Extended Data Fig. 5: eIF1A and eIF5B reside simultaneously on initiation complexes when the 60S subunit joins.

a. Schematic of single-molecule experiments that examined eIF1A and eIF5B dynamics on the 48S initiation complex at equilibrium. Pre-formed 48S initiation complexes on β-globin mRNA were tethered at equilibrium within ZMWs in the presence of 1 mM ATP and GTP. After removal of untethered components, data acquisition began via excitation with a 532 nm laser, and an imaging mix that contained (final concentrations) 10 nM eIF1A-Cy5 (red) and 20 nM eIF5B-Cy3.5 (orange) was present at 30 °C. ZMWs with tethered 48S PICs were identified by the initial presence of 40S-Cy3 fluorescence signal (green). b. Rudimentary structural comparison where a low-resolution model of human eIF5B (PDB: 4UJC) was docked onto a high-resolution model of a mammalian 48S PIC post-recognition of the start codon (PDB: 6YAL). From these crude analyses, eIF1A was predicted to be within FRET distance (< 80 Å) of the N-terminus of truncated eIF5B, consistent with the eIF5B(Cy3.5, donor)-eIF1A(Cy5,acceptor) FRET signal observed in our single-molecule assays. c. Example single-molecule fluorescence data that depicts either: top, a complex with 40S(Cy3,donor)-eIF1A(Cy5,acceptor) FRET in the absence of eIF5B signal (‘eIF5B absent’); bottom, a complex with both 40S(Cy3,donor)-eIF1A(Cy5,acceptor) and eIF5B(Cy3.5,donor)-eIF1A(Cy5,acceptor) FRET, (‘eIF5B present’). Focused analyses were conducted on both forms of the 48S PIC to derive kinetic parameters. d. Cumulative probability plots of observed eIF1A reassociation times with (left) or eIF1A lifetimes on (right) the 48S initiation complex at 30 °C at equilibrium. eIF1A-Cy5 was present at 10 nM. The lines represent fits of the observed data to double-exponential functions, which yielded the indicated rates. All errors represent 95% C.I. 831 and 589 eIF1A binding events were analyzed when eIF5B was present or absent, respectively. e. Schematic of the four-color single-molecule experiment. The doubly labeled 43S PIC (10 nM by 40S-Cy3; eIF1A-Cy5), 40 nM eIF5B-Cy3.5, and 200 nM 60S-Cy5.5 subunit were added to b-globin mRNA tethered within ZMWs in the presence of saturating concentrations of eIFs 4A, 4B, 4G, and 4E, and 1 mM ATP and GTP at 30 °C. During imaging, eIF1A-Cy5 was present at 4.5-fold molar excess relative to the 40S subunit. Fluorescence data were acquired for 600 s with excitation via the 532 nm laser. The potential FRET signals are indicated in the box. f, g. Theoretical (panel F) and example (panel G) four-color single-molecule fluorescence data from a ZMW where a loaded doubly labeled 43S PIC (40S-Cy3, green; eIF1A-Cy5, red) was bound by eIF5B-Cy3.5 (orange), which was followed by 60S-Cy5.5 (purple) subunit joining. The 43S PIC was recruited in a 40S(Cy3, donor)-eIF1A(Cy5, acceptor) FRET state. Once eIF5B bound, the eIF1A-Cy5 signal increased due to eIF5B(Cy3.5, donor)-eIF1A(Cy5, acceptor) FRET. Joining of the 60S subunit was indicated by appearance of Cy5.5 fluorescence signal due to 40S(Cy3, donor)-60S(Cy5.5, acceptor) FRET. Departure of eIF1A-Cy5 and eIF5B-Cy3.5 was indicated by loss of Cy5 and Cy3.5 fluorescence signals, respectively. In panel G, the full experimental window is depicted on the left, and the middle and right panels represent zoomed views of the indicated time windows. Given bleed through across the four fluorescent channels, the fluorescence signals in each channel were made transparent before relevant events for presentation here. h. Quantification of eIF1A and eIF5B occupancy upon 60S subunit joining in the four-color single-molecule experiment. The potentially convoluted FRET signals and fluorescence bleed through among channels and factors precluded rigorous kinetic analyses, as exact frames for association and dissociation were extremely challenging to assign. However, the experiment did allow the presence of eIF1A and eIF5B upon 60S subunit joining to be quantified unmistakably. On a large majority of 48S initiation complexes (183/271), eIF1A-Cy5 signal was present when the 60S subunit joined, which corresponded to an estimated eIF1A occupancy of about 85 ± 5 % after correction for eIF1A labeling efficiency (~80 %). Of those eIF1A-bound 48S complexes, nearly half (88/183) also contained eIF5B-Cy3.5 signal when the 60S subunit joined, which indicated that about 90 ± 10 % of the 48S PICs contained both eIF1A and eIF5B, after correction for eIF5B labeling efficiency (~45%). eIF1A preceded eIF5B association on nearly all (79/88; 90 ± 20 %) 48S complexes that contained both labeled proteins when the 60S subunit joined.