Ligand interactions with eukaryotic translation initiation factor 2: role of the gamma-subunit

EMBO J. 1996 Nov 15;15(22):6311-20.


Eukaryotic translation initiation factor 2 (eIF-2) comprises three non-identical subunits alpha, beta and gamma. In vitro, eIF-2 binds the initiator methionyl-tRNA in a GTP-dependent fashion. Based on similarities between eukaryotic eIF-2gamma proteins and eubacterial EF-Tu proteins, we previously proposed a major role for the gamma-subunit in binding guanine nucleotide and tRNA. We have tested this hypothesis by examining the biochemical activities of yeast eIF-2 purified from wild-type strains and strains harboring mutations in the eIF-2gamma structural gene (GCD11) predicted to alter ligand binding by eIF-2. The alteration of tyrosine 142 in yeast eIF-2gamma, corresponding to histidine 66 in Escherichia coli EF-Tu, dramatically reduced the affinity of eIF-2 for Met-tRNAi(Met) without affecting the k(off) value for guanine nucleotides. In contrast, non-lethal substitutions at a conserved lysine residue (K250) in the putative guanine ring-binding loop increased the off-rate for GDP, thereby mimicking the function of the guanine nucleotide exchange factor eIF-2B, without altering the apparent dissociation constant for Met-tRNAi(Met). For eIF-2[gamma-K250R], the increased off-rate also seen for GTP was masked by the presence of Met-tRNAi(Met) in vitro. In vivo, increasing the dose of the yeast initiator tRNA gene suppressed the slow-growth phenotype and reduced GCN4 expression in gcd11-K250R and gcd11-Y142H strains. These studies indicate that the gamma-subunit of eIF-2 does indeed provide EF-Tu-like function to the eIF-2 complex, and further suggest that the level of Met-tRNAi(Met) is critical for maintaining wild-type rates of initiation in vivo.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Electrophoresis, Polyacrylamide Gel
  • Eukaryotic Initiation Factor-2 / chemistry
  • Eukaryotic Initiation Factor-2 / isolation & purification
  • Eukaryotic Initiation Factor-2 / metabolism*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Gene Expression / genetics
  • Guanosine Diphosphate / metabolism
  • Guanosine Triphosphate / metabolism
  • Lac Operon / genetics
  • Mutagenesis, Site-Directed / genetics
  • Peptide Elongation Factor Tu / metabolism
  • RNA, Transfer, Met / metabolism
  • RNA-Binding Proteins / metabolism*
  • Saccharomyces cerevisiae / chemistry
  • Transformation, Genetic / genetics
  • beta-Galactosidase / metabolism


  • Eukaryotic Initiation Factor-2
  • Fungal Proteins
  • RNA, Transfer, Met
  • RNA-Binding Proteins
  • Guanosine Diphosphate
  • Guanosine Triphosphate
  • beta-Galactosidase
  • Peptide Elongation Factor Tu