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Figure 1: Discrepancy between experimental FS rates and ribosomal occupancy. (a) Schematic of FS reporters based on a bicistronic expression construct containing CrPV IRES. For the enlarged region of flag-Rluc, all three reading frames are color coded with stop sites shown as perpendicular lines. The FS windows are highlighted in red shade. The right panel shows the relative Rluc activities in transfected cells. Data are shown as means ± SEM, n = 3. (b) The top panel shows the gene structure of Rluc with all three reading frames and stop sites color coded. The middle panel shows the predicted ribosome occupancy along the Rluc coding region based on the +1 FS rate of 0.03% and –1 FS of 0.02%. The bottom panel shows the density of RPFs mapped to the Rluc coding region in transfected HEK293 cells. The line plot represents average ribosome density within a window of 200 nt. (c) Using ribosome profiling data in HEK293 cells, average ribosome density is plotted for the first and second half of each ORF. Genes with no less than 50 read counts in at least half of the CDS are shown in dark red. (d) Using ribosome profiling data in HEK293 cells, the ratio of average ribosome density between first and second half of each ORF is plotted against the ORF size. Genes with no less than 50 read counts in at least half of the CDS are shown in dark red.