Molecular cloning and characterization of cDNA encoding the α subunit of the rat protein synthesis initiation factor eIF-2B

Kevin M. Flowers, Scot R. Kimball, Richard C. Feldhoffi, Alan G. Hinnebusch, Leonard S. Jefferson

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)4274-4278
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number10
DOIs
StatePublished - May 9 1995

All Science Journal Classification (ASJC) codes

  • General

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