转录共组装和天然纯化用于结构分析的大型 RNA-RNA 复合物。
Cotranscriptional Assembly and Native Purification of Large RNA-RNA Complexes for Structural Analyses.
机构信息
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, USA.
Structural Biochemistry Unit, National Institute of Dental and Craniofacial Research, Bethesda, MD, USA.
出版信息
Methods Mol Biol. 2023;2568:1-12. doi: 10.1007/978-1-0716-2687-0_1.
Recent technological developments such as cryogenic electron microscopy (Cryo-EM) and X-ray free electron lasers (XFEL) have significantly expanded the available toolkit to visualize large, complex noncoding RNAs and their complexes. Consequently, the quality of the RNA sample, as measured by its chemical monodispersity and conformational homogeneity, has become the bottleneck that frequently precludes effective structural analyses. Here we describe a general RNA sample preparation protocol that combines cotranscriptional RNA folding and RNA-RNA complex assembly, followed by native purification of stoichiometric complexes. We illustrate and discuss the utility of this versatile method in overcoming RNA misfolding and enabling the structural and mechanistic elucidations of the T-box riboswitch-tRNA complexes.
最近的技术发展，如低温电子显微镜（Cryo-EM）和 X 射线自由电子激光（XFEL），极大地扩展了可用的工具包，用于可视化大型、复杂的非编码 RNA 及其复合物。因此，RNA 样品的质量，如通过其化学单分散性和构象均一性来衡量，已成为经常阻碍有效结构分析的瓶颈。在这里，我们描述了一种通用的 RNA 样品制备方案，该方案结合了转录过程中的 RNA 折叠和 RNA-RNA 复合物组装，然后进行化学计量比复合物的天然纯化。我们说明了并讨论了这种通用方法的实用性，它克服了 RNA 错误折叠，使 T 框核糖体开关-tRNA 复合物的结构和机制得以阐明。
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