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 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 alpha 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 approximately 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 alpha subunit. Expression of this cDNA in vitro yields a peptide which comigrates with natural eIF-2B alpha 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-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.
Flowers
K M
KM
Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University, Hershey 17033, USA.
Kimball
S R
SR
Feldhoff
R C
RC
Hinnebusch
A G
AG
Jefferson
L S
LS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D001921:Brain / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004331:Drosophila melanogaster / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D005816:Genetic Complementation Test; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D011817:Rabbits; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D020662:Guanine Nucleotide Exchange Factors; D046911:Macromolecular Substances; D011506:Proteins