Abstract
The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation.
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Acknowledgements
We thank members of the Qian laboratory for critical reading of the manuscript and Dr Jonathan Yewdell (NIAID, NIH) for providing anti-puromycin monoclonal antibody. B L is partly supported by the Genomics Scholar's Award from Center for Vertebrate Genomics at Cornell. This work was supported by grants from National Institutes of Health (1 DP2 OD006449-01), Ellison Medical Foundation (AG-NS-0605-09), and DOD Exploration-Hypothesis Development Award (W81XWH-11-1-0236) to S-B Q.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
Reproducibility of post-initiation pausing in HEK293 cells. (PDF 266 kb)
Supplementary information, Figure S2
Reproducibility of post-initiation pausing in HeLa (A) and MEF cells (B). (PDF 87 kb)
Supplementary information, Figure S3
Length distribution of RPF reads mapped to the 1st (AUG) codon, 5th codon, or the entire CDS. (PDF 117 kb)
Supplementary information, Figure S4
Characterizing RPFs starting with AUG. (PDF 172 kb)
Supplementary information, Figure S5
Sequence composition for the 1st (AUG) codon, 5th codon, and the entire CDS region in human genome. (PDF 147 kb)
Supplementary information, Figure S6
GO term analysis for gene groups with weak (A) and strong (B) post-initiation pausing. (PDF 105 kb)
Supplementary information, Figure S7
Monosome fraction maintains post-initiation pausing. (PDF 161 kb)
Supplementary information, Figure S8
Post-initiation pausing in other eukaryotic cell species, including budding yeast S. cerevisiae (A), nematode C. elegans (B), and zebrafish D. rerio (C). (PDF 132 kb)
Supplementary information, Figure S9
Evolutionary conservation analysis of RPL4 in both eukaryotes and prokaryotes. (PDF 461 kb)
Supplementary information, Figure S10
Examples of transcripts whose post-initiation pausing is reduced when translated by ribosomes bearing RPL4(Δloop). (PDF 263 kb)
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Han, Y., Gao, X., Liu, B. et al. Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation. Cell Res 24, 842–851 (2014). https://doi.org/10.1038/cr.2014.74
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DOI: https://doi.org/10.1038/cr.2014.74
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