Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases^1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.

翻译起始定义了合成蛋白质的特性和数量。在许多人类疾病中，这一过程是失调的^1,2 。关键的承诺步骤是核糖体亚基在信使 RNA 上的翻译起始位点处连接形成功能性核糖体。在这里，我们使用体外重建系统结合单分子光谱和结构方法来检查人类核糖体亚基如何连接。单分子荧光揭示了普遍保守的真核起始因子 eIF1A 和 eIF5B 何时与起始复合物结合或分离。在单分子动力学的指导下，我们使用单粒子冷冻电子显微镜观察了包含 eIF1A 和 eIF5B 的起始复合物。由此产生的结构揭示了两种蛋白质之间的真核生物特异性接触如何重塑起始复合物，以将起始氨酰-tRNA定向为与核糖体亚基连接相容的构象。总的来说，我们的研究结果为人类翻译启动过程中 eIF1A 和 eIF5B 精心编排的分子编排提供了定量和架构框架。