NRF-1, AN ACTIVATOR INVOLVED IN NUCLEAR-MITOCHONDRIAL INTERACTIONS, UTILIZES A NEW DNA-BINDING DOMAIN CONSERVED IN A FAMILY OF DEVELOPMENTAL REGULATORS

Nuclear respiratory factor 1 (NRF-1) was first discovered as an activa tor of the cytochrome c gene and was subsequently found to play a broa der role in nuclear-mitochondrial interactions. We have now cloned a H eLa cDNA encoding NRF-1 using degenerate oligomers derived from trypti c peptide sequences for PCR amplification. The cDNA-encoded protein wa s indistinguishable from the authentic HeLa cell factor on denaturing gels, displayed the expected NRF-1 DNA-binding specificity, and made t he same guanine nucleotide contacts as HeLa NRF-1 on binding known NRF -1 recognition sites. Antiserum raised against the highly purified rec ombinant protein recognized the identical DNA-protein complex formed u sing either a crude nuclear fraction or nearly homogeneous HeLa NRF-1. Recombinant NRF-1 also activated transcription through specific sites from several NRF-1-responsive promoters, confirming both the transcri ptional activity and specificity of the cDNA product. Portions of NRF- 1 are closely related to sea urchin P3A2 and the erect wing (EWG) prot ein of Drosophila. Both are recently identified developmental regulato ry factors. The region of highest sequence identity with P3A2 and EWG was in the amino-terminal half of the molecule, which was found by del etion mapping to contain the DNA-binding domain, whereas the carboxy-t erminal half of NRF-1 was highly divergent from both proteins. The DNA -binding domain in these molecules is unrelated to motifs found common ly in DNA-binding proteins; thus, NRF-1, P3A2, and EWG represent the f ounding members of a new class of highly conserved sequence-specific r egulatory factors.