Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal the mechanism of its mRNA-dependent and nascent peptide−dependent translational stalling. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent polyubiquitination of collided disomes and, preferentially, trisomes. The distinct trisome architecture, visualized using cryo-EM, provides the structural basis for the more-efficient recognition by Hel2 compared with that of disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled polyubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.

核糖体相关质量控制（RQC）代表真核细胞中的抢救途径，该途径在翻译停滞后触发。碰撞的核糖体被识别用于随后的解离，随后降解新生肽。然而，内源性RQC诱导序列和泛素依赖性核糖体解离的基础机制仍然知之甚少。在这里，我们从酿酒酵母中鉴定了SDD1信使RNA作为内源性RQC底物，并揭示了其mRNA依赖性和新生肽依赖性翻译失速的机制。SDD1的体外翻译mRNA使冲突的二聚体（最好是三体）的Hel2依赖性多泛素化得以重建。使用cryo-EM可视化的独特的三重体体系结构为Hel2与二聚体相比更有效的识别提供了结构基础。随后，RQC触发器（RQT）复合体的Slh1解旋酶亚基以ATP依赖的方式优先解离第一个停滞的多泛素化核糖体。总之，这些发现为RQC及其在维持细胞蛋白质稳态中的生理作用提供了基本的机械见解。