Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt Universität zu Berlin, Berlin, Germany.
Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Medical Physics and Biophysics, Group Protein X-ray Crystallography and Signal Transduction, Charitéplatz 1, D-10117, Berlin, Germany.
Nat Commun. 2023 Feb 17;14(1):898. doi: 10.1038/s41467-023-36607-9.
Ribosome biogenesis is a fundamental multi-step cellular process in all domains of life that involves the production, processing, folding, and modification of ribosomal RNAs (rRNAs) and ribosomal proteins. To obtain insights into the still unexplored early assembly phase of the bacterial 50S subunit, we exploited a minimal in vitro reconstitution system using purified ribosomal components and scalable reaction conditions. Time-limited assembly assays combined with cryo-EM analysis visualizes the structurally complex assembly pathway starting with a particle consisting of ordered density for only ~500 nucleotides of 23S rRNA domain I and three ribosomal proteins. In addition, our structural analysis reveals that early 50S assembly occurs in a domain-wise fashion, while late 50S assembly proceeds incrementally. Furthermore, we find that both ribosomal proteins and folded rRNA helices, occupying surface exposed regions on pre-50S particles, induce, or stabilize rRNA folds within adjacent regions, thereby creating cooperativity.
核糖体生物发生是所有生命领域中一个基本的多步骤细胞过程，涉及核糖体 RNA(rRNA)和核糖体蛋白的产生、加工、折叠和修饰。为了深入了解细菌 50S 亚基仍未被探索的早期组装阶段，我们利用纯化的核糖体成分和可扩展的反应条件，开发了一个最小的体外重组系统。限时组装测定与 cryo-EM 分析相结合，可视化了从仅包含约 23S rRNA 结构域 I 的有序密度的~500 个核苷酸和三个核糖体蛋白组成的粒子开始的结构复杂的组装途径。此外，我们的结构分析表明，早期 50S 组装以域为单位进行，而晚期 50S 组装则逐步进行。此外，我们发现核糖体蛋白和折叠的 rRNA 螺旋，占据前 50S 颗粒表面暴露的区域，诱导或稳定相邻区域内的 rRNA 折叠，从而产生协同作用。
