Genetic Identification of Nascent Peptides That Induce Ribosome Stalling

Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2009; 284(50):34809-18. DOI: 10.1074/jbc.M109.039040
Source: PubMed


Several nascent peptides stall ribosomes during their own translation in both prokaryotes and eukaryotes. Leader peptides that induce stalling can regulate downstream gene expression. Interestingly, stalling peptides show little sequence similarity and interact with the ribosome through distinct mechanisms. To explore the scope of regulation by stalling peptides and to better understand the mechanism of stalling, we identified and characterized new examples from random libraries. We created a genetic selection that ties the life of Escherichia coli cells to stalling at a specific site. This selection relies on the natural bacterial system that rescues arrested ribosomes. We altered transfer-messenger RNA, a key component of this rescue system, to direct the completion of a necessary protein if and only if stalling occurs. We identified three classes of stalling peptides: C-terminal Pro residues, SecM-like peptides, and the novel stalling sequence FXXYXIWPP. Like the leader peptides SecM and TnaC, the FXXYXIWPP peptide induces stalling efficiently by inhibiting peptidyl transfer. The nascent peptide exit tunnel and peptidyltransferase center are implicated in this stalling event, although mutations in the ribosome affect stalling on SecM and FXXYXIWPP differently. We conclude that ribosome stalling can be caused by numerous sequences and is more common than previously believed.

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    • "Accordingly, it is not surprising that proline was identified to be a crucial amino acid in many peptides that cause translational arrest (Hayes, 2002; Tanner et al., 2009; Ito et al., 2010). Stalling is especially evident when translating distinct diprolylcomprising sequences (Tanner et al., 2009; Doerfel et al., 2013; Hersch et al., 2013; Peil et al., 2013; Ude et al., 2013). As such sequences occur frequently within proteins (Mandal et al., 2014; Starosta et al., 2014b), almost all living organisms have evolved a specialized translation elongation factor, referred to as EF-P in bacteria or IF-5A in eukaryotes (eIF-5A, recently renamed EF5 (Dever et al., 2014)) and archaea (aIF-5A), that alleviates such ribosome stalling thereby allowing protein synthesis to continue (Doerfel et al., 2013; Gutierrez et al., 2013; Ude et al., 2013) "
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    ABSTRACT: Translation of polyproline proteins leads to translation arrest. To overcome this ribosome stalling effect, bacteria depend on a specialized translation elongation factor P (EF-P), being orthologous and functional identical to eukaryotic/archaeal elongation factor e/aIF-5A (recently renamed "EF5"). EF-P binds to the stalled ribosome between the peptidyl-tRNA binding and tRNA-exiting sites, and stimulates peptidyl-transferase activity thus allowing translation to resume. In their active form both EF-P and e/aIF-5A are post-translationally modified at a positively charged residue, which protrudes towards the peptidyl-transferase center when bound to the ribosome. While archaeal and eukaryotic IF-5A strictly depend on (deoxy-) hypusination (hypusinylation) of a conserved lysine, bacteria have evolved diverse analogous modification strategies to activate EF-P. In Escherichia coli and Salmonella enterica a lysine is extended by β-lysinylation and subsequently hydroxylated, whereas in Pseudomonas aeruginosa and Shewanella oneidensis an arginine in the equivalent position is rhamnosylated. Inactivation of EF-P, or the corresponding modification systems, reduces not only bacterial fitness but also impairs virulence. Here we review the function of EF-P and IF-5A and their unusual posttranslational protein modifications.
    No preview · Article · Nov 2015 · Molecular Microbiology
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    • "For example, in the absence of EFP, the ribosome density is strongly enriched at the RPPP sequence in recG, with a pause score of 381 in the Defp1 dataset and 475 in the Defp2 dataset (Figure 2A). The density is highest at the first nucleotide of the second Pro codon (Figure 2A, inset), consistent with previous observations that stalling occurs with the second Pro codon in the P site and the third in the A site (Doerfel et al., 2013; Peil et al., 2013; Tanner et al., 2009; Woolstenhulme et al., 2013). sequences is seen in the corresponding RNA-seq data, confirming that the high pause scores are not due to cloning bias (Figure S1). "
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    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. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Preview · Article · Apr 2015 · Cell Reports
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    • "Molecular Cell 51, 35–45, July 11, 2013 ª2013 Elsevier Inc. 39 the third Pro codon in the A site of the ribosome (Tanner et al., 2009; Woolstenhulme et al., 2013). "
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    ABSTRACT: Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stimulate Met-puromycin synthesis, a model assay for peptide bond formation. More recently, eIF5A was shown to promote translation elongation; however, its precise requirement in protein synthesis remains elusive. We use in vivo assays in yeast and in vitro reconstituted translation assays to reveal a specific requirement for eIF5A to promote peptide bond formation between consecutive Pro residues. Addition of eIF5A relieves ribosomal stalling during translation of three consecutive Pro residues in vitro, and loss of eIF5A function impairs translation of polyproline-containing proteins in vivo. Hydroxyl radical probing experiments localized eIF5A near the E site of the ribosome with its hypusine residue adjacent to the acceptor stem of the P site tRNA. Thus, eIF5A, like its bacterial ortholog EFP, is proposed to stimulate the peptidyl transferase activity of the ribosome and facilitate the reactivity of poor substrates like Pro.
    Preview · Article · May 2013 · Molecular cell
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