A synthetic substrate for tRNA splicing

A novel method for the synthesis of precursor tRNA as substrate for in vitro splicing is reported. A construct consisting of the Saccharomyces cerevisiae pre-tRNAPhe gene under the control of a bacteriophage T7 promoter was assembled from a set of synthetic oligonucleotides and cloned into an M13 vector. By the use of T7 RNA polymerase, BstNI-runoff transcripts were produced. The resulting pre-tRNA was shown to possess mature termini and was accurately spliced by highly purified yeast tRNA-splicing endonuclease and ligase. Using this synthetic pre-tRNA, the kinetic parameters of the tRNA-splicing endonuclease were also determined. Use of this system provides several advantages for the study of tRNA-splicing mechanisms. Mutant tRNA precursors can be readily synthesized. It is also possible to synthesize large quantities of pre-tRNA for structural studies.