Abstract
RNA helicases are the largest class of enzymes in eukaryotic RNA metabolism. In cells, protein cofactors regulate RNA helicase functions and impact biochemical helicase activities. Understanding how cofactors affect enzymatic activities of RNA helicases is thus critical for delineating physical roles and regulation of RNA helicases in cells. Here, we discuss approaches and conceptual considerations for the design of experiments to interrogate cofactor effects on RNA helicase activities in vitro. We outline the mechanistic frame for helicase reactions, discuss optimization of experimental setup and reaction parameters for measuring cofactor effects on RNA helicase activities, and provide basic guides to data analysis and interpretation. The described approaches are also instructive for determining the impact of small molecule inhibitors of RNA helicases.