a, The dwell times for the transition from initiation to elongation (open circles) were fitted to single-exponential distributions to estimate the average transition dwell times (Δt values, with 95% confidence intervals, red) from experiments performed with unlabelled eIF5B and cap-RPL30 mRNA or model mRNA-Kozak at 20 °C in the presence of 1–10 mM Mg2+ (data for 3 mM Mg2+ were taken from Fig. 1d). For cap-RPL30, unstable 80S formation was observed at 1 mM Mg2+ (f) and thus no Δt values were obtained. From bottom to top for each group: n = 108, n = 189, n = 124, n = 150, n = 144, n = 118 and n = 195. b, The dwell times for the transition from initiation to elongation (open circles) were fitted to single-exponential distributions to estimate the average transition dwell times (Δt values, with 95% confidence intervals, red) from experiments performed with unlabelled eIF5B and cap-RPL30 or model mRNA-Kozak, under the indicated conditions. From left to right for each group: n = 195, n = 152, n = 118, n = 130, n = 150, n = 132, n = 189 and n = 159. c, The dwell times (open circles) for the transition from initiation to elongation, eIF5B lifetimes on 80S, and tRNA arrival times after eIF5B departure were fitted to single-exponential distributions to estimate the average dwell times (with 95% confidence intervals, black). Experiments were performed with cap-RPL30 mRNA at 3 mM free Mg2+ and 20 °C in the presence of unlabelled eIF5B (n = 195; data taken from Fig. 1d) or Cy5.5–eIF5B (n = 164; data taken from Fig. 2c); or at 2 mM free Mg2+ and 30 °C in the presence of unlabelled eIF5B (n = 152; data taken from b) or Cy5.5–eIF5B (n = 150). d, The dwell times for 60S joining (open circles) from the same experiments as in a were fitted to single-exponential distributions (for model mRNA-Kozak) or double- exponential distributions (for cap-RPL30; the average times for the fast phase were plotted here) to estimate the average dwell time (with 95% confidence intervals, red). e, The cumulative probability distribution of the 60S joining times, showing the compromised rate of 60S joining in experiments that were performed with the model mRNA-Kozak at 1 mM Mg2+ and 20 °C as in a (n = 150). However, we still observed that the arrival of tRNA to the A site occurred readily after 80S formation. The kinetics of 60S joining under these reaction conditions were not fitted well by single- or double-exponential distributions and therefore no average time was deduced for the bar plot in d. f, Sample trace from experiments performed with cap-RPL30 mRNA at 1 mM Mg2+ and 20 °C as described in a (n = 200). g, h, Dot plots for Fig. 2c, e, showing the single-exponential distributions of the dwell times, with the average dwell times (with 95% confidence interval) in black. Notes: (1) increasing free Mg2+ concentrations increased the Δt values (that is, higher Mg2+ concentration favours the eIF5B-bound semi-rotated 80S conformation (Extended Data Fig. 10)). This seems contrary to the known effects of free Mg2+ concentrations on the conformation of the bacterial pre-translocation 70S elongation complex50: lower Mg2+ concentration favours the rotated state and higher Mg2+ concentration favours the non-rotated state. The apparent discrepancy might be a result of the different compositions of the complexes: in our case, the semi-rotated state of the 80S contains an acylated Met-tRNAMet i in the P site and a protein factor eIF5B bound in the A site; whereas in the pre-translocation 70S complex, the P-site tRNA is deacylated and a peptidyl-tRNA is bound in the A site. The differences in the energy landscape between these two types of complexes can also be explained by the observation that there are no apparent fluctuations between semi-rotated and non-rotated 80S conformations with eIF5B bound, whereas there are frequent fluctuations between rotated and non-rotated 70S conformations in the pre-translocation complexes. (2) The different magnitudes of dependence of Δt values on temperature (Fig. 1d) and Mg2+ concentration for different mRNAs suggest that there are mRNA sequence-context differences in the thermodynamics that govern the transition from initiation to elongation. Similarly, the minimal Mg2+ concentrations required for stable 80S assembly on different mRNAs are different. Source data