Extended Data Fig. 3: eIF5B associated more rapidly with pre-equilibrated 48S initiation complexes.

a. Schematic of an alternative experimental setup (‘tethered 48S’) used to examine eIF5B association with pre-equilibrated 48S initiation complexes. In this setup, a pre-formed 48S initiation complex at equilibrium on the β-globin mRNA was tethered to the ZMW imaging surface 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 (final concentrations) 20 nM eIF5B-Cy3.5, 100 nM 60S-Cy5 subunits, 1 µM eIF1A, and 1 mM ATP and GTP were added. ZMWs with tethered 48S PICs were identified by the initial presence of 40S-Cy3 fluorescence signal (green). b. Example single-molecule fluorescence data from a tethered 48S PIC experiment where eIF5B association led to 60S subunit joining and rapid departure of eIF5B from the 80S initiation complex at 30 °C. The dwells were defined as in the ‘tethered mRNA’ experimental setup (see, Extended Data Fig. 2d). c. Comparison of observed eIF5B association times (t₁) in tethered mRNA versus tethered 48S PIC experimental setups at 30 °C. The pre-equilibration of the 48S PIC prior to tethering allowed the slow upstream step to proceed, which enabled rapid and concentration-dependent eIF5B association. Fits of the observed association times are represented by the lines, which yielded the indicated rates (n = 172 and 111 for 20 & 40 nM, respectively). In one experiment (magenta, n = 113), the 48S PIC was formed and pre-equilibrated in the absence of eIF5, and then 2.5 nM eIF5 was added simultaneously with labeled eIF5B and 60S subunits to the tethered 48S PIC. The ‘tethered mRNA’ data were replotted from Extended Data Fig. 2e to facilitate comparisons. All errors represent 95% C.I. d. Example single-molecule fluorescence data from the tethered 48S complex experimental setup where wild-type eIF5B-Cy3.5 (20 nM) was added to pre-equilibrated 48S initiation complxes either in the absence of 60S subunits (top) or when 60S subunits (100 nM) were present but GTP was replaced with 1 mM GDPNP in free solution (bottom). e. Plot of the total lifetime of wild-type eIF5B-Cy3.5 on tethered 48S complexes in the indicated conditions. In the presence of 60S subunits and 1 mM GTP, eIF5B remained on initiation complexes for a total of ~15 s (t₂ + t₃). The total lifetime of eIF5B was lengthened approximately 34-fold to ~510 s when GTP was replaced with 1 mM of the non-hydrolyzable analog, GDPNP. eIF5B total lifetime also was lengthened dramatically (to ~ 130 s) when 60S subunits were omitted and 1 mM GTP was present. f. Table of 60S subunit joining and eIF5B association efficiencies when eIF2–\({\text{Met-tRNA}}_{{\rm{i}}}^{{\rm{Met}}}\)–GTP or eIF2–\({\text{Met-tRNA}}_{{\rm{i}}}^{{\rm{Met}}}\)–GDPNP were present at 30 °C. 60S subunit joining efficiency was quantified in the tethered mRNA experimental setup with 1 µM unlabeled eIF5B present. The 95% CI of the observed efficiencies are indicated, with 200 or 204 40S loading events or 48S complexes analyzed in each experiment, respectively. Observed efficiencies were corrected to account for the 40S and 60S subunit labeling efficiencies, which were 80% and 38%, respectively, with errors propagated from the observed 95% CIs. eIF5B association efficiency (45% labeled) was quantified in the tethered 48S PIC setup (to facilitate analyses) in the absence of 60S subunits.