Dynamics of ribosome scanning and recycling revealed by translation complex profiling

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Regulation of messenger RNA translation is central to eukaryotic gene expression control1. Regulatory inputs are specified by the mRNA untranslated regions (UTRs) and often target translation initiation. Initiation involves binding of the 40S ribosomal small subunit (SSU) and associated eukaryotic initiation factors (eIFs) near the mRNA 5′ cap; the SSU then scans in the 3′ direction until it detects the start codon and is joined by the 60S ribosomal large subunit (LSU)2,3,4,5 to form the 80S ribosome. Scanning and other dynamic aspects of the initiation model have remained as conjectures because methods to trap early intermediates were lacking. Here we uncover the dynamics of the complete translation cycle in live yeast cells using translation complex profile sequencing (TCP-seq), a method developed from the ribosome profiling6 approach. We document scanning by observing SSU footprints along 5′ UTRs. Scanning SSU have 5′-extended footprints (up to ~75 nucleotides), indicative of additional interactions with mRNA emerging from the exit channel, promoting forward movement. We visualized changes in initiation complex conformation as SSU footprints coalesced into three major sizes at start codons (19, 29 and 37 nucleotides). These share the same 5′ start site but differ at the 3′ end, reflecting successive changes at the entry channel from an open to a closed state following start codon recognition. We also observe SSU ‘lingering’ at stop codons after LSU departure. Our results underpin mechanistic models of translation initiation and termination, built on decades of biochemical and structural investigation, with direct genome-wide in vivo evidence. Our approach captures ribosomal complexes at all phases of translation and will aid in studying translation dynamics in diverse cellular contexts. Dysregulation of translation is common in disease and, for example, SSU scanning is a target of anti-cancer drug development7. TCP-seq will prove useful in discerning differences in mRNA-specific initiation in pathologies and their response to treatment.

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Data deposits

The sequences of reads with poly(A) tracts were deposited to the Sequence Read Archive, under accession code SRP074093. An online interface for browsing and analysing the TCP-seq data is available at (http://bioapps.erc.monash.edu/TCP/) along with (http://dx.doi.org/10.6084/m9.figshare.3206725). The underlying mapped dataset is also available (http://dx.doi.org/10.6084/m9.figshare.3206698).


  1. 1.

    et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011)

  2. 2.

    The scanning mechanism of eukaryotic translation initiation. Annu. Rev. Biochem. 83, 779–812 (2014)

  3. 3.

    , & The mechanism of eukaryotic translation initiation and principles of its regulation. Nature Rev. Mol. Cell Biol. 11, 113–127 (2010)

  4. 4.

    , & Principles of translational control: an overview. Cold Spring Harb. Perspect. Biol . 4, a011528 (2012)

  5. 5.

    Eukaryotic protein synthesis: still a mystery. J. Biol. Chem. 285, 21197–21201 (2010)

  6. 6.

    Ribosome profiling: new views of translation, from single codons to genome scale. Nature Rev. Genet. 15, 205–213 (2014)

  7. 7.

    et al. Targeting the translation machinery in cancer. Nature Rev. Drug Discov . 14, 261–278 (2015)

  8. 8.

    How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell 15, 1109–1123 (1978)

  9. 9.

    et al. Translation initiation mediated by RNA looping. Proc. Natl. Acad. Sci. USA 112, 1041–1046 (2015)

  10. 10.

    , , & In vivo stabilization of preinitiation complexes by formaldehyde cross-linking. Methods Enzymol. 429, 163–183 (2007)

  11. 11.

    et al. Rational extension of the ribosome biogenesis pathway using network-guided genetics. PLoS Biol. 7, e1000213 (2009)

  12. 12.

    , , & Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments. eLife 3, e01257 (2014)

  13. 13.

    & Migration of 40S ribosomal subunits on messenger RNA in the presence of edeine. J. Biol. Chem. 253, 6568–6577 (1978)

  14. 14.

    , , , & Structural roles for human translation factor eIF3 in initiation of protein synthesis. Science 310, 1513–1515 (2005)

  15. 15.

    How does a scanning ribosomal particle move along the 5′-untranslated region of eukaryotic mRNA? Brownian Ratchet model. Biochemistry 48, 10688–10692 (2009)

  16. 16.

    , & What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame? Genes Dev. 18, 62–75 (2004)

  17. 17.

    et al. Genome-wide measurement of RNA secondary structure in yeast. Nature 467, 103–107 (2010)

  18. 18.

    , , & Cap-independent translation is required for starvation-induced differentiation in yeast. Science 317, 1224–1227 (2007)

  19. 19.

    et al. Conformational differences between open and closed states of the eukaryotic translation initiation complex. Mol. Cell 59, 399–412 (2015)

  20. 20.

    et al. Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex. Nucleic Acids Res. 42, 9623–9640 (2014)

  21. 21.

    & The initiation of mammalian protein synthesis and mRNA scanning mechanism. Nature 500, 307–311 (2013)

  22. 22.

    et al. The C-terminal domain of eukaryotic initiation factor 5 promotes start codon recognition by its dynamic interplay with eIF1 and eIF2β. Cell Rep. 1, 689–702 (2012)

  23. 23.

    et al. Structure of the mammalian 80S initiation complex with initiation factor 5B on HCV-IRES RNA. Nature Struct. Mol. Biol . 21, 721–727 (2014)

  24. 24.

    et al. Molecular architecture of a eukaryotic translational initiation complex. Science 342, 1240585 (2013)

  25. 25.

    et al. Structural basis of highly conserved ribosome recycling in eukaryotes and archaea. Nature 482, 501–506 (2012)

  26. 26.

    et al. The essential vertebrate ABCE1 protein interacts with eukaryotic initiation factors. J. Biol. Chem. 281, 7452–7457 (2006)

  27. 27.

    et al. The iron-sulphur protein RNase L inhibitor functions in translation termination. EMBO Rep. 11, 214–219 (2010)

  28. 28.

    et al. Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae. RNA 21, 124–134 (2015)

  29. 29.

    , & Termination and post-termination events in eukaryotic translation. Adv. Protein Chem. Struct. Biol. 86, 45–93 (2012)

  30. 30.

    et al. Possible steps of complete disassembly of post-termination complex by yeast eEF3 deduced from inhibition by translocation inhibitors. Nucleic Acids Res. 41, 264–276 (2013)

  31. 31.

    et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003)

  32. 32.

    et al. Synchronizing nuclear import of ribosomal proteins with ribosome assembly. Science 338, 666–671 (2012)

  33. 33.

    , , , & Probing the closed-loop model of mRNA translation in living cells. RNA Biol. 12, 248–254 (2015)

  34. 34.

    , & Probe-directed degradation (PDD) for flexible removal of unwanted cDNA sequences from RNA-seq libraries. Curr. Protoc. Hum. Genet. 85, 11 15 11–11 15 36 (2015)

  35. 35.

    , & Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage. BMC Genomics 15, 401 (2014)

  36. 36.

    & Fast gapped-read alignment with Bowtie 2. Nature Methods 9, 357–359 (2012)

  37. 37.

    , & Genome-wide identification of spliced introns using a tiling microarray. Genome Res. 17, 503–509 (2007)

  38. 38.

    , & Surveying the relative impact of mRNA features on local ribosome profiling read density in 28 datasets. bioRxiv (2015)

  39. 39.

    & Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation. Genome Res. 24, 2011–2021 (2014)

  40. 40.

    , , & Ribosome profiling reveals post-transcriptional buffering of divergent gene expression in yeast. Genome Res. 24, 422–430 (2014)

  41. 41.

    & GtRNAdb: a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Res. 37, D93–D97 (2009)

  42. 42.

    , & edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010)

  43. 43.

    et al. Causal signals between codon bias, mRNA structure, and the efficiency of translation and elongation. Mol. Syst. Biol. 10, 770 (2014)

  44. 44.

    , , & WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004)

  45. 45.

    et al. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320, 1344–1349 (2008)

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This work was supported by an ARC Discovery Grant (DP1300101928) and an NHMRC Senior Research Fellowship (514904) awarded to T.P. N.E.S. was supported by a Go8 European Fellowship. We are grateful to A. G. Hinnebusch, C. G. Proud and R. D. Hannan for discussions and suggestions for this work. We acknowledge technical support from the Australian Cancer Research Foundation Biomolecular Resource Facility (JCSMR, ANU), D. Powell and S. Androulakis at the Monash Bioinformatics Platform.

Author information

Author notes

    • Stuart K. Archer
    •  & Nikolay E. Shirokikh

    These authors contributed equally to this work.


  1. EMBL–Australia Collaborating Group, Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory 2601, Australia

    • Stuart K. Archer
    • , Nikolay E. Shirokikh
    •  & Thomas Preiss
  2. Monash Bioinformatics Platform, Monash University, Melbourne, Victoria 3800, Australia

    • Stuart K. Archer
  3. Moscow Regional State Institute of Humanities and Social Studies, Kolomna 140410, Russia

    • Nikolay E. Shirokikh
  4. Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia

    • Traude H. Beilharz
  5. Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010, Australia

    • Thomas Preiss


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S.K.A., T.H.B. and T.P. designed the research, S.K.A. and N.E.S. performed the experiments, S.K.A, N.E.S., T.H.B. and T.P. analysed the data, discussed the results and wrote the paper.

Corresponding author

Correspondence to Thomas Preiss.

Reviewer Information Nature thanks E. Alkalaeva, P. Baranov and Y. Mechulam for their contribution to the peer review of this work.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    Summary of sequencing read counts for each of the RNA fragment libraries, mapped step-wise to different RNA biotypes (rRNA, other non-coding RNAs, mRNAs). The libraries included RNA fragments from either ribosomal small subunit (SSU), full ribosome (RS) and unseparated total input (TI) fractions of the wild-type or SYO1-TAP yeast strains.

  2. 2.

    Supplementary Table 2

    Ranking of Saccharomyces cerevisiae mRNAs by the degree of disrupted ribosomal scanning. To derive a measure for scanning disruption, for each mRNA a ratio was calculated of SSU peak coverage in the 5'UTR over SSU peak coverage in the start codon region.

  3. 3.

    Supplementary Table 3

    List of sequences of DNA probes used to deplete libraries for cDNA molecules containing ribosomal RNA (rRNA) sequences by the probe-directed degradation method.

PDF files

  1. 1.

    Supplementary Discussion

    The introduction provides an in-depth and referenced account of eukaryotic translation initiation focusing on the major questions addressed in this work. Results and discussion further integrate particulars of the TCP-Seq method, and the findings obtained with it, into the current knowledge of eukaryotic protein synthesis.


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