Mapping tRNA structure in solution using double-strand-specific ribonuclease V1from cobra venom

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Abstract
A method for mapping all base-paired stems in both elongation and initiator tRNAs is described using double-strand-specific ribonuclease V 1 from the venom of the cobra Naja naja oxiana . 32 p-end-labeled RNA is first partially digested with double-strand-specific V 1 nuclease under near physiological conditions, and the resultant fragments are then electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel run in 90% formamide. After autoradiography, the base-paired nucleotides are definitively located by comparing V 1 generated bands with fragments of known length produced by both Neurospora endonuclease and base-specific ribonucleases. Using the substrates yeast tRNA Phe and E. coli tRNA Met of known three-dimensional structure, we find V 1 nuclease to cleave entirely within every base-paired stem. Our studies also reveal that nuclease V 1 will digest paired nucleotides not hydrogen-bonded by standard Watson-Crick base-pairing. In yeast tRNA Phe cleavage of both wobble base-pairs and nucleotides involved in tertiary base-base hydrogen bonding is demonstrated.