A simple fluorescence microplate assay to monitor RNA-DNA hybrid unwinding by the bacterial transcription termination factor Rho

Isabelle Simon , Marc Boudvillain (1)

1 CBM - Centre de biophysique moléculaire

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 ATPdependent 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.

Mots clés: Rho; transcription; termination; helicase; RNA; fluorescence

Domaines: Biologie moléculaire; Biochimie [q-bio.BM]

Isabelle Simon, Marc Boudvillain. A simple fluorescence microplate assay to monitor RNA-DNA hybrid unwinding by the bacterial transcription termination factor Rho. Marc Boudvillain. RNA remodeling proteins : Methods and Protocols, 2209, Humana press, pp.143-161, 2021, Methods in Molecular Biology, 978-1-0716-0937-8. ⟨10.1007/978-1-0716-0935-4_10⟩. ⟨hal-03036494⟩