Ribosome A and P sites revealed by length analysis of ribosome profiling data.
Bottom Line: Yet the location of the codon being decoded in ribosome footprints is still unknown, and has been complicated by the recent observation of footprints with non-canonical lengths.Here we show how taking into account the variations in ribosome footprint lengths can reveal the ribosome aminoacyl (A) and peptidyl (P) site locations.We also show that GC-rich motifs at the 5' ends of footprints are found in yeast, calling into question the anti-Shine-Dalgarno effect's role in ribosome pausing.
Affiliation: Department of Biology, Johns Hopkins University, 3400 N. Charles St Baltimore, MD 21218, USA.Show MeSH
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Mentions: We tallied the occurrences of proline codons at all positions in ribosome footprints, separated by footprint length (Figure 1) and normalized these frequencies by the background frequencies from a parallel mRNA-seq experiment (14). The resulting data can be visualized as a 2D heat map, where hotter colors represent enrichment in ribosome footprints relative to the mRNA background. We confirm that, as previously described in yeast, all four proline codons (CCN) are enriched (Figure 3). Overall, prolines are about 1.5–2.5-fold enriched, with CCA being the most and CCG the least (Figure 3). Furthermore, we find that longer ribosome footprints show similar codon frequencies as shorter footprints, if the footprints are aligned to the 5′ end. The positions which are most enriched in prolines are conserved between the lengths in vertical patterns. For example, proline codon CCA is most highly enriched 4 codons downstream the 5′ end and enrichment is less at the other positions. Since proline incorporation is thought to retard ribosome processivity at the P site, during peptidyl transfer (13), we infer that the greatest enrichment at position four indicates the P site location.
Affiliation: Department of Biology, Johns Hopkins University, 3400 N. Charles St Baltimore, MD 21218, USA.