Synthesis of peptidyl aminoacyl transfer RNA: A chemical method for the purification of transfer RNA's

Abstract

A general chemical method for the purification of amino acid-specific tRNA's is described. The desired tRNA species are enzymatically esterified with a specific aminoacyl-tRNA synthetase. The resulting aminoacyl-tRNA is then separated from the bulk of unesterified tRNA by reacting this mixture in alkaline dioxane-water (0.6:1, v/v) with N- carboxy-β- benzyl- l-aspartate anhydride to yield the corresponding (β-benzyl- l-aspartyl) n-aminoacyl-tRNA((Bzl-Asp) n-aminoacyl-tRNA)). The latter precipitates out from the reaction mixture as a coadsorbate with free poly-β-benzyl- l-aspartate—produced as a result of the action of water—whereas uncharged tRNA chains, which do not react with N- carboxy-β- benzyl- l-aspartate anhydride, remain in solution. A substantial purification of the reacted aminoacyl-tRNA is thus achieved: between 50 and 70% of the starting aminoacyl-tRNA is recovered in the precipitate with a 10–25-fold purification. The (Bzl-Asp) n-aminoacyl-tRNA is separated from the free poly-β-benzyl- l-aspartate by dissolution in phenol and subsequent extraction with a mixture of aq. NaClO 4 and chloroform. Once the free poly-β-benzyl- l-aspartate is removed, remaining (Bzl-Asp) n-aminoacyl-tRNA becomes soluble in water. The preferential precipitation of aminoacyl-tRNA is due to specific interaction (during poly-amino acid chain synthesis) between the peptidyl moiety of (Bzl-Asp) n-aminoacyl-tRNA and free poly-β-benzyl- l-aspartate. It was also shown that the peptidyl moiety of (Bzl-Asp) n-Val-tRNA must reach a minimum chain length of about five Bzl-Asp residues before any part of it can be entrapped in the dioxane-water insoluble fraction. The polypeptidyl ester is hydrolyzed by pronase to liberate purified tRNA. About 70% of the biological activity of the enriched tRNA is recovered. The success of the purification depends on various requirements, including the availability of tRNA preparations completely free of nuclease activity and devoid of polypeptide contamination. A procedure for the isolation of tRNA that meets these requirements is described.