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Nucleic Acids Research, 1981, Vol. 9, No. 19 5125-5140
© 1981

MOLECULAR BIOLOGY

Mapping tRNA structure in solution using double-strand-specific ribonuclease V1 from cobra venom

Raymond E. Lockard and Ajit Kumar

Department of Biochemistry, The George Washington University School of Medicine and Health Sciences 2300 Eye Street, N.W., Washington, DC 20037, USA

A method for mapping all base-paired stems in both elongation and initiator tRNAs is described using double-strand-specific ribonuclease V1 from the venom of the cobra Naja naja oxiana. 32p-end-labeled RNA is first partially digested with double-strand-specific V1 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 V1 generated bands with fragments of known length produced by both Neurospora endonuclease and base-specific ribonucleases. Using the substrates yeast tRNAPhe and E. coli tRNAMet of known three-dimensional structure, we find V1 nuclease to cleave entirely within every base-paired stem. Our studies also reveal that nuclease V1 will digest paired nucleotides not hydrogen-bonded by standard Watson-Crick base-pairing. In yeast tRNAPhe cleavage of both wobble base-pairs and nucleotides involved in tertiary base-base hydrogen bonding is demonstrated.


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