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

Abstract

Summary 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 identify features that explain this phenomenon. These features allow us to predict which proteins likely have reduced output in the efp knockout strain.

Dual DNA rulers reveal an ‘mRNA looping’ intermediate state during ribosome translocation

“…The residue at the 3ʹ end of the mRNA exiting the ribosome is deduced by the cDNA length that was limited by the clash between the reserve transcriptase and the ribosome. The ribosome profiling method maps ribosome-covered mRNA with high throughput sequencing, which reveals the global ribosome distribution but lacks single codon precision [7]. Among indirect translocation assays, the tandem LC/MS/MS analysis of oligopeptide composition deduces the ribosome reading frame based on the synthesized peptide [8].…”

Mapping the non-standardized biases of ribosome profiling

“…A disadvantage of MNase is its preferential cleavage at A or T nucleotides (Dingwall et al, 1981) and consequently, the MNase-generated ribosome footprints might be enriched in A or T nucleotides at their 5′ ends. Compared to fragments derived from yeast lysates treated with RNase I, the MNase-generated footprints are more heterogeneous in length (Becker et al, 2013) due to steric effects and less precise 5′ cleavage (Woolstenhulme et al, 2015). In contrast, MNase cleaves precisely at the 3′ end contour of the ribosome, thus the calibration of the reads in bacterial system should be preferably done using the 3′ ends of the reads (Woolstenhulme et al, 2015) (see section 'Analysis of the sequencing data').…”

“…Compared to fragments derived from yeast lysates treated with RNase I, the MNase-generated footprints are more heterogeneous in length (Becker et al, 2013) due to steric effects and less precise 5′ cleavage (Woolstenhulme et al, 2015). In contrast, MNase cleaves precisely at the 3′ end contour of the ribosome, thus the calibration of the reads in bacterial system should be preferably done using the 3′ ends of the reads (Woolstenhulme et al, 2015) (see section 'Analysis of the sequencing data'). RNase I cleavages are precise at both 5′ and 3′ ends, enabling calibration using both termini.…”

“…Pulverized bacteria or monocellular eukaryotes are homogenized in a mill with liquid nitrogen (Oh et al, 2011;Guydosh and Green, 2014;Woolstenhulme et al, 2015). This method is transferrable to any cell type and frozen tissue and should be the preferred lysis approach as it allows treatment of the sample at very low temperatures.…”

“…During the homogenization, local temperature fluctuations in the sample should be avoided by careful choice of the conditions, i.e. short homogenization pulses and pre-cooling the grinder jar before and after each homogenization cycle (Oh et al, 2011;Guydosh and Green, 2014;Woolstenhulme et al, 2015).…”