Lawson Michael R, Berger James M
Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Methods Mol Biol. 2021;2209:133-142. doi: 10.1007/978-1-0716-0935-4_9.
The bacterial Rho protein is an exemplar RecA-family hexameric helicase that assists with the termination of RNA polymerase activity on a variety of transcripts. During its catalytic cycle, Rho both loads onto and translocates along RNA through a series of tightly regulated, ligand-dependent conformational changes. Here we describe an assay to track Rho as it switches from an open-ring (RNA-loading) to a closed-ring (RNA-translocation) configuration by monitoring the association of a fluorescein-labeled RNA to Rho's central pore as a change in fluorescence anisotropy. The assay, which is in principle adaptable to the study of ligand-dependent isomerization events in other ring-shaped translocases, is readily amenable to 384-well format plates and small-molecule screening efforts.
细菌Rho蛋白是RecA家族六聚体解旋酶的一个典范，它有助于终止RNA聚合酶对多种转录本的活性。在其催化循环中，Rho通过一系列严格调控的、依赖配体的构象变化，既加载到RNA上，又沿着RNA进行易位。在这里，我们描述了一种检测方法，通过监测荧光素标记的RNA与Rho中心孔的结合导致的荧光各向异性变化，来追踪Rho从开环（RNA加载）到闭环（RNA易位）构型的转变。该检测方法原则上适用于研究其他环形转位酶中依赖配体的异构化事件，易于应用于384孔板和小分子筛选研究。
