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Isolation of Ribosome Stalling Motifs from Random Libraries

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Regulatory Nascent Polypeptides

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Abstract

Although we tend to think that the ribosome can link together amino acids in any order, we have found that certain sequences interfere with protein synthesis. Characterization of stalling peptides found in bacteria has revealed multiple mechanisms of translational inhibition. Given that natural motifs differ in sequence and contain only three or four essential residues, peptide-mediated stalling may be quite widespread. To examine the scope of this phenomenon, we performed genetic selections in Escherichia coli to identify additional stalling motifs from random libraries. We characterized the mechanism of stalling with purified components using pre-steady-state kinetic methods. Some motifs block termination by inhibiting catalysis by release factors. In others, peptidyl transfer to certain aminoacyl-tRNAs is inhibited. Residues upstream of a stalling motif can either enhance or suppress these effects. One theme that emerges from these studies is the poor reactivity of proline, both as a peptidyl donor, at the C-terminus of the nascent peptide, and as a peptidyl acceptor, as prolyl-tRNA. This effect is compounded at three or more consecutive Pro codons. The translation factor EF-P alleviates stalling at polyproline sequences but has little or no effect on the motifs identified in our selections. Although our stalling motifs are in some sense artificial, several are underrepresented in bacterial proteomes, suggesting that they have been selected against, and analysis of ribosome profiling datasets reveals evidence of stalling where they occur in endogenous E. coli proteins.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Brigham Young University, C203 BNSN, Provo, UT, 84602, USA

    Christopher J. Woolstenhulme & Allen R. Buskirk

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  1. Christopher J. Woolstenhulme
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  2. Allen R. Buskirk
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Correspondence to Allen R. Buskirk .

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  1. Kyoto Sangyo University, Kita-ku, Kyoto, Japan

    Koreaki Ito

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Woolstenhulme, C.J., Buskirk, A.R. (2014). Isolation of Ribosome Stalling Motifs from Random Libraries. In: Ito, K. (eds) Regulatory Nascent Polypeptides. Springer, Tokyo. https://doi.org/10.1007/978-4-431-55052-5_13

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