title: Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P.
abstract: Ribosomes are the protein synthesizing factories of the cell, polymerizing polypeptide chains from their constituent amino acids. However, distinct combinations of amino acids, such as polyproline stretches, cannot be efficiently polymerized by ribosomes, leading to translational stalling. The stalled ribosomes are rescued by the translational elongation factor P (EF-P), which by stimulating peptide-bond formation allows translation to resume. Using metabolic stable isotope labeling and mass spectrometry, we demonstrate in vivo that EF-P is important for expression of not only polyproline-containing proteins, but also for specific subsets of proteins containing diprolyl motifs (XPP/PPX). Together with a systematic in vitro and in vivo analysis, we provide a distinct hierarchy of stalling triplets, ranging from strong stallers, such as PPP, DPP, and PPN to weak stallers, such as CPP, PPR, and PPH, all of which are substrates for EF-P. These findings provide mechanistic insight into how the characteristics of the specific amino acid substrates influence the fundamentals of peptide bond formation.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.
authors.forename: Lauri
authors.initials: L
authors.lastname: Peil
authors.forename: Agata L
authors.initials: AL
authors.lastname: Starosta
authors.forename: Jürgen
authors.initials: J
authors.lastname: Lassak
authors.forename: Gemma C
authors.initials: GC
authors.lastname: Atkinson
authors.forename: Kai
authors.initials: K
authors.lastname: Virumäe
authors.forename: Michaela
authors.initials: M
authors.lastname: Spitzer
authors.forename: Tanel
authors.initials: T
authors.lastname: Tenson
authors.forename: Kirsten
authors.initials: K
authors.lastname: Jung
authors.forename: Jaanus
authors.initials: J
authors.lastname: Remme
authors.forename: Daniel N
authors.initials: DN
authors.lastname: Wilson
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D002853:Chromatography, Liquid; D048168:Escherichia coli K12 / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000502:physiology*; D040901:Proteomics; D012270:Ribosomes / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D001616:beta-Galactosidase
chemical_list: D010445:Peptide Elongation Factors; C020746:factor EF-P; D011392:Proline; D001616:beta-Galactosidase