Purification of yeast transfer RNA ligase

Abstract

The splicing of yeast tRNA precursors requires the action of at least two proteins, an endonuclease which cleaves the two splice junctions to release the linear intron and a ligase which joins the resulting tRNA half molecules together. Each of the activities of tRNA ligase can be assayed independent of the overall reaction. Researchers have routinely assayed the enzyme activity during purification by the ligation of tRNA half-molecules produced by the action of tRNA endonuclease. The pre-tRNA phe substrate can now conveniently be synthesized in vitro by T7 RNA polymerase transcription of a synthetic gene. One microliter of each reaction mixture is applied to a cellulose plate which is developed in solvent containing saturated ammonium sulfate, 1 M sodium acetate, 2-propanol (40:9: 1) and then analyzed by autoradiography. The amounts of cyclic GMP and 2′-GMP are quantitated by cutting the spots from the thin-layer plate and counting them in a scintillation counter.