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. 2019 Feb 6;8:e42591. doi: 10.7554/eLife.42591

Figure 1. Comparison of ribosome profiling data from yeast and E.coli.

(A) Average ribosome density on genes aligned at the start codon using the 5’-end of reads in yeast (library SRR1042864), or the center or 3’-end of reads from E. coli (library SRR1734438). (B) Length distribution of yeast and E. coli ribosome-protected fragments mapping uniquely to coding sequences. (C) The fraction of reads at the first, second, or third nt within codons in yeast profiling data (blue), E. coli profiling data (grey), RNA-seq from total RNA digested with MNase (yellow), and profiling data in which nucleases RelE and MNase were used to generate ribosome-protected footprints (red). See also Figure 1—figure supplement 1.

Figure 1.

Figure 1—figure supplement 1. Preferential isolation of long RPFs increases ribosome density at SD-like motifs within open reading frames.

Figure 1—figure supplement 1.

Cross-correlation plots were generated by first, calculating a genome-wide map of SD affinity using an eight nt sliding window, and then taking the correlation of the SD-affinity map with ribosome density at different offset values (Li et al., 2012). The strong peak at −22 in L18 (black) indicates a positive correlation between SD affinity and ribosome density as would be expected if SD motifs caused ribosomes to pause in ORFs. The −22 offset is consistent with the known spacing between the SD motif and the 3’-boundary of the ribosome during initiation (top). In contrast to the strong positive correlation at −22 seen in L18, a negative correlation is observed in L17 (blue). This difference arises from how footprints were isolated: L18 contains exclusively long RPFs (>28 nt) whereas L17 contains exclusively short RPFs (20–30 nt). Given that footprints that interact with rRNA tend to be longer (30–35 nt), isolating only long RPFs leads to artificial enrichment of ribosome density at SD-like motifs. No SD pauses are observed in our libraries (e.g. L26, red) that capture the whole distribution of footprint sizes, nor were they observed in samples prepared using our new methods with high MgCl2 lysis buffers with cells harvested by either filtration (L29) or direct freezing (L33) (purple and green respectively). In addition to the peaks at −22 initially attributed to pauses on SD motifs, two other peaks are also observed. The peak at −15 arises from pauses at Gly codons (Figure 3 and Mohammad et al., 2016) because Gly codons are G-rich, giving a spurious but strong SD affinity. In a similar fashion, the peak at 0 arises from cloning bias because the nucleotide G is enriched at the 3’-ends of reads.