time from loss of the final eIF1-Cy5 signal to appearance of eIF5B-Cy3.5 signal is defined as the ‘eIF5B binding time’. (b) Cumulative probability plot of the eIF5B binding time measured relative to initial loading of the 43S initiation complex, which was signified by either appearance of tRNAi-Cy3 to eIF1-Cy5 FRET (this study) or initial appearance of 40S-Cy3 signal. The overlapped plots indicate that labeled tRNAi-Cy3 and eIF1-Cy5 function analogously as their unlabeled versions. (c) Plot of the percentage of loaded 43S initiation complexes that contain multiple eIF1 binding events on β-globin mRNA in the indicated conditions. ‘Productive’ indicates complexes that progressed to eIF5B binding, whereas ‘any’ indicates that all complexes were analyzed regardless of whether eIF5B bound. (d) Cumulative probability plots of the indicated parameters at differing Cy5-eIF1 concentrations, observed on any loaded initiation complexes (both successful and unsuccessful). Lines represent fits to exponential functions. (e) Schematic of the real-time single-molecule assay with direct excitation of all fluorophores present using dual 532 nm and 640 nm lasers. This excitation scheme examined whether eIF1-Cy5 fully departed initiation complexes. (f) Example single-molecule data of the direct excitation assay showing termination of eIF1 binding is correlated to loss of FRET. The total number of initiation complexes analyzed is indicated (n = 115) and all 744 eIF1-Cy5 binding events ended with complete loss of eIF1-Cy5 signal. See Supplementary Table 1 for the number of complexes and binding events analyzed in each experiment.
