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

Dragana Korencić·Dieter Söll·Alexandre Ambrogelly
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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.

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DNADNA, Single-StrandedDNA-Directed RNA PolymerasesGenetic TechniquesRNA, TransferTemplates, GeneticTranscription, GeneticViral ProteinsJournal ArticleResearch Support, U.S. Gov't, P.H.S.

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Dragana Korencić, Dieter Söll, Alexandre Ambrogelly. 2002-10-15. A one-step method for in vitro production of tRNA transcripts.. https://doi.org/10.1093/nar%2Fgnf104

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