A one-step method for in vitro production of tRNA transcripts.
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.
Korencić
Dragana
D
Department of Molecular Biophysics and Biochemistry and. Department of Chemistry, Yale University, New Haven, CT 06520-8114, USA.
Söll
Dieter
D
Ambrogelly
Alexandre
A
D004247:DNA / Q000737:chemistry / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005821:Genetic Techniques; D012343:RNA, Transfer / Q000096:biosynthesis* / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins
D004277:DNA, Single-Stranded; D014764:Viral Proteins; D004247:DNA; D012343:RNA, Transfer; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases