Molecular Cloning and Characterization of cDNA Encoding the α Subunit of the Rat Protein Synthesis Initiation Factor eIF-2B

Eukaryotic initiation factor 2B (eIF-2B) is an essential component of the pathway of peptide-chain initiation in mammalian cells, yet little is known about its molecular structure and regulation. To investigate the structure, regulation, and interactions of the individual subunits of eIF-2B, we have begun to clone, characterize, and express the corresponding cDNAs. We report here the cloning and characterization of a 1510-bp cDNA encoding the α subunit of eIF-2B from a rat brain cDNA library. The cDNA contains an open reading frame of 918 bp encoding a polypeptide of 305 aa with a predicted molecular mass of 33.7 kDa. This cDNA recognizes a single RNA species ≈1.6 kb in length on Northern blots of RNA from rat liver. The predicted amino acid sequence contains regions identical to the sequences of peptides derived from bovine liver eIF-2B α subunit. Expression of this cDNA in vitro yields a peptide which comigrates with natural eIF-2Bα in SDS/polyacrylamide gels. The predicted amino acid sequence exhibits 42% identity to that deduced for the Saccharomyces cerevisiae GCN3 protein, the smallest subunit of yeast eIF-2B. In addition, expression of the rat cDNA in yeast functionally complements a gcn3 deletion for the inability to induce histidine biosynthetic genes under the control of GCN4. These results strongly support the hypothesis that mammalian eIF-2α and GCN3 are homologues. Southern blots indicate that the eIF-2Bα cDNA also recognizes genomic DNA fragments from several other species, suggesting significant homology between the rat eIF-2Bα gene and that from other species.