A Fluorescent Assay to Monitor Ligand-Dependent Closure of the Hexameric Rho Helicase Ring

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.