Mapping tRNA structure in solution using double-strand-specific ribonuclease V1 from cobra venom.
A method for mapping all base-paired stems in both elongation and initiator tRNAs is described using double-stranded-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 than 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 an E, coli tRNAfMet 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.
D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D004546:Elapid Venoms; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases; D004926:Escherichia coli; D006801:Humans; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000032:analysis*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012358:RNA, Transfer, Met; D012260:Ribonucleases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae
D004546:Elapid Venoms; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D012343:RNA, Transfer; D004722:Endoribonucleases; D012260:Ribonucleases; C032825:ribonuclease V(1)