The selection of an open reading frame (ORF) for translation of eukaryotic mRNA relies on remodeling of the scanning 48S initiation complex into an elongation-ready 80S ribosome. Using cryo-electron microscopy, we visualize the key commitment steps orchestrating 48S remodeling in humans. The mRNA Kozak sequence facilitates mRNA scanning in the 48S open state and stabilizes the 48S closed state by organizing the contacts of eukaryotic initiation factors (eIFs) and ribosomal proteins and by reconfiguring mRNA structure. GTPase-triggered large-scale fluctuations of 48S-bound eIF2 facilitate eIF5B recruitment, transfer of initiator tRNA from eIF2 to eIF5B and the release of eIF5 and eIF2. The 48S-bound multisubunit eIF3 complex controls ribosomal subunit joining by coupling eIF exchange to gradual displacement of the eIF3c N-terminal domain from the intersubunit interface. These findings reveal the structural mechanism of ORF selection in human cells and explain how eIF3 could function in the context of the 80S ribosome.

用于真核 mRNA 翻译的开放阅读框 (ORF) 的选择依赖于将扫描 48S 起始复合物重塑为准备延伸的 80S 核糖体。使用冷冻电子显微镜，我们可视化了人类 48S 重塑的关键承诺步骤。 mRNA Kozak 序列通过组织真核起始因子 (eIF) 和核糖体蛋白的接触以及重新配置 mRNA 结构，促进 48S 打开状态下的 mRNA 扫描并稳定 48S 关闭状态。 GTPase 触发的 48S 结合 eIF2 的大规模波动促进 eIF5B 招募、起始 tRNA 从 eIF2 转移到 eIF5B 以及 eIF5 和 eIF2 的释放。 48S 结合的多亚基 eIF3 复合物通过耦合 eIF 交换与 eIF3c N 端结构域从亚基间界面的逐渐位移来控制核糖体亚基连接。这些发现揭示了人类细胞中 ORF 选择的结构机制，并解释了 eIF3 如何在 80S 核糖体背景下发挥作用。