METHODS Preparation and Assay of Double - Strand - Specific Nucleic Acids Ribonuclease Vi from Research Naja

A method for mapping all base-paired stems in both elongation and initiator tRNAs is described using double-strand-specific ribonuclease V^ from the venom of the cobra Naia naja oxiana. 32p_eneie(j RNA j s first partially digested with double-strand-specific V]_ 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^ generated bands with fragments of known length produced by both Neurospora endonuclease and base-specific ribonucleases. Using the substrates yeast tRNA and E. coli tRNA^ of known three-dimensional structure, we find V^ nuclease to cleave entirely within every base-paired stem. Our studies also reveal that nuclease V^ will digest paired nucleotides not hydrogen-bonded by standard Watson-Crick base-pairing. In yeast tRNA cleavage of both wobble base-pairs and nucleotides involved in tertiary base-base hydrogen bonding is demonstrated.