Extended Data Fig. 1: Real-time monitoring of human ribosomal subunit recruitment to β-globin mRNA.

a. Human 40S and 60S ribosomal subunits were labeled with fluorescent dyes on the N-terminus of uS19 or C-terminus of uL18, respectively. These labeling positions yield an inter-subunit FRET signal upon formation of translation-competent 80S ribosomes. The structural model was obtained from PDB: 4UG0. Of note, the first nine N-terminal amino acids of uS19 and last two C-terminal acids of uL18 were unresolved in the structure, which also does not account for the fused ybbR tags (11 amino acids). Thus, the reported distance between the 40S and 60S labeling sites is only an approximation. b. A real-time single molecule fluorescence assay using zero-mode waveguides (ZMWs) on a custom PacBio RSII DNA sequencer. Components of interest are tethered to the imaging surface within individual ZMWs, and reaction components are added directly to the surface. Time-resolved, real-time 4-color fluorescence emission was monitored across ~150,000 ZMWs after excitation with a 532 nm laser. The order and time elapsed between relevant fluorescence signals (e.g., green & orange) were determined in ~100–200 ZMWs with the desired signals. Rates of association and dissociation were determined using probability based statistical models; cumulative distribution functions of the observed times were calculated and subsequently fit to exponential functions to yield association or dissociation rates, as appropriate. c. Schematic of a real-time single-molecule assay to monitor ribosomal subunit joining on tethered β-globin mRNA (the ‘tethered mRNA’ setup). β-globin mRNA with a 5' m⁷G cap, poly(A)₃₀ tail, and 3'-terminal biotin moiety was tethered to the neutravidin-coated ZMW imaging surface. Immediately after tethering, eIFs 4A, 4B, 4G, and 4E were added. After start of data acquisition and excitation via the 532 nm laser at 30 °C, the 43S PIC (5 nM via the labeled 40S-Cy3), unlabeled eIF5B (1 µM), 60S-Cy5 subunits (100 nM), and 1 mM of ATP and GTP were added. Unlabeled 43S PIC components were present at \(\ge \) 1.5-fold excess during each step of the experiment. Fluorescence data were acquired for 600 s. d. Cartoon schematic of theoretical single-molecule fluorescence data where 40S and 60S subunits were recruited to an mRNA to form 80S ribosomes. The 40S subunit association time (t_40S) was defined as the time elapsed from addition of the 43S PIC until appearance of the Cy3 signal. The 60S subunit association time (t_60S) was defined as the time elapsed from 40S subunit association until appearance of the 40S(Cy3,donor)-60S(Cy5,acceptor) FRET signal. e. Example single-molecule fluorescence trace with FRET efficiency (E_FRET) plot. Recruitment of the 40S subunit (as the 43S PIC) was indicated by a burst of Cy3 (green) fluorescence intensity. 60S subunit joining was indicated by appearance of the 40S-60S FRET signal. f. Density maps of normalized 40S-Cy3 and 60S-Cy5 fluorescence intensities post-synced to 60S subunit joining (n = 128). As in the individual trace shown in panel D, joining of the 60S subunit led to an anti-correlated decrease in Cy3 and increase in Cy5 signals, indicative of intersubunit FRET when the 80S ribosome formed. g. Histogram and single gaussian function fit (line) of the observed E_FRET in the real-time initiation assay. The mean E_FRET (µ) was 0.65 ± 0.1 and the standard deviation (σ) was 0.17 ± 0.1 (n = 168), consistent with structural predictions for translation-competent 80S ribosomes. h. Table of 40S loading and 60S joining efficiencies (left) and a plot of observed 40S association times (right) in the indicated conditions. 40S subunit loading efficiency (80% labeling efficiency in all experiments) was defined as the fraction of 1,000 analyzed ZMWs with at least one stable (> 10 s) 40S subunit association event. The concentration of 40S subunits (5 nM) was optimized to yield a single recruitment event per ZMW, which is most probable when < 30% of ZMWs contain a recruitment event, as predicted by Poisson distribution statistics. 60S subunit joining efficiency (with errors propagated from 95% CIs) was defined the fraction of recruited 40S subunits (300 analyzed) with a 60S subunit joining event (indicated by Cy3-Cy5 FRET), which was normalized to account for relative 60S subunit labeling efficiencies (35 or 80% labeled). 40S association time (t_40S) was defined as above (n = 135, 128, 131, 155, and 137, from left to right), and the median observed times are represented by the orange lines. i. Cumulative probability plot of 40S and 60S subunit association times and the fits to single-exponential functions, which yielded apparent association rates of k_on,40S ≈ 0.049 ± 0.002 s⁻¹ and k_on,60S ≈ 0.033 ± 0.001 s⁻¹ when added at 5 nM and 100 nM (final concentration), respectively (n = 128).