Charité - Univesitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Medical Physics and Biophysics, Berlin, Germany.
Max Planck Institute for Molecular Genetics, Microscopy and Cryo-Electron Microscopy Service Group, Berlin, Germany.
Nat Commun. 2024 Feb 26;15(1):1756. doi: 10.1038/s41467-024-46092-3.
Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts.
传统的翻译核糖体结构研究依赖于体外组装和核糖体在特定状态下的停滞。为了全面可视化细菌翻译，我们在 PURE 体外翻译系统中重新激活了体外衍生的大肠杆菌多核糖体，并通过 cryo-EM 分析了正在伸长的多核糖体。我们发现，70S 核糖体中有 31%组装成二联体复合物，代表包括解码和终止中间态在内的 8 种不同的功能状态，以及亲核攻击前状态。二联体复合物的功能多样性以及 RNase 消化实验表明，在延伸过程中，暂停的二联体复合物会短暂形成。结构分析揭示了领先和排队核糖体之间的五个二联体界面，当领先核糖体穿过延伸循环时，这些界面会发生重排。我们的研究结果从分子水平揭示了 bL9 的 CTD 如何阻碍排队核糖体的因子结合位点，以阻止有害碰撞，并说明了翻译动力学如何重塑核糖体间的接触。
