Translation initiation is often attributed as the rate-determining step of eukaryotic protein synthesis and key to gene expression control. Despite this centrality, the series of steps involved in this process is poorly understood. Here, we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5′ UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features that regulate the recruitment and processivity of scanning ribosomes and redefine optimal initiation contexts. Our approach enables deconvoluting translation initiation into separate stages and identifying regulators at each step.
Bibliographical noteFunding Information:
We wish to thank members of the Valen and Thompson labs (University of Bergen, Norway), Ignatova and Duncan labs (University of Hamburg, Germany), and Preiss lab (Australian National University, Australia) for helpful discussions of methodologies. We wish to thank Andrea Pauli (IMP, Austria), Sushma Nagaraja-Grellscheid, and Eric Thompson (University of Bergen, Norway) for providing valuable feedback on the manuscript. The project was funded by the Trond Mohn Foundation, the Norwegian Research Council (#250049) and core funding from the Sars International Centre for Marine Molecular Biology. A.G. Y.N.T.C. and E.V. designed the research. Y.N.T.C. M.K. T.T.B. and S.H. performed the experiments. A.G. Y.N.T.C. H.T. and E.V. analyzed the data. A.O. and C.R.W. designed and synthesized the 4Ei-10 compound. All authors discussed the results and contributed to writing the paper. The authors declare no competing interests.
The project was funded by the Trond Mohn Foundation , the Norwegian Research Council (# 250049 ) and core funding from the Sars International Centre for Marine Molecular Biology .
© 2020 The Author(s)
- Kozak sequence
- next-generation sequencing
- ribosome profiling
- ribosome recruitment
- scanning processivity
- small ribosomal subunit (SSU)
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't