Dynamics of ribosome scanning and recycling revealed by translation complex profiling

Abstract

Regulation of messenger RNA translation is central to eukaryotic gene expression control. Regulatory inputs are specified by them RNA 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) 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 profiling 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 development. TCP-seq will prove useful in discerning differences in mRNA-specific initiation in pathologies and their response to treatment.

Ribosome signatures aid bacterial translation initiation site identification

“…Typhimurium samples (Additional file 1: Figure S8). These shorter reads were also consistent with recent reports of ribosomal subunits in a variety of distinct configurations, observed from translation complex profiling in the eukaryote Saccharomyces cerevisiae [42]. Whether similar patterns of read length distributions can be observed in eukaryotic ribo-seq datasets remains to be determined, although conceptually the method and metrics described herein are fully extendable to eukaryotic datasets.…”

Transcriptome-wide measurement of translation by ribosome profiling

“…Rather our hope lies in the systematic examination of the influence of pertinent experimental parameters exemplified by Heyer et al [21], and the power of approaches such as that developed by O’Connor et al [64] to infer the sequence biases of different protocols. Also, a diversity of protocols can yield exciting results, such as the combination of fixation and serial sucrose gradient ultracentrifugation that was recently used to sequence the footprints of scanning initiation complexes [67]. …”

The Growing Toolbox for Protein Synthesis Studies

“…However, ribosome profiling does not provide information about scanning ribosomes, since the interaction between the 40S ribosome and the mRNA is weak, and so any footprint would be susceptible to dissociation from the 40S [78]. Formaldehyde cross-linking of the scanning 40S and mRNA freezes this fragile complex, and subsequent 40S isolation allows profiling of the scanning pre-initiation complex – a methodology called translation complex profiling sequencing, or TCP-Seq [79] (Figure 4e). Three distinct populations of 40S footprints sizes (19, 29, 37 nt) reflect the sequential changes in conformation and/or assembly/dissociation of factors on the translation initiation complex.…”