Fig. 1
From: Disome-seq reveals widespread ribosome collisions that promote cotranslational protein folding
Disome-seq detects ribosome collisions. a A schematic explaining why traditional ribo-seq could miss the information on ribosome collisions and why the investigation of disome footprints may provide unique information on translation. b The disome persisted after RNase I digestion. Sucrose gradient profiles of the ribosome-mRNA complexes without (black) and with (green) RNase I digestion are shown. The x-axis displays the positions in the 5–50% sucrose gradient. The y-axis indicates the RNA abundance inferred from UV absorption (OD254). RNP, free ribonucleoprotein. c The schematic of disome-seq for 3-AT treated yeast cells. Briefly, ribosomes were extracted in a lysis buffer and digested with RNase I. Extracted RNA was separated on a polyacrylamide gel. RNA fragments with the length of approximate 20–30 nts or 50–80 nts were subjected to high-throughput sequencing. d The length distribution of disome footprints obtained from 3-AT treated yeast cells. Footprints are shown in different colors according to the coding frame of its 5′-end (the top panel). The average footprint abundance of two replicates is shown, in the unit of reads per million (RPM). e Determination of the conformation for the 58-nt disome footprints. Aggregated abundance profiles of the 5′-end of monosome (top) and disome footprints (bottom) are plotted around histidine (His) codons. Footprints were aligned by the first nucleotide of His codons (set at position 0). P and A represent the P-site and A-site of a ribosome, respectively. The 5′-end of the monosome (disome) footprints exhibited the main peak at 15-nt (45-nt) upstream of the histidine codons in the yeast genome, indicating that the A-site of the ribosome (the leading ribosome in a disome) locates at the 16th–18th (46th–48th) nts in the 28-nt monosome (58-nt disome) footprints. The average footprint abundance of two replicates is shown