Freie Universität Berlin, Institute of Chemistry and Biochemistry, Laboratory of Structural Biochemistry, Berlin, Germany.
MRC Laboratory of Molecular Biology, Structural Studies Division, Cambridge, UK.
Methods Mol Biol. 2021;2209:193-215. doi: 10.1007/978-1-0716-0935-4_13.
Functional aspects of nucleic acid helicases can be interrogated by various in vitro methods, using purified components, including nucleic acid binding and unwinding assays. Here we describe detailed protocols for the production and purification of the spliceosomal Ski2-like RNA helicase, Brr2, and one of its regulatory factors, the Jab1 domain of the Prp8 protein from yeast. Furthermore, we include a production protocol for radioactively labeled yeast U4/U6 di-snRNA substrate. We describe polyacrylamide gel-based assays to investigate Brr2's RNA binding and unwinding activities. The purification protocols and activity assays can be easily adapted for the purification and functional interrogation of other helicases, cofactors, and RNA substrates.
核酸解旋酶的功能方面可以通过各种体外方法进行研究，使用纯化的组件，包括核酸结合和解旋测定。在这里，我们描述了详细的方案，用于生产和纯化剪接体 Ski2 样 RNA 解旋酶 Brr2 及其调节因子之一，来自酵母的 Prp8 蛋白的 Jab1 结构域。此外，我们还包括放射性标记的酵母 U4/U6 双 snRNA 底物的生产方案。我们描述了基于聚丙烯酰胺凝胶的测定法来研究 Brr2 的 RNA 结合和解旋活性。这些纯化方案和活性测定方法可以很容易地适应其他解旋酶、辅助因子和 RNA 底物的纯化和功能研究。
