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

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.

[1]  Runjun D. Kumar,et al.  PoxA, yjeK, and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica. , 2010, Molecular cell.

[2]  E. O’Shea,et al.  An Integrated Approach Reveals Regulatory Controls on Bacterial Translation Elongation , 2014, Cell.

[3]  R. Parker,et al.  Endonucleolytic cleavage of eukaryotic mRNAs with stalls in translation elongation , 2006, Nature.

[4]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Anthony C. Forster,et al.  Slow peptide bond formation by proline and other N-alkylamino acids in translation , 2009, Proceedings of the National Academy of Sciences.

[6]  Hunter B. Fraser,et al.  Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation , 2014, Genome research.

[7]  Harry F. Noller,et al.  The Path of Messenger RNA through the Ribosome , 2001, Cell.

[8]  M. Ibba,et al.  Translation Initiation Rate Determines the Impact of Ribosome Stalling on Bacterial Protein Synthesis* , 2014, The Journal of Biological Chemistry.

[9]  C. J. Woolstenhulme,et al.  eIF5A promotes translation of polyproline motifs. , 2013, Molecular cell.

[10]  Daniel N. Wilson,et al.  Nascent peptides that block protein synthesis in bacteria , 2013, Proceedings of the National Academy of Sciences.

[11]  R. Bundschuh,et al.  The conserved GTPase LepA contributes mainly to translation initiation in Escherichia coli , 2014, Nucleic acids research.

[12]  Kirsten Jung,et al.  Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P , 2013, Proceedings of the National Academy of Sciences.

[13]  Gene-Wei Li,et al.  The anti-Shine-Dalgarno sequence drives translational pausing and codon choice in bacteria , 2012, Nature.

[14]  T. Inada,et al.  Nascent Peptide-dependent Translation Arrest Leads to Not4p-mediated Protein Degradation by the Proteasome* , 2009, Journal of Biological Chemistry.

[15]  J. Plotkin,et al.  Synonymous but not the same: the causes and consequences of codon bias , 2011, Nature Reviews Genetics.

[16]  Jonathan S. Weissman,et al.  rRNA:mRNA pairing alters the length and the symmetry of mRNA-protected fragments in ribosome profiling experiments , 2013, Bioinform..

[17]  Henning Urlaub,et al.  EF-P Is Essential for Rapid Synthesis of Proteins Containing Consecutive Proline Residues , 2013, Science.

[18]  M. Rodnina,et al.  Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates* , 2008, Journal of Biological Chemistry.

[19]  L. Gold,et al.  [27] Extension inhibition analysis of translation initiation complexes☆ , 1988 .

[20]  Shashi Bhushan,et al.  SecM-Stalled Ribosomes Adopt an Altered Geometry at the Peptidyl Transferase Center , 2011, PLoS biology.

[21]  Koreaki Ito,et al.  Arrest peptides: cis-acting modulators of translation. , 2013, Annual review of biochemistry.

[22]  Peter White,et al.  EF-P Dependent Pauses Integrate Proximal and Distal Signals during Translation , 2014, PLoS genetics.

[23]  Nicholas T. Ingolia,et al.  Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity and Dynamics of Mammalian Proteomes , 2011, Cell.

[24]  Nicholas T. Ingolia,et al.  Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling , 2009, Science.

[25]  C. J. Woolstenhulme,et al.  Genetic Identification of Nascent Peptides That Induce Ribosome Stalling* , 2009, The Journal of Biological Chemistry.

[26]  J. Weissman,et al.  Selective Ribosome Profiling Reveals the Cotranslational Chaperone Action of Trigger Factor In Vivo , 2011, Cell.

[27]  Koreaki Ito,et al.  Genetically encoded but nonpolypeptide prolyl-tRNA functions in the A site for SecM-mediated ribosomal stall. , 2006, Molecular cell.

[28]  William Wiley Navarre,et al.  Loss of Elongation Factor P Disrupts Bacterial Outer Membrane Integrity , 2011, Journal of bacteriology.

[29]  Koreaki Ito,et al.  The Ribosomal Exit Tunnel Functions as a Discriminating Gate , 2002, Cell.

[30]  Ryohei Ishii,et al.  A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P , 2010, Nature Structural &Molecular Biology.

[31]  R A Laskey,et al.  High sequence specificity of micrococcal nuclease. , 1981, Nucleic acids research.

[32]  Leonard J. Foster,et al.  Divergent Protein Motifs Direct Elongation Factor P-Mediated Translational Regulation in Salmonella enterica and Escherichia coli , 2013, mBio.

[33]  Daniel N. Wilson,et al.  Lys34 of translation elongation factor EF-P is hydroxylated by YfcM. , 2012, Nature chemical biology.

[34]  Zoya Ignatova,et al.  Transient ribosomal attenuation coordinates protein synthesis and co-translational folding , 2009, Nature Structural &Molecular Biology.

[35]  R. Sauer,et al.  SsrA‐mediated peptide tagging caused by rare codons and tRNA scarcity , 1999, The EMBO journal.

[36]  Kirsten Jung,et al.  Translation Elongation Factor EF-P Alleviates Ribosome Stalling at Polyproline Stretches , 2013, Science.

[37]  D. P. Burma,et al.  Association of ribonuclease I with ribosomes and their subunits. , 1972, The Journal of biological chemistry.

[38]  L. Gold,et al.  Extension inhibition analysis of translation initiation complexes. , 1988, Methods in enzymology.

[39]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[40]  Kirsten Jung,et al.  A conserved proline triplet in Val-tRNA synthetase and the origin of elongation factor P. , 2014, Cell reports.

[41]  Kirsten Jung,et al.  Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site , 2014, Nucleic acids research.