Large-scale purification of the 3'-OH-terminal tRNA-like sequence (n = 159) of turnip-yellow-mosaic-virus RNA.

In order 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, we have developed large-scale methods for purifying the tRNA-like sequence. Several experimental approaches were tested. One procedure was retained enabling us to purify large quantities of the homogeneous tRNA-like fragment. Starting from 1.5 g turnip yellow mosaic virus, one obtains 400 mg RNA, which is partially digested by T1 ribonuclease and which yields 1-2 mg pure tRNA-like fragment after three chromatographic steps: two filtrations on Ultrogel ACA 54 and one reverse-phase chromatography (RPC 5) in the presence of urea. A method has been worked out allowing preparation of 10 mg of the fragment per month. The purified RNA material appeared homogeneous upon 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.