Large‐Scale Purification of the 3'‐OH‐Terminal tRNA‐Like Sequence (n= 159) of Turnip‐Yellow‐Mosaic‐Virus RNA

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Abstract
To undertake structural and functional studies on the 3''-terminal part of turnip yellow mosaic virus RNA, a structure which can be specifically aminoacylated by valyl-tRNA synthetase, large-scale methods for purifying the tRNA-like sequence were developed. Several experimental approaches were tested. One procedure was retained enabling purification of large quantities of the homogeneous tRNA-like fragment. Starting from 1.5 g turnip yellow mosaic virus, 400 mg RNA was obtained, which is partially digested by T1 RNase and which yields 1-2 mg pure tRNA-like fragment after 3 chromatographic steps: 2 filtrations on Ultrogel ACA 54 and 1 reverse-phase chromatography (RPC5) in the presence of urea. A method was worked out allowing preparation of 10 mg of the fragment per month. The purified RNA material appeared homogeneous on polyacrylamide gel electrophoresis under denaturing conditions. The isolated tRNA-like structure can be valylated to an extent of 100% in the presence of purified yeast valyl-tRNA synthetase with kinetic parameters resembling those of the tRNAVal aminoacylation.