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

Thomas J. Barrett, Nicole P. Sandhu, Andy J. Tomlinson, Linda M. Benson, Malayannan Subramaniam, Stephen Naylor, Thomas C. Spelsberg

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

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 β-sheet DNA binding motif. This complex is flanked by nuclear matrix attachment sites.

Original languageEnglish (US)
Pages (from-to)753-762
Number of pages10
JournalBiochemistry
Volume39
Issue number4
DOIs
StatePublished - Feb 1 2000

ASJC Scopus subject areas

  • Biochemistry

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    Barrett, T. J., Sandhu, N. P., Tomlinson, A. J., Benson, L. M., Subramaniam, M., Naylor, S., & Spelsberg, T. C. (2000). Interactions of the nuclear matrix-associated steroid receptor binding factor with its DNA binding element in the c-myc gene promoter. Biochemistry, 39(4), 753-762. https://doi.org/10.1021/bi991809v