CNRS, UMR 8015, Laboratoire de Cristallographie et RMN biologiques, 4 avenue de l'Observatoire, 75006 Paris, France and Université Paris Descartes, Sorbonne Paris Cité, UMR 8015, Laboratoire de Cristallographie et RMN biologiques, 4 avenue de l'Observatoire, 75006 Paris, France.
Nucleic Acids Res. 2013 Aug;41(15):e150. doi: 10.1093/nar/gkt576. Epub 2013 Jun 26.
RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA-protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA-His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.
RNA 已成为许多细胞过程中的主要参与者。要在分子水平上理解这些过程,就需要对结构、生化和药理学研究使用同质的 RNA 样本。我们之前设计了一种通用方法,可以在大肠杆菌中高效表达重组 RNA。在这项工作中,我们将这种方法扩展到了 RNA/蛋白质的共表达。我们设计了几个质粒,可以在大肠杆菌中过表达 RNA-蛋白质复合物。我们研究了这些工具在许多应用中的潜力,包括在病毒蛋白假颗粒中封装的核酸酶敏感 RNA 的生产、与伴侣蛋白共生产非编码 RNA、通过与合适的修饰酶共生产来掺入转录后 RNA 修饰,以及最后通过镍亲和层析生产和纯化 RNA-His 标记的蛋白质复合物。我们表明,最后一种应用可以轻松提供用于晶体学研究的纯材料。我们报告的新工具将为 RNA 功能以及与蛋白质相互作用的大规模结构和分子研究铺平道路。