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  • Title: A synthetic substrate for tRNA splicing.
    Author: Reyes VM, Abelson J.
    Journal: Anal Biochem; 1987 Oct; 166(1):90-106. PubMed ID: 2823642.
    Abstract:
    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.
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