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 oviduct nuclear matrix. When bound to avian genomic DNA, RBF generates saturable high-affinity binding sites for the avian progesterone receptor (PR). Recent studies have shown that RBF binds to a 54 bp element in the 5'-flanking region of the progesterone-regulated avian c-myc gene, and nuclear matrix-like attachment sites flank the RBF element [Lauber et al. (1997) J. Biol. Chem. 272, 24657-24665]. In this paper, electrophoretic mobility shift assays (EMSAs) and S1 nuclease treatment are used to demonstrate that the RBF-maltose binding protei (MBP) fusion protein binds to single-stranded DNA of its element. Only the N-terminal domain of RBF binds the RBF DNA element as demonstrated by southwestern blot analyses, and by competition EMSAs between RBF-MBP and the N-terminal domain. Mass spectrometric analysis of the C-terminal domain of RBF demonstrates its potential to form noncovalent protein-protein interactions via a potential leucine-isoleucine zipperlike structure, suggesting a homo- and/or possible heterodimer structure in solution. 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 specific single-stranded DNA element. The dimers of RBF are generated by C-terminal leucine zipper and the DNA binding occurs at the N-terminal parallel beta-sheet DNA binding motif. This complex is flanked by nuclear matrix attachment sites.
Barrett
T J
TJ
Department of Biochemistry, Mayo Clinic, 200 First Street, S.W., Rochester, Minnesota 55905, USA.
Sandhu
N P
NP
Tomlinson
A J
AJ
Benson
L M
LM
Subramaniam
M
M
Naylor
S
S
Spelsberg
T C
TC
D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D016259:Genes, myc; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015530:Nuclear Matrix / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D030161:Avian Proteins; D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; C085646:RBF-1 protein, Gallus gallus; D011980:Receptors, Progesterone; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases