CLONING AND CHARACTERIZATION OF COMPLEMENTARY AND GENOMIC DNAS ENCODING THE EPSILON-SUBUNIT OF RAT TRANSLATION INITIATION FACTOR-2B

Eukaryotic initiation factor-2B (eIF-2B) is a guanine nucleotide-excha nge protein involved in the recycling of eIF-2 during peptide-chain in itiation. Regulation of eIF-2B activity occurs under a wide range of c onditions by diverse mechanisms. To better understand the regulation o f eIF-2B activity as well as the coordinate expression of its five sub units, we have begun to clone and characterize the cDNAs and genes enc oding these proteins. In the present study, complementary and genomic DNAs encoding the epsilon-subunit of rat eIF-2B were cloned and charac terized. The cDNA is 2517 bp in length, including a 30 nt poly(A) tail , and recognizes both 2.7 and 3.5 kb mRNA species on Northern blots of rat RNA. The cDNA contains a 2151 bp open reading frame encoding 716 amino acids producing a protein with a predicted molecular mass of 80 kDa. The derived amino acid sequence contains regions identical to thr ee peptides obtained from bovine liver eIF-2B epsilon and is 31% ident ical to Gcd6, the putative yeast eIF-2B epsilon. Examination of the de rived amino acid sequence of rat eIF-2B epsilon reveals phosphorylatio n site motifs for several protein kinases which have been implicated i n regulation of guanine nucleotide exchange activity. The mRNA for eIF -2B epsilon is expressed to a similar extent in most rat tissues exami ned with the exception of testis, where its expression is approx three -fold greater. We have also isolated and sequenced the coding and 5'-f lanking region of the rat eIF-2B epsilon gene. The 16 exons encoding r at eIF-2B epsilon art: contained within 9.5 kb of genomic DNA. Examina tion of the promoter region of the gene reveals a consensus binding si te for the alpha-Pd transcription factor as well as possible cytokine- response elements and binding sites for testis-specific transcription factors.