4 STRUCTURALLY DISTINCT, NON-DNA-BINDING SUBUNITS OF HUMAN NUCLEAR RESPIRATORY FACTOR-2 SHARE A CONSERVED TRANSCRIPTIONAL ACTIVATION DOMAIN

Nuclear respiratory factor 2 (NRF-2) was previously purified to near h omogeneity from HeLa cells on the basis of its ability to bind tandem recognition sites in the rat cytochrome oxidase subunit IV (RC04) prom oter. It consisted of five subunits, alpha, beta(1), beta(2), gamma(1) , and gamma(2). Sequencing of tryptic peptides from alpha and from mix tures of the two beta or two gamma subunits revealed sequence identiti es with subunits of the mouse GA-binding protein (GABP), a ubiquitousl y expressed ETS domain activator composed of three subunits, alpha, be ta(1), and beta(2). To understand the precise relationship between NRF -2 and GABP, cDNAs for all five NRF-2 subunits have now been cloned an d their products have been overexpressed. The results establish that t he two additional NRF-2 subunits are molecular variants that differ fr om GABP beta(1) and beta(2) by having a 12-amino-acid insertion contai ning two serine doublets. PCR and RNase protection assays show that mR NAs for these variants are expressed in the human but not the rodent c ells and tissues examined. The insertion did not alter the ability of the beta and gamma subunits to associate with alpha, the DNA-binding s ubunit, nor did it affect the ability of NRF-2 beta(1) or beta(2) to d irect high-affinity binding of alpha to tandem sites in the RC04 promo ter. In addition, the four NRF-2 beta and gamma subunits were equally proficient in activating transcription in transfected cells when fused to a GAL4 DNA-binding domain. The domain responsible for this transcr iptional activation was localized by deletion mapping to a region of a pproximately 70 amino acids that is conserved in all four NRF-2 beta a nd gamma subunits. The repeated glutamine-containing hydrophobic clust ers within this region bear a strong resemblance to those recently imp licated in protein-protein interactions within the transcriptional app aratus.