High-precision analysis of translational pausing by ribosome profiling in bacteria lacking EFP

Abstract

Ribosome 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.

Figure 1

Three strategies for assigning ribosome occupancy. A) Average ribosome occupancy aligned at the start codon. Reads from the Δefp3 dataset were assigned using the center-assignment strategy (grey) or the 5′ or 3′-ends of the reads (black and blue, respectively). B) Average ribosome occupancy aligned at the stop codon. C) Expanded view at the start codon using the 3′-assignment method. D) Average density aligned at start codons using the 5′-assignment strategy. Each plot represents reads of a single length; eleven plots are shown in different colors for 25 – 35 nt reads. E) Comparison of the three assignment strategies at the stalling motif in secM, where the ribosome stalls with the Gly codon in the P site.

Figure 2

Ribosome pausing at Pro-Pro motifs. A) Ribosome density in the recG gene for two biological replicates of wild-type (black) and efp knockout cells (red). Inset: expanded view of the strong pause site in the Δefp2 data. B) Correlation of pause scores at 1537 individual instances of Pro-Pro motifs in two biological replicates of the efp knockout strain. The red line represents y = x. C) Pause scores at individual instances of Pro-Pro motifs in the wt2 and Δefp2 data. D) Fraction of Pro-Pro instances with a pause score above a given strength, calculated from the average of the Δefp1 and Δefp2 data (red), wt1 and wt2 data (black), or from the ΔepmA1 (blue), ΔepmB1 (green), or ΔepmC1 data (yellow).

Figure 3

Effect of protein context on proline pauses. A) Pause scores for PP(X) where X is the codon in the ribosomal A site. These numbers represent the average of the scores computed for each of the four efp knockout datasets, with standard error of the mean. B) Effect of the −3 residue (Z) in the nascent polypeptide on pausing at ZPP(X). C) Effect of the −2 residue in the nascent peptide on pausing at Pro-Pro, with the second Pro codon in the A site. D) Average ribosome density at one, two, or three Pro codons in the combined Δefp3 and Δefp4 data. Inset: close-up of the density 30 – 60 nt downstream of the pause site. See also Figure S1 and Table S2.

Figure 4

Reduced ribosome density downstream of pause sites. A) Ribosome occupancy on the cysQ and katE genes in wild-type (black) and efp mutant data (red). B) For a subset of 300 genes containing strong pausing motifs, we calculated an asymmetry score (AS), the density upstream of the pause divided by the density downstream. The fraction of genes above a given AS is plotted for the combined four wild-type (black) or efp mutant datasets (red). C) For 30 genes with the highest or lowest AS in the efp knockout, we plotted the fraction of genes with more than the given translational efficiency. D) For 30 genes with the highest AS in the efp knockout, the fraction of genes with the pausing motif after a specific position was plotted. E) Correlation of the relative protein abundance in MS data vs the relative ribosome occupancy downstream of the pause site for genes with strong pausing motifs. See also Figure S2 and Table S3.

Figure 5

Three factors that determine how pausing affects protein output. Translational efficiency is represented by a scale of low to high initiation levels; pause strength by the size of the pause button; and 5′-polarity by the position along the gene. Red represents reduced protein levels and green represents normal levels in the efp mutant.