Guanosine modifications in runoff transcripts of synthetic transfer RNA-Phe genes microinjected into Xenopus oocytes

Henri Grosjean*, Louis Droogmans, Richard Giégé, Olke C. Uhlenbeck

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

41 Scopus citations

Abstract

We have investigated whether unmodified yeast phenylalanine transfer RNA as well as one of its precursors containing an intron of nineteen nucleotides in the anticodon (pre-tRNA-Phe) can become substrates for selected tRNA modification enzymes present in a eukaryotic cell. This study was done by microinjecting into the cytoplasm of Xenopus laevis oocytes transcripts completely deprived of the naturally occurring modified nucleotides; these were obtained in vitro from appropriate synthetic genes under the control of bacteriophage T7 promoter. During the in vitro transcription, 32P labels were introduced with the guanosine triphosphate thus allowing easy detection of guanosine modifications in tRNA by two-dimensional chromatography after complete digestion into 5′-mononucleotides by nuclease P1. Results indicate that modifications occur on five guanosines (at positions 10, 26, 34, 37 and 46) in yeast tRNA-Phe and only on three guanosines (at 10, 26 and 46) in yeast precursor tRNA-Phe. These are the modifications expected from the known nucleotide sequences of naturally occurring Xenopus and yeast tRNA-Phe, i.e. N2-methyl-G10, N2,N2-dimethyl-G26, 2′-O-methyl-G34, N1-methyl-G37 or Y nucleoside-37 and N7-methyl-G46. The rates of modifications occurring in the two kinds of tRNA-Phe are faster in the intron-less tRNA-Phe than in the intron-containing tRNA-Phe. However quantitative modifications are only observed after as long as 75 h incubation in the oocytes.

Original languageEnglish (US)
Pages (from-to)267-273
Number of pages7
JournalBBA - Gene Structure and Expression
Volume1050
Issue number1-3
DOIs
StatePublished - Aug 27 1990

Keywords

  • (Xenopus oocytes)
  • Modified nucleoside
  • T7 transcript
  • Yeast tRNA-Phe
  • tRNA maturation

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Genetics

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