Dynamics of ribosome scanning and recycling revealed by translation complex profiling

Regulation of mRNA translation is central to eukaryotic gene expression control. Translation initiation involves binding of the 40S ribosomal small subunit (SSU) and associated initiation factors 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) 2-5 to form the 80S ribosome (RS). Scanning and other dynamic aspects of the initiation model remain conjecture as methods to trap early intermediates are 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 profiling approach. We document scanning by observing SSU footprints along 5''UTRs. SSU footprint sizes and locations reveal stark conformational changes over the course of translation initiation. Further, positional information on SSUs in individual mRNAs are suggestive of cis-regulation at the level of scanning. Our approach captures ribosomal complexes at all phases of translation and will aid in studying translation dynamics in diverse cellular contexts.