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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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).
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.
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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.