A new method to monitor the rate of conformational transitions in RNA

Abstract

Many RNAs need Mg2+ to produce stable tertiary structures. Here we describe a simple method to measure the rate and activation parameters of tertiary structure unfolding that exploits this Mg2+ dependence. Our approach is based on mixing an RNA solution with excess EDTA in a stopped-flow instrument equipped with an absorbance detector, under conditions of temperature and ionic strength where, after chelation of Mg2+, tertiary structure unfolds. We have demonstrated the utility of this method by studying phenylalanine-specific transfer RNA from yeast (tRNA(Phe)) because the unfolding rates and the corresponding activation parameters have been determined previously and provide a benchmark for our technique. We find that within error, our stopped-flow method reproduces both the rate and activation enthalpy for tertiary unfolding of yeast tRNA(Phe) measured previously by temperature-jump relaxation kinetics. Since many different RNAs require divalent magnesium for tertiary structure stabilization, this technique should be applicable to study the folding of other RNAs.