RNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 368 Plantation St., Worcester, MA 01605, USA.
RNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 368 Plantation St., Worcester, MA 01605, USA.
Methods. 2018 Mar 15;137:55-66. doi: 10.1016/j.ymeth.2017.12.004. Epub 2017 Dec 14.
Bacterial ribosomal protein S1 is the largest and highly flexible protein of the 30S subunit, and one of a few core ribosomal proteins for which a complete structure is lacking. S1 is thought to participate in transcription and translation. Best understood is the role of S1 in facilitating translation of mRNAs with structured 5' UTRs. Here, we present cryo-EM analyses of the 70S ribosome that reveal multiple conformations of S1. Based on comparison of several 3D maximum likelihood classification approaches in Frealign, we propose a streamlined strategy for visualizing a highly dynamic component of a large macromolecular assembly that itself exhibits high compositional and conformational heterogeneity. The resulting maps show how S1 docks at the ribosomal protein S2 near the mRNA exit channel. The globular OB-fold domains sample a wide area around the mRNA exit channel and interact with mobile tails of proteins S6 and S18. S1 also interacts with the mRNA entrance channel, where an OB-fold domain can be localized near S3 and S5. Our analyses suggest that S1 cooperates with other ribosomal proteins to form a dynamic mesh near the mRNA exit and entrance channels to modulate the binding, folding and movement of mRNA.
细菌核糖体蛋白 S1 是 30S 亚基中最大且高度灵活的蛋白质，也是少数几个完整结构缺失的核心核糖体蛋白之一。S1 被认为参与转录和翻译。人们对 S1 在促进具有结构化 5'UTR 的 mRNA 翻译中的作用了解最多。在这里，我们呈现了对 70S 核糖体的冷冻电镜分析，揭示了 S1 的多种构象。基于在 Frealign 中比较几种 3D 最大似然分类方法，我们提出了一种用于可视化大型大分子组装中高度动态组件的简化策略，该组件本身表现出高组成和构象异质性。得到的图谱显示了 S1 如何在核糖体蛋白 S2 附近停靠在 mRNA 出口通道。球形 OB 折叠结构域在 mRNA 出口通道周围的广阔区域中采样，并与蛋白质 S6 和 S18 的移动尾部相互作用。S1 还与 mRNA 入口通道相互作用，在那里可以在 S3 和 S5 附近定位 OB 折叠结构域。我们的分析表明，S1 与其他核糖体蛋白合作，在 mRNA 出口和入口通道附近形成一个动态网格，以调节 mRNA 的结合、折叠和运动。
