Clarifying the Translational Pausing Landscape in Bacteria by Ribosome Profiling

• Ribosome footprints containing Shine-Dalgarno motifs are longer than other footprints
• An earlier study reporting pauses at SD motifs preferentially isolated long footprints
• Pauses at SD motifs within open reading frames are not observed in vitro

SummaryThe rate of protein synthesis varies according to the mRNA sequence in ways that affect gene expression. Global analysis of translational pausing is now possible with ribosome profiling. Here, we revisit an earlier report that Shine-Dalgarno sequences are the major determinant of translational pausing in bacteria. Using refinements in the profiling method as well as biochemical assays, we find that SD motifs have little (if any) effect on elongation rates. We argue that earlier evidence of pausing arose from two factors. First, in previous analyses, pauses at Gly codons were difficult to distinguish from pauses at SD motifs. Second, and more importantly, the initial study preferentially isolated long ribosome-protected mRNA fragments that are enriched in SD motifs. These findings clarify the landscape of translational pausing in bacteria as observed by ribosome profiling.

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