In Vitro Assay of Bacterial RNA Helicases Liquid-Liquid Phase Separation.

DEAD-box RNA helicases are widespread ATP-driven enzymes that reshape RNA structures and RNA-protein complexes during many steps of RNA metabolism. In bacteria, DEAD-box RNA helicases support cellular growth and responses to stress by contributing to ribosome biogenesis, RNA degradation, and translation initiation. Growing evidence indicates that several DEAD-box RNA helicases can also form liquid-liquid phase-separated condensates that concentrate RNA and partner proteins. This behavior adds a new layer of control to RNA regulation, although the molecular factors that promote or tune liquid-liquid phase separation (LLPS) are still unclear. Here, we describe an in vitro microscopy-based assay to characterize the LLPS behavior of bacterial DEAD-box RNA helicases. The protocol includes step-by-step procedures to evaluate condensate formation, salt sensitivity and dissolution, RNA dependency and specificity, and ATP effects. We further provide detailed instructions for quantitative analysis of microscope images, including size and assessment of protein enrichment within condensates. Altogether, this workflow enables systematic dissection of key physicochemical parameters governing DEAD-box helicase LLPS and provides a standardised framework for comparative studies across bacterial DEAD-box proteins or variants.