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.
Reyes
V M
VM
Division of Chemistry, California Institute of Technology, Pasadena 91125.
Abelson
J
J
D001483:Base Sequence; D004262:DNA Restriction Enzymes / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics; D012322:RNA Precursors / Q000096:biosynthesis* / Q000235:genetics; D012326:RNA Splicing; D012345:RNA, Transfer, Amino Acid-Specific / Q000096:biosynthesis*; D012360:RNA, Transfer, Phe / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013604:T-Phages / Q000235:genetics; D014158:Transcription, Genetic
D009841:Oligonucleotides; D012322:RNA Precursors; D012345:RNA, Transfer, Amino Acid-Specific; D012360:RNA, Transfer, Phe; C041032:pre-tRNA(Phe); D004262:DNA Restriction Enzymes