EF-P dependent pauses integrate proximal and distal signals during translation

Elongation factor P (EF-P) is required for the efficient synthesis of proteins with stretches of consecutive prolines and other motifs that would otherwise lead to ribosome pausing. However, previous reports also demonstrated that levels of most diprolyl-containing proteins are not altered by the deletion of efp. To define the particular sequences that trigger ribosome stalling at diprolyl (PPX) motifs, we used ribosome profiling to monitor global ribosome occupancy in Escherichia coli strains lacking EF-P. Only 2.8% of PPX motifs caused significant ribosomal pausing in the Δefp strain, with up to a 45-fold increase in ribosome density observed at the pausing site. The unexpectedly low fraction of PPX motifs that produce a pause in translation led us to investigate the possible role of sequences upstream of PPX. Our data indicate that EF-P dependent pauses are strongly affected by sequences upstream of the PPX pattern. We found that residues as far as 3 codons upstream of the ribosomal peptidyl-tRNA site had a dramatic effect on whether or not a particular PPX motif triggered a ribosomal pause, while internal Shine Dalgarno sequences upstream of the motif had no effect on EF-P dependent translation efficiency. Increased ribosome occupancy at particular stall sites did not reliably correlate with a decrease in total protein levels, suggesting that in many cases other factors compensate for the potentially deleterious effects of stalling on protein synthesis. These findings indicate that the ability of a given PPX motif to initiate an EF-P-alleviated stall is strongly influenced by its local context, and that other indirect post-transcriptional effects determine the influence of such stalls on protein levels within the cell.