The C-terminus of eRF1 defines a functionally important domain for translation termination in Saccharomyces cerevisiae

L Eurwilaichitr, F M Graves, I Stansfield, M F Tuite

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Abstract

Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3, which interact to form a heterodimer that mediates termination at all three stop codons. By C-terminal deletion analysis of eRF1 from the yeast Saccharomyces cerevisiae, we show that the extreme C-terminus of this 437-amino-acid protein defines a functionally important domain for translation termination. A strain encoding eRF1 lacking the C-terminal 32 amino acids is not viable, whereas deletion of the C-terminal 19 amino acids is viable but shows a termination defect in vivo causing an enhancement of nonsense suppression. Using a combination of two-hybrid analysis and in vitro binding studies, we demonstrate that deletions encompassing the C-terminus of eRF1 cause a significant reduction in eRF3 binding to eRF1. All of the C-terminally truncated eRF1 still bind the ribosome, suggesting that the C-terminus does not constitute a ribosome-binding domain and eRF1 does not need to form a stable complex with eRF3 in order to bind the ribosome. These data, together with previously published data, suggest that the region between amino acids 411 and 418 of yeast eRF1 defines an essential functional domain that is part of the major site of interaction with eRF3. However, a stable eRF1:eRF3 complex does not have to be formed to maintain viability or efficient translation termination. Alignment of the seven known eukaryotic eRF1 sequences indicates that a highly conserved motif, GFGGIGG/A is present within the region of the C-terminus, although our deletion studies suggest that it is sequences C-terminal to this region that are functionally important.

Original languageEnglish
Pages (from-to)485-96
Number of pages12
JournalMolecular Microbiology
Volume32
Issue number3
Publication statusPublished - May 1999

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Keywords

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Conserved Sequence
  • Genes, Suppressor
  • Molecular Sequence Data
  • Peptide Fragments
  • Peptide Termination Factors
  • Phenotype
  • Protein Biosynthesis
  • Recombinant Proteins
  • Ribosomes
  • Saccharomyces cerevisiae
  • Xenopus Proteins
  • Xenopus laevis
  • Journal Article
  • Research Support, Non-U.S. Gov't

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