DNA binding properties of a chemically synthesized DNA binding domain of hRFX1.
The RFX DNA binding domain (DBD) is a novel highly conserved motif belonging to a large number of dimeric DNA binding proteins which have diverse regulatory functions in eukaryotic organisms, ranging from yeasts to human. To characterize this novel motif, solid phase synthesis of a 76mer polypeptide corresponding to the DBD of human hRFX1 (hRFX1/DBD), a prototypical member of the RFX family, has been optimized to yield large quantities (approximately 90 mg) of pure compound. Preliminary two-dimensional1H NMR experiments suggested the presence of helical regions in this sequence in agreement with previously reported secondary structure predictions. In gel mobility shift assays, this synthetic peptide was shown to bind in a cooperative manner the 23mer duplex oligodeoxynucleotide corresponding to the binding site of hRFX1, with a 2:1 stoichoimetry due to an inverse repeat present in the 23mer. The stoichiometry of this complex was reduced to 1:1 by decreasing the length of the DNA sequence to a 13mer oligonucleotide containing a single half-site. Surface plasmon resonance measurements were achieved using this 5'-biotylinated 13mer oligonucleotide immobilized on an avidin-coated sensor chip. Using this method an association constant (K a = 4 x 10(5)/M/s), a dissociation constant (K d = 6 x 10(-2)/s) and an equilibrium dissociation constant (K D = 153 nM) were determined for binding of hRFX1/DBD to the double-stranded 13mer oligonucleotide. In the presence of hRFX1/DBD the melting temperature of the 13mer DNA was increased by 16 degreesC, illustrating stabilization of the double-stranded conformation induced by the peptide.
Cornille
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F
Département de Pharmacochimie Moléculaire et Structurale, INSERM U266, CNRS URA D1500, UFR des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire, 75270 Paris Cedex 06, France. cornille@pharmacie.univ-paris5.fr
Emery
P
P
Schüler
W
W
Lenoir
C
C
Mach
B
B
Roques
B P
BP
Reith
W
W
D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D004268:DNA-Binding Proteins / Q000138:chemical synthesis / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009691:Nucleic Acid Denaturation; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors / Q000138:chemical synthesis / Q000378:metabolism*
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors