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Additional file 3. Peer review history.

Additional file 1: Fig. S1. High-throughput quantification of GFP intensities by FACS-seq. Fig. S2. Frame- and context-dependent inhibitory effects on protein synthesis by proximal dAUGs. Fig. S3. Codon adaptation index (CAI) and minimum free energy (MFE) do not significantly vary among Duo variants that contain dATGs at different positions. Fig. S4. The mRNA levels of dual-frame reporters and their respective controls measured by quantitative PCR. Fig. S5. High-throughput measurement of mRNA levels for dATG variants. Fig. S6. GFP intensities of dATG variants in the background of upf1Δ. Fig. S7. GFP intensity and mRNA level of the dATG variants in the 2A-inserted library. Fig. S8. Scatter plots showing GFP intensity and mRNA level in two biological replicates for yeast libraries. Fig. S9. GFP intensity and mRNA level for uATG variants in the background of hoΔ. Fig. S10. GFP intensity and mRNA level for uATG variants in the background of upf1Δ. Fig. S11. The optimization of the parameters in the Brownian ratchet scanning model using the MCMC algorithms. Fig. S12. The observed GFP intensities of dATG variants in FACS-seq experiments and the simulated GFP intensities under the Brownian ratchet scanning model. Fig. S13. Numbers of genes that harbor frame 0 or frame +2 dATGs at individual positions downstream of the aATG in the yeast or human genomes. Fig. S14. Numbers of genes harboring out-of-frame dATG for highly/broadly and lowly/narrowly expressed genes.

Additional file 2. Review history.

Additional file 1: Fig. S1. Disomes persisted after RNase I digestion. Fig. S2. Correlations between libraries of disome-seq, monosome-seq, and mRNA-seq. Fig. S3. The distribution of monosome and disome footprints. Fig. S4. The A-site pausing scores between replicates or between data processing approaches. Fig. S5. Highly translated mRNAs are crowded with ribosomes. Fig. S6. The contact interfaces of disomes (cycloheximide omitted in the lysate) and di-ribosomes. Fig. S7. The identification of disome-associated proteins. Fig. S8. Ribosome footprints in the two-ribosome-containing transcripts. Fig. S9. The detection of the 5′-ligation bias during the library preparation for monosome-seq and disome-seq. Fig. S10. The pausing scores estimated using the codon/amino-acid frequency in the genome as the background. Fig. S11. Additional cryo-EM structures of disomes. Fig. S12. On the 61-nt disome footprints. Fig. S13. On the 53-nt disome footprints. Fig. S14. The “inverse ramp” of disome footprints on the CDS. Fig. S15. The mRNA secondary structure downstream of disome footprints. Fig. S16. The presumed conformation and the putative A-site for disome footprints of various lengths Fig. S17. Cryo-EM data processing for disome particles that were collected with cycloheximide omitted in the lysis buffer. Fig. S18. Fourier shell correlation (FSC) curves for the final 3D density maps after RELION-based post-processing. Table S1. Summary of the monosome-seq libraries. Table S2. Summary of the disome-seq libraries. Table S3. Summary of the mRNA-seq libraries. Table S4. Disome-associated proteins (replicate 1: heavy isotope labeled disome proteins, disome/monosome intensity ratio > 1.5). Table S5. Disome-associated proteins (replicate 2: light isotope labeled disome proteins, disome/monosome intensity ratio > 1.5). Table S6. Primers used for tagging 4×FLAG to the C-terminus of each chaperone.