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Nucleic Acids Research, Vol 25, Issue 16 3297-3301, Copyright © 1997 by Oxford University Press

ARTICLES

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

EJ Maglott and GD Glick
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109- 1055, USA.

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 (tRNAPhe) 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 tRNAPhe 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.
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