Centre de Biophysique Moléculaire (UPR 4301), CNRS, Orléans, France.
Ecole doctorale Santé, Sciences Biologiques et Chimie du Vivant (ED 549), Université d'Orléans, Orléans Cedex 2, France.
Methods Mol Biol. 2021;2209:143-161. doi: 10.1007/978-1-0716-0935-4_10.
Transcription termination factor Rho contributes to shape the transcriptomes of many bacteria and is essential in a large subset of them. Although the transcription termination function of Rho is not always easy to reconstitute and to study in vitro, assays based on the ATP-dependent RNA-DNA hybrid unwinding activity of the factor can prove useful to dissect Rho mechanisms or to seek new antibiotics targeting Rho. However, current in vitro assays of Rho helicase activity are time-consuming, as they usually require radiolabeling of the hybrid substrates and analysis of reaction products by gel electrophoresis. Here, we describe a fluorescence-based microplate assay that informs on Rho helicase activity in a matter of minutes and allows the multiplexed analysis of conditions required for primary biochemical characterization or for drug screening.
转录终止因子 Rho 有助于塑造许多细菌的转录组，并且对其中很大一部分细菌是必需的。尽管 Rho 的转录终止功能并不总是易于在体外重组和研究，但基于该因子的 ATP 依赖性 RNA-DNA 杂交解旋活性的测定可以证明有助于剖析 Rho 机制或寻找针对 Rho 的新抗生素。然而，目前 Rho 解旋酶活性的体外测定耗时较长，因为它们通常需要对杂交底物进行放射性标记，并通过凝胶电泳分析反应产物。在这里，我们描述了一种基于荧光的微孔板测定法，该测定法在几分钟内提供 Rho 解旋酶活性的信息，并允许对用于初步生化特性分析或药物筛选的条件进行多重分析。
