| Summary: | Summary: 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. : Giess et al. introduce RCP-seq, a method to study small ribosomal subunit (SSU) dynamics in zebrafish. It reveals threading as the main mode of recruitment to the mRNA and quantifies the impact of sequence features that affect SSU processivity. By contrasting scanning to translating ribosomes, the study calculates initiation rates and redefines the optimal translation initiation context for zebrafish. Keywords: translation, RNA, ribosome profiling, next-generation sequencing, small ribosomal subunit (SSU), scanning processivity, initiation, ribosome recruitment, uORF, Kozak sequence
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