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KMID : 0578319940040050003
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Molecules and Cells
1994 Volume.4 No. 5 p.3 ~ p.7
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Enzymatic synthesis of Unmodified E. coli tRNAPhe in Vitro
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Kim Ick-Young
Lee Se-Yong
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Abstract
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E. soli tRNA Ph" 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 MI RNA used was also obtained by in vitro transcription with phage T7 RNA polymerise from a plasmid DNA containing rnpB gene of E coll. The obtained transcript tRNAlh" lacks all modifications, but maintains its activity for aminoacylation by phenylalanyl tRNA synthetase.
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KEYWORD
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