A new method to monitor the rate of conformational transitions in RNA.
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.
Maglott
E J
EJ
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Glick
G D
GD
D004492:Edetic Acid / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D008722:Methods; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009695:Nucleic Acid Renaturation; D012331:RNA, Fungal / Q000737:chemistry; D012360:RNA, Transfer, Phe / Q000737:chemistry*; D012441:Saccharomyces cerevisiae; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D012331:RNA, Fungal; D012360:RNA, Transfer, Phe; D004492:Edetic Acid; D008274:Magnesium