Dynamics of ribosome scanning and recycling revealed by translation complex profiling

Stuart K. Archer, Nikolay E. Shirokikh, Traude H. Beilharz, Thomas Preiss

Research output: Contribution to journalLetterResearchpeer-review

85 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)570-574
Number of pages5
Issue number7613
Publication statusPublished - 28 Jul 2016


  • RNA
  • proteome
  • ribosome
  • next-generation sequencing
  • RNA sequencing

Cite this