Cloning of wheat germ eukaryotic initiation factor eIF2
Eukaryotic Initiation Factor 2 (eIF2) is a protein complex found in eukaryotes involved in the initiation of translation. eIF2 consists of three non-identical subunits (α, β, and γ) and is required for the translation of virtually all mRNA transcripts. eIF2 forms a ternary complex with GTP and Met-tRNAi Met, which then associates with the 40S ribosomal subunit. This 43S preinitiation complex scans the mRNA transcript for the AUG initiation codon, at which time eIF2’s GTP is hydrolyzed to GDP and eIF2-GDP dissociates from the 40S subunit. A guanine nucleotide exchange factor called eIF2B is required to reform eIF2-GTP, which can now facilitate a new round of translation initiation. In many eukaryotic systems, eIF2 has been documented as a site of regulation of translation via phosphorylation at a conserved serine residue on the α subunit. These studies have found that phosphorylated eIF2 (eIF2[αP]) binds eIF2B with a much higher affinity than non-phosphorylated eIF2. Since eIF2B is typically present at less than half the cellular concentration of eIF2, the result of this strong binding is sequestration of eIF2B and inhibition of its nucleotide exchange activity. Because only eIF2-GTP can bind Met-tRNAi Met, the result is inhibition of eIF2’s ternary complex formation, and thus inhibition of protein synthesis. Most studies exploring this mechanism of translational control have focused on yeast and mammalian systems. However, in higher plants, regulation of translation by phosphorylation of eIF2α has not been shown to occur in vivo to date. Although homologous genes of all five eIF2B subunits have been identified in plants, the eIF2B protein complex has not yet been isolated from plants. Similarly, though a gene with homology to an eIF2α-kinase from yeast (gcn2) has been identified in plants, phosphorylation of eIF2 in a plant translational lysate system has not yet been shown to inhibit translation. The difference of the binding affinity of eIF2 for GDP and GTP is not nearly as severe as that found in other eukaryotic systems, suggesting that a recycling factor (eIF2B) and phosphorylation of eIF2α may play a less critical regulatory role in plants. There are significant differences between plants and animals in their responses to environmental stress, and therefore methods of translational regulation may also be different. In order to explore the extent of regulation via phosphorylation of eIF2α in plants, and to uncover other plant-specific regulation on eIF2, it is necessary to be able to express the eIF2 complex in a manipulable bacterial system. This will allow mutagenesis of the three subunits to probe structure and function. This thesis reports the use of recombinant methods to construct a single expression vector containing all three genes of eIF2 from T. aestivum (wheat), with the goal of subsequent expression of eIF2α, β, and γ as a functional complex in E. coli.