Higher-order Structure of Yeast tRNA^Tyr and Its Chimeric Mutants in Aqueous Solution As Studied by Hydroxyl Radical “Footprinting”

Abstract

Hydroxyl radical, generated by reduction of hydrogen peroxide by Fe (II)-EDTA, was used to investigate the higher-order structure of yeast tRNA^Tyr and its chimeric mutants in aqueous solution. Exposure of free tRNA^Tyr to this reagent gave cleavage patterns consistent with the three-dimensional structure of yeast tRNA^Phe established by X-ray crystallography. This probing method was applied to detect the base-mismatches in the anticodon stem region of a chimeric tRNA^Tyr mutant, which was constructed by ligating the 5′-half fragment of T. utilis tRNA^Tyr and the 3′-half fragment of baker's yeast tRNA^Tyr with T4 RNA ligase. The result of this analysis clearly showed that this probing method is sensitive enough to detect even a minute variation of the secondary structure of RNA molecules.