Ribosome-targeting antibiotics represent an important class of antimicrobial drugs. Chloramphenicol (Cm) is a well-studied ribosomal peptidyl transferase center (PTC) binder and growing evidence suggests that its inhibitory action depends on the sequence of the nascent peptide. How such selective inhibition on the molecular scale manifests on the cellular level remains unclear. Here, we use cryo-electron tomography to analyze the impact of Cm inside the bacterium Mycoplasma pneumoniae. By resolving the Cm-bound ribosomes to 3.0 Å, we elucidate Cm’s coordination with natural nascent peptides and transfer RNAs in the PTC. We find that Cm leads to the accumulation of a number of translation elongation states, indicating ongoing futile accommodation cycles, and to extensive ribosome collisions. We, thus, suggest that, beyond its direct inhibition of protein synthesis, the action of Cm may involve the activation of cellular stress responses. This work exemplifies how in-cell structural biology can expand the understanding of mechanisms of action for extensively studied antibiotics.

核糖体靶向抗生素是一类重要的抗菌药物。氯霉素 （Cm） 是一种经过充分研究的核糖体肽基转移酶中心 （PTC） 结合剂，越来越多的证据表明其抑制作用取决于新生肽的序列。这种分子水平上的选择性抑制如何在细胞水平上表现出来尚不清楚。在这里，我们使用冷冻电子断层扫描来分析 Cm 对肺炎支原体细菌的影响。通过将 Cm 结合的核糖体解析为 3.0 Å，我们阐明了 Cm 与 PTC 中天然新生肽和转移 RNA 的配位。我们发现 Cm 导致许多翻译伸长态的积累，表明正在进行的徒劳的调节循环，并导致广泛的核糖体碰撞。因此，我们认为，除了直接抑制蛋白质合成之外，Cm 的作用还可能涉及细胞应激反应的激活。这项工作举例说明了细胞内结构生物学如何扩大对广泛研究的抗生素的作用机制的理解。