In vitro synthesis of selenocysteinyl-tRNA(UCA) from seryl-tRNA(UCA): involvement and characterization of the selD gene product

Proc Natl Acad Sci U S A. 1990 Jan;87(2):543-7. doi: 10.1073/pnas.87.2.543.

Abstract

The selD gene from Escherichia coli, whose product is involved in selenium metabolism, has been cloned and sequenced. selD codes for a protein of 347 amino acids with a calculated molecular weight of 36,687. Analysis of the selD gene product through expression of the gene in the phage T7 promoter/polymerase system confirmed the predicted molecular weight of the protein. Gene disruption experiments demonstrated that the SelD protein is required both for the incorporation of selenium into the modified nucleoside 5-methylaminomethyl-2-selenouridine of tRNA and for the biosynthesis of selenocysteine from an L-serine residue esterbonded to tRNA(Ser)(UCA). tRNA(Ser)(UCA) has been purified, aminoacylated with L-serine, and used as a substrate for the development of an in vitro system for selenocysteine biosynthesis. Efficient formation of selenocysteinyl-tRNA(Ser)(UCA) was achieved by using extracts in which both the selD and the selA gene products were overproduced. The results demonstrate that selenocysteine is synthesized from L-serine bound to tRNA(UCA) and they are in accord with SelD functioning as a donor of reduced selenium.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics*
  • Base Sequence
  • Codon / genetics
  • Drosophila Proteins*
  • Escherichia coli / genetics*
  • Genes, Bacterial*
  • Genotype
  • Molecular Sequence Data
  • Mutation
  • Phosphotransferases*
  • Plasmids
  • RNA, Transfer, Amino Acid-Specific / metabolism*
  • RNA, Transfer, Amino Acyl / biosynthesis*
  • RNA, Transfer, Amino Acyl / genetics
  • RNA, Transfer, Ser / metabolism*
  • Restriction Mapping
  • Selenium / metabolism

Substances

  • Bacterial Proteins
  • Codon
  • Drosophila Proteins
  • RNA, Transfer, Amino Acid-Specific
  • RNA, Transfer, Amino Acyl
  • RNA, Transfer, Ser
  • selenocysteinyl-tRNA
  • Phosphotransferases
  • selenophosphate synthetase
  • Selenium

Associated data

  • GENBANK/M30184