Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Science. 2010 Oct 29;330(6004):673-7. doi: 10.1126/science.1193220.
Ribosomes are self-assembling macromolecular machines that translate DNA into proteins, and an understanding of ribosome biogenesis is central to cellular physiology. Previous studies on the Escherichia coli 30S subunit suggest that ribosome assembly occurs via multiple parallel pathways rather than through a single rate-limiting step, but little mechanistic information is known about this process. Discovery single-particle profiling (DSP), an application of time-resolved electron microscopy, was used to obtain more than 1 million snapshots of assembling 30S subunits, identify and visualize the structures of 14 assembly intermediates, and monitor the population flux of these intermediates over time. DSP results were integrated with mass spectrometry data to construct the first ribosome-assembly mechanism that incorporates binding dependencies, rate constants, and structural characterization of populated intermediates.
核糖体是自我组装的大分子机器，可将 DNA 翻译成蛋白质，对核糖体生物发生的理解是细胞生理学的核心。先前对大肠杆菌 30S 亚基的研究表明，核糖体的组装是通过多个平行途径而不是通过单个限速步骤进行的，但对该过程的机制信息知之甚少。发现单颗粒分析（DSP）是时间分辨电子显微镜的一种应用，它被用来获取超过 100 万个组装中的 30S 亚基的快照，识别和可视化 14 个组装中间体的结构，并监测这些中间体随时间的群体通量。DSP 结果与质谱数据相结合，构建了第一个包含结合依赖性、速率常数和 populated 中间体结构特征的核糖体组装机制。
