Influence of Hydrostatic Pressure and Cosolutes on RNA Tertiary Structure

Abstract

RNAs fold into complex three-dimensional structures that are critical to their various biological functions. These structures often form independently of the RNA secondary structure, and the three-dimensional fold is stabilized by specific tertiary interactions between various motifs. However, the detailed molecular mechanisms that drive formation of these RNA tertiary interactions are not well understood. The most commonly used variables for probing the thermodynamics or kinetics of RNA−RNA interactions are temperature, metal ion concentration, and chemical denaturant concentration. In this study, the effects of hydrostatic pressure and nondenaturing cosolutes were examined. The commonly occurring GAAA tetraloop−receptor RNA tertiary motif was used as a model system, with formation of this interaction assayed by fluorescence resonance energy transfer. The results showed that the GAAA tetraloop−receptor interaction is slightly destabilized by hydrostatic pressure, and analysis of the pressure data yielded the change in partial molar volume for this RNA−RNA interaction. Polyethylene glycol and dextran cosolutes were both shown to favor formation of the tertiary structure, whereas sucrose and glycerol had little effect on the folding of the RNA. These results demonstrate that hydrostatic pressure and nondenaturing cosolute concentration can be useful variables for investigating tertiary or intermolecular RNA−RNA interactions.