Department of Integrative Structural and Computational Biology, Department of Chemistry, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA.
Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037, USA.
Nat Commun. 2023 Aug 26;14(1):5220. doi: 10.1038/s41467-023-40859-w.
Assembly of ribosomes in bacteria is highly efficient, taking ~2-3 min, but this makes the abundance of assembly intermediates very low, which is a challenge for mechanistic understanding. Genetic perturbations of the assembly process create bottlenecks where intermediates accumulate, facilitating structural characterization. We use cryo-electron microscopy, with iterative subclassification to identify intermediates in the assembly of the 50S ribosomal subunit from E. coli. The analysis of the ensemble of intermediates that spans the entire biogenesis pathway for the 50 S subunit was facilitated by a dimensionality reduction and cluster picking approach using PCA-UMAP-HDBSCAN. The identity of the cooperative folding units in the RNA with associated proteins is revealed, and the hierarchy of these units reveals a complete assembly map for all RNA and protein components. The assembly generally proceeds co-transcriptionally, with some flexibility in the landscape to ensure efficiency for this central cellular process under a variety of growth conditions.
细菌中核糖体的组装效率非常高，大约需要 2-3 分钟，但这使得组装中间体的丰度非常低，这给机制理解带来了挑战。对组装过程的遗传干扰会产生中间体积累的瓶颈，从而有利于结构表征。我们使用冷冻电子显微镜，通过迭代子分类来鉴定来自大肠杆菌的 50S 核糖体亚基组装过程中的中间体。使用 PCA-UMAP-HDBSCAN 进行降维和聚类选择的方法，有助于分析跨越 50S 亚基生物发生途径的整个中间体集合。揭示了 RNA 与相关蛋白中协同折叠单元的身份，并且这些单元的层次结构揭示了所有 RNA 和蛋白质成分的完整组装图。组装通常是共转录进行的，在景观中有一定的灵活性，以确保在各种生长条件下这个核心细胞过程的效率。
