High-Precision Analysis of Translational Pausing by Ribosome Profiling in Bacteria Lacking EFP

• The 3′ end of footprints best defines occupancy in bacterial ribosome profiling
• Though pausing at Pro-Pro motifs is widespread, context determines the intensity
• Strong pauses near the 5′ end of genes reduce downstream ribosome density

SummaryRibosome profiling is a powerful method for globally assessing the activity of ribosomes in a cell. Despite its application in many organisms, ribosome profiling studies in bacteria have struggled to obtain the resolution necessary to precisely define translational pauses. Here, we report improvements that yield much higher resolution in E. coli profiling data, enabling us to more accurately assess ribosome pausing and refine earlier studies of the impact of polyproline motifs on elongation. We comprehensively characterize pausing at proline-rich motifs in the absence of elongation factor EFP. We find that only a small fraction of genes with strong pausing motifs have reduced ribosome density downstream, and we identify features that explain this phenomenon. These features allow us to predict which proteins likely have reduced output in the efp-knockout strain.

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