The mouse elongation factor-2 gene: isolation and characterization of the promoter

Elongation factor 2 (EF-2) is a protein involved in peptide chain elongation in eukaryotes. We isolated the mouse EF-2 gene and characterized its promoter. We showed that the majority of enhancer elements were located within 500 bp of the flanking sequence and identified a factor binding site sequence (CGTCACGTGACGC) located between nucleotides -58 and -47 containing two CGTCA motifs separated by two nucleotides. The motif represents a half-site for binding of the cAMP response element (CRE) binding protein (CREB). Mutation analysis indicated that the presence of one CGTCA site alone conferred cAMP inducibility, but the presence of one or two CGTCA sites and spacing nucleotides elicited cAMP-independent, constitutive expression. UV cross-linking and DNA affinity chromatography revealed that three 40-, 43-, and 65-kD proteins bound to the CRE-like element. Of these, the 65-kD protein was unique to the CRE-like element. The 40-kD protein was ATF1 and the 43-kD protein with the molecular size of CREB was not CREB, on the basis of reactivity to their respective antibodies. Because ATF1 responds poorly to cAMP induction, it is likely the contributor to the constitutive expression rather than inductive expression of the CRE-like element, and, thus, the EF-2 gene.