Identification of a selenocysteine-specific aminoacyl transfer RNA from rat liver

Wayne C. Hawkes, David E. Lyons, Al L. Tappel

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

The aminoacylation of rat liver tRNA with selenocysteine was studied in tissue slices and in a cell-free system with [75Se]selenocysteine and [75Se]selenite as substrates. [75Se]Selenocysteyl tRNA was isolated via phenol extraction, 1 M NaCl extraction and chromatography on DEAE-cellulose. [75Se]Selenocysteyl tRNA was purified on columns of DEAE-Sephacel, benzoylated DEAE-cellulose and Sepharose 4B. In a dual-label aminoacylation with [35S]cysteme, the most highly purified 75Se-fractions were > 100-fold purified relative to 35S. These fractions contained < 0.7% of the [35S]cysteine originally present in the total tRNA. When [35Se]selenocysteyl tRNA was purified from a mixture of 14C-labeled amino acids, over 97% of the [14C]aminoacyl tRNA was removed. The [75Se]selenocysteine was associated with the tRNA via an aminoacyl linkage. Criteria used for identification included alkaline hydrolysis and recovery of [75Se]selenocysteine, reaction with hydroxylamine and recovery of [75Se]selenocysteyl hydroxamic acid and release of 75Se by ribonuclease. The specificity of [75Se]selenocysteine aminoacylation was demonstrated by resistance to competition by a 125-fold molar excess of either unlabeled cysteine or a mixture of the other 19 amino acids in the cell-free selenocysteine aminoacylation system.

Original languageEnglish (US)
Pages (from-to)183-191
Number of pages9
JournalBBA - Gene Structure and Expression
Volume699
Issue number3
DOIs
StatePublished - Dec 31 1982

Keywords

  • (Rat liver)
  • Aminoacylation
  • Selenocysteine
  • tRNA

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Genetics
  • Structural Biology
  • Medicine(all)

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