Institut für Medizinische Physik und Biophysik, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.
UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Berlin, Germany.
Mol Cell. 2018 Jun 7;70(5):881-893.e3. doi: 10.1016/j.molcel.2018.05.003.
The assembly of ribosomal subunits is an essential prerequisite for protein biosynthesis in all domains of life. Although biochemical and biophysical approaches have advanced our understanding of ribosome assembly, our mechanistic comprehension of this process is still limited. Here, we perform an in vitro reconstitution of the Escherichia coli 50S ribosomal subunit. Late reconstitution products were subjected to high-resolution cryo-electron microscopy and multiparticle refinement analysis to reconstruct five distinct precursors of the 50S subunit with 4.3-3.8 Å resolution. These assembly intermediates define a progressive maturation pathway culminating in a late assembly particle, whose structure is more than 96% identical to a mature 50S subunit. Our structures monitor the formation and stabilization of structural elements in a nascent particle in unprecedented detail and identify the maturation of the rRNA-based peptidyl transferase center as the final critical step along the 50S assembly pathway.
核糖体亚基的组装是所有生命领域蛋白质生物合成的必要前提。尽管生化和生物物理方法已经提高了我们对核糖体组装的理解，但我们对这个过程的机制理解仍然有限。在这里，我们进行了大肠杆菌 50S 核糖体亚基的体外重构。对晚期重构产物进行高分辨率 cryo-电子显微镜和多颗粒精修分析，以重建 50S 亚基的五个不同的前体，分辨率为 4.3-3.8Å。这些组装中间体定义了一个渐进的成熟途径，最终形成一个晚期组装颗粒，其结构与成熟的 50S 亚基超过 96%相同。我们的结构以前所未有的细节监测新生颗粒中结构元素的形成和稳定，并确定基于 rRNA 的肽基转移酶中心的成熟是沿着 50S 组装途径的最后一个关键步骤。
