Reconstitution of yeast translation initiation

Methods Enzymol. 2007:430:111-45. doi: 10.1016/S0076-6879(07)30006-2.

Abstract

To facilitate the mechanistic dissection of eukaryotic translation initiation we have reconstituted the steps of this process using purified Saccharomyces cerevisiae components. This system provides a bridge between biochemical studies in vitro and powerful yeast genetic techniques, and complements existing reconstituted mammalian translation systems (Benne and Hershey, 1978; Pestova and Hellen, 2000; Pestova et al., 1998; Trachsel et al., 1977). The following describes methods for synthesizing and purifying the components of the yeast initiation system and assays useful for its characterization.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Eukaryotic Initiation Factor-1 / isolation & purification
  • Eukaryotic Initiation Factor-1 / metabolism
  • Eukaryotic Initiation Factor-2 / isolation & purification
  • Eukaryotic Initiation Factor-2 / metabolism
  • Methionine / metabolism
  • Methionine-tRNA Ligase / isolation & purification
  • Methionine-tRNA Ligase / metabolism
  • Protein Biosynthesis*
  • Protein Isoforms / isolation & purification
  • Protein Isoforms / metabolism
  • RNA, Fungal / genetics
  • RNA, Fungal / isolation & purification
  • RNA, Fungal / metabolism
  • RNA, Ribosomal / isolation & purification
  • RNA, Ribosomal / metabolism
  • RNA, Transfer, Met / metabolism
  • Ribosome Subunits, Large, Eukaryotic / chemistry
  • Ribosome Subunits, Large, Eukaryotic / genetics
  • Ribosome Subunits, Large, Eukaryotic / metabolism
  • Ribosome Subunits, Small, Eukaryotic / chemistry
  • Ribosome Subunits, Small, Eukaryotic / genetics
  • Ribosome Subunits, Small, Eukaryotic / metabolism
  • Saccharomyces cerevisiae Proteins* / genetics
  • Saccharomyces cerevisiae Proteins* / isolation & purification
  • Saccharomyces cerevisiae Proteins* / metabolism
  • Saccharomyces cerevisiae* / genetics
  • Saccharomyces cerevisiae* / metabolism

Substances

  • Eukaryotic Initiation Factor-1
  • Eukaryotic Initiation Factor-2
  • Protein Isoforms
  • RNA, Fungal
  • RNA, Ribosomal
  • RNA, Transfer, Met
  • Saccharomyces cerevisiae Proteins
  • Methionine
  • Methionine-tRNA Ligase