MOLECULAR-CLONING AND CHARACTERIZATION OF CDNA-ENCODING THE ALPHA-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 subunit s of eIF-2B, we have begun to clone, characterize, and express the cor responding cDNAs, We report here the cloning and characterization of a 1510-bp cDNA encoding the alpha subunit of eIF-2B from a rat brain cD NA 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 approximate to 1.6 kb in len gth on Northern blots of RNA from rat liver, The predicted amino acid sequence contains regions identical to the sequences of peptides deriv ed from bovine liver eIF-2B alpha subunit, Expression of this cDNA in vitro yields a peptide which comigrates with natural eIF-2B alpha in S DS/polyacrylamide gels, The predicted amino acid sequence exhibits 42% identity to that deduced for the Saccharomyces cerevisiae GCN3 protei n, the smallest subunit of yeast eIF-2B. In addition, expression of th e rat cDNA in yeast functionally complements a gcn3 deletion for the i nability to induce histidine biosynthetic genes under the control of G CN4. These results strongly support the hypothesis that mammalian eIF- 2 alpha and GCN3 are homologues, Southern blots indicate that the eIF- 2B alpha cDNA also recognizes genomic DNA fragments from several other species, suggesting significant homology between the rat eIF-2B alpha gene and that from other species.