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. 2014 Aug 21;10(8):e1004553.
doi: 10.1371/journal.pgen.1004553. eCollection 2014 Aug.

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

Sara Elgamal  1 , Assaf Katz  2 , Steven J Hersch  3 , David Newsom  4 , Peter White  5 , William Wiley Navarre  3 , Michael Ibba  1
Affiliations

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

Sara Elgamal et al. PLoS Genet. .

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Translation pausing at PPX sequences.
A) Histogram of pausing index of PPX sequences in WT (dark gray) and Δefp strains (light gray). B) Histogram of changes in pausing index of PPX after efp deletion. C) Example of two genes bearing different ribosome occupancies for a similar PPX sequence. Left, malE contains a PNPPK sequence that does not produce translation pausing and right ubiD contains a PEPPK sequence that produces a pause in translation. Insets show the ribosome occupancies for the full genes. Black and red are used for the two WT strain replicates, green and blue for the Δefp replicates, while cyan and magenta are used for the complemented strain replicates. Values in the figure are normalized to the corresponding gene ribosome occupancy average.
Figure 2
Figure 2. Effects on translation pausing of aSD affinity and the identity of the amino acid preceding PPX.
A) Diverse sequences coding for similar amino acid patterns, but with varying aSD affinities, were introduced at the N-terminus of GFP. GFP production in WT and Δefp strains was measured and normalized against mCherry, which is cloned as a transcription fusion. aSD affinities 6 positions upstream of the third nucleotide of the X codon are shown. B) Distribution of the effects of all the clones that code for the same amino acid pattern (irrespective of their specific aSD affinities) are represented as box plots in dark lines (average as a small square, mean as the middle line of the box, box limits represent 25th and 75th percentiles). Average values for each specific clone are shown in light gray. The mean of at least three biological replicates is shown and error bars (which indicate one standard deviation) are only shown in A for clarity.
Figure 3
Figure 3. Effect of amino acids preceding PPX on pausing efficiency.
Various sequences were introduced at the area coding for the N-terminus of GFP. When possible, sequences of genes found to either pause or not pause in E. coli were used (names in parenthesis). A) PP-basic, B) PP-acid and PP-hydrophobic, C) PP-amide, D) PP-hydroxy and E) PP-Pro. The mean of at least three biological replicates is shown and error bars indicate one standard deviation.
Figure 4
Figure 4. Dependence on EF-P of AtpA and AtpD synthesis.
A) Ribosome profiling and SILAC ratios comparing atpD and atpA translational stalling and AtpD and AtpA protein levels in E. coli and Salmonella Typhimurium. Ribosome profiling is from this work; † E. coli SILAC data from ; ‡ S. Typhimurium SILAC data from . B) Representative western blot showing AtpD levels in strains of Salmonella. DnaK was included as a loading control. C) Densitometry quantification of western blots showing mean AtpD/DnaK ratio relative to WT across three biological replicates. Error bars indicate one standard deviation. D) Fluorescence ratios comparing synthesis of plasmid-encoded AtpD- and AtpA-GFP translational fusions in wild-type (WT) and efp mutant Salmonella. LacZ is included as a control and specific point mutations affecting the PPG motifs are indicated. Ratios show WT/Δefp for GFP fluorescence at 10 hours post-inoculation normalized to optical density (600 nm). The mean of at least three biological replicates is shown and error bars indicate one standard deviation.
Figure 5
Figure 5. Effect of upstream residues on the EF-P dependence of AtpD and AtpA synthesis.
A) Sequences (Salmonella Typhimurium) of AtpD and AtpA in proximity to their PPG motifs (bold). The relative position when the glycine of PPG occupies the A site is shown above. The amino acid position of the second proline of the PPG motif in each protein is indicated below. B) Fluorescence ratios comparing expression of plasmid-borne AtpD-GFP translational fusions in wild-type (WT) and efp mutant Salmonella. ‘Swap’ constructs indicate swap-in of AtpA sequence for the specified number of amino acids upstream of the PPG motif. LacZ, unmodified (wt), P214L and R279P constructs from Figure 4 are included for comparison. Ratios show WT/Δefp for GFP fluorescence at 10 hours post-inoculation normalized to optical density (600 nm). The mean of at least three biological replicates is shown and error bars indicate one standard deviation. C) As in B, but with AtpA-GFP translational fusion constructs with swap-in of AtpD upstream sequence.
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