Interactions of the nuclear matrix-associated steroid receptor binding factor with its DNA binding element in the c-myc gene promoter

Steroid receptor binding factor (RBF) was originally isolated from avian ov iduct nuclear matrix. When bound to avian genomic DNA, RBF generates satura ble high-affinity binding sites for the avian progesterone receptor (PR). R ecent studies have shown that RBF binds to a 54 bp element in the 5'-flanki ng region of the progesterone-regulated avian c-myc gene, and nuclear matri x-like attachment sites flank the RBF element [Lauber et al. (1997) J. Biol . Chem. 272, 24657-24665]. In this paper, electrophoretic mobility shift as says (EMSAs) and Si nuclease treatment are used to demonstrate that the RBF -maltose binding protei (MBP) fusion protein binds to single-stranded DNA o f its element. Only the N-terminal domain of RBF binds the RBF DNA element as demonstrated by southwestern blot analyses, and by competition EMSAs bet ween RBF-MBP and the N-terminal domain. Mass spectrometric analysis of the C-terminal domain of RBF demonstrates its potential to form noncovalent pro tein-protein interactions via a potential leucine-isoleucine zipperlike str ucture, suggesting a homo- and/or possible heterodimer structure in solutio n. These data support that the nuclear matrix binding site (acceptor site) for PR in the c-myc gene promoter is composed of RBF dimers bound to a spec ific single-stranded DNA element. The dimers of RBF are generated by C-term inal leucine zipper and the DNA binding occurs at the N-terminal parallel b eta-sheet DNA binding motif. This complex is flanked by nuclear matrix atta chment sites.