tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cm⁵U₃₄) in eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood. In this study, we present a series of cryo-EM structures of human ELP123 in complex with tRNA and cofactors at four different stages of the reaction. The structures at resolutions of up to 2.9 Å together with complementary functional analyses reveal the molecular mechanism of the modification reaction. Our results show that tRNA binding exposes a universally conserved uridine at position 33 (U₃₃), which triggers acetyl-CoA hydrolysis. We identify a series of conserved residues that are crucial for the radical-based acetylation of U₃₄ and profile the molecular effects of patient-derived mutations. Together, we provide the high-resolution view of human Elongator and reveal its detailed mechanism of action.

tRNA 修饰影响核糖体延伸速度和共翻译折叠动力学。 Elongator 复合物负责在真核 tRNA 中的摆动尿苷碱基 (cm ⁵ U ₃₄ ) 处引入 5-羧甲基。然而，人类伸长器的结构和功能仍然知之甚少。在这项研究中，我们展示了人类 ELP123 与 tRNA 和辅因子复合物在四个不同反应阶段的一系列冷冻电镜结构。分辨率高达 2.9 Å 的结构与互补的功能分析一起揭示了修饰反应的分子机制。我们的结果表明，tRNA 结合暴露了第 33 位普遍保守的尿苷 (U ₃₃ )，从而引发乙酰辅酶 A 水解。我们鉴定了一系列对 U ₃₄的自由基乙酰化至关重要的保守残基，并分析了患者衍生突变的分子效应。我们共同提供人体伸长器的高分辨率视图并揭示其详细的作用机制。