Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro.

J. R. Sampson*, O. C. Uhlenbeck

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

598 Scopus citations

Abstract

A recombinant plasmid was constructed with six synthetic DNA oligomers such that the DNA sequence corresponding to yeast tRNA(Phe) is flanked by a T7 promoter and a BstNI restriction site. Runoff transcription of the BstNI-digested plasmid with T7 RNA polymerase gives an unmodified tRNA of the expected sequence having correct 5' and 3' termini. This tRNA(Phe) transcript can be specifically aminoacylated by yeast phenylalanyl-tRNA synthetase and has a Km only 4-fold higher than that of the native yeast tRNA(Phe). The Km is independent of Mg2+ concentration, whereas the Vmax is very dependent on Mg2+ concentration. Comparison of the melting profiles of the native and the unmodified tRNA(Phe) at different Mg2+ concentrations suggests that the unmodified tRNA(Phe) has a less stable tertiary structure. Using one additional DNA oligomer, a mutant plasmid was constructed having a guanosine to thymidine change at position 20 in the tRNA gene. A decrease in Vmax/Km by a factor of 14 for aminoacylation of the mutant tRNA(Phe) transcript is observed.

Original languageEnglish (US)
Pages (from-to)1033-1037
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number4
DOIs
StatePublished - Feb 1988

ASJC Scopus subject areas

  • General

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