Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

Australian Cancer Research Foundation Department of Cancer Biology and Therapeutics John Curtin School of Medical Research Australian National University Canberra ACT 2601 Australia

EMBL Australia Collaborating Group Department of Genome Sciences John Curtin School of Medical Research Australian National University Canberra ACT 2601 Australia

Laboratory of Gene Regulation and Development Eunice Kennedy Shriver National Institute of Child Health and Human Development National Institutes of Health Bethesda MD 20892 USA

Laboratory of Regulation of Gene Expression Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 142 20 Prague Czech Republic

Victor Chang Cardiac Research Institute Darlinghurst NSW 2010 Australia

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