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Enzymatic Synthesis of Unmodified E. coli tRNAPhe in Vitro

Molecules and Cells 1990년 1권 1호 p.3 ~ 7
 ( Kim Ick-Young ) - Korea University Department of Agricultural Chemistry

 ( Lee Se-Yong ) - Korea University Department of Agricultural Chemistry


E. coli tRNAPhc completely devoid of modified nucleosides was synthesized by in vitro transcription from a pheW gene. The gene was modified to introduce a BstNI restriction endonuclease site at the 3' end by oligonucleotide-directed site-specific mutagenesis. The plasmid DNA containing the mutated gene was linearized with BstNI, so that its run-off transcription produces the mature CCA 3' end of tRNA. The in vitro transcripts thus obtained with phage SP6 RNA polymerase were then processed with M1 RNA, the catalytic subunit of RNase P, to remove the 5' flanking sequence and produce the mature length tRNA. The M1 RNA used was also obtained by in vitro transcription with phage T7 RNA polymerase from a plasmid DNA containing rnpB gene of E. coli. The obtained transcript tRNAPhc lacks all modifications, but maintains its activity for aminoacylation by phenylalanyl tRNA synthetase.


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