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.

针对起始阶段的翻译控制是基因表达调控的核心。要了解其所有方面，就需要技术上的重大进步。在这里，我们修改了与核糖体谱分析相关的酵母翻译复合物谱测序（TCP-seq），并使其适用于哺乳动物细胞。人类TCP-seq除了能够捕获80S核糖体（80Ss）之外，还能捕获40S亚基（40Ss）的足迹，它揭示出哺乳动物和酵母40Ss在5'TRs中的分布相似，表明了相当大的进化保守性。我们进一步开发了酵母和人类选择性TCP-seq（Sel-TCP-seq），可以选择与免疫靶向因子相关的40S和80S。Sel-TCP-seq证明eIF2和eIF3沿5'UTR沿40S扫描，在AUG识别后相继解离。值得注意的是 在初始延伸周期中，有一部分eIF3持续存在。着重介绍Sel-TCP-seq的多功能性，我们还鉴定了四种起始的48S构象中间体，提供了有关ATF4 和GCN4 mRNA的翻译控制，并证明了起始因子复合物的共翻译组装。