A one-step method for in vitro production of tRNA transcripts

Nucleic Acids Res. 2002 Oct 15;30(20):e105. doi: 10.1093/nar/gnf104.

Abstract

Sequencing of a large number of microbial genomes has led to the discovery of new enzymes involved in tRNA biosynthesis and tRNA function. Preparation of a great variety of RNA molecules is, therefore, of major interest for biochemical characterization of these proteins. We describe a fast, cost-effective and efficient method for in vitro production of tRNA transcripts. T7 RNA polymerase requires a double-stranded DNA promoter in order to initiate transcription; however, elongation does not require a double-stranded DNA template. A partially double-stranded transcription template formed by annealing of a short oligonucleotide, complementary to the T7 promoter, to a larger oligonucleotide is shown to be a good substrate for in vitro transcription. This method allows rapid production of a variety of tRNA transcripts which can be aminoacylated well. This eliminates the need for cloning of tRNA genes, large-scale plasmid preparation and enzymatic digestion.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • DNA / chemistry
  • DNA / genetics
  • DNA, Single-Stranded / chemistry
  • DNA-Directed RNA Polymerases / metabolism
  • Genetic Techniques*
  • RNA, Transfer / biosynthesis*
  • RNA, Transfer / metabolism
  • Templates, Genetic
  • Transcription, Genetic*
  • Viral Proteins

Substances

  • DNA, Single-Stranded
  • Viral Proteins
  • DNA
  • RNA, Transfer
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases