title: DNA binding domain of RFX5: interactions with X-box DNA and RFXANK.
abstract: Regulatory factor X (RFX) is a heterotrimeric protein complex having RFX5, RFXANK and RFXAP as its three subunits. It is involved in the regulation of the transcription of MHCII molecules in antigen presenting cells. The RFX complex binds to X-box DNA, using the DNA binding domain, present in RFX5. The DNA binding domain (DBD) of RFX5 (12kD) and intact RFXANK (35 kD) were subcloned, expressed and purified. The associations of RFX5DBD with the X-box DNA and between RFX5DBD and RFXANK were measured in this study. The interaction of RFX5DBD and X-box DNA was studied using steady state fluorescence quenching and circular dichroism. The binding dissociation constant (K(d)) of the DNA-protein complex was determined from fluorescence measurements. The van't Hoff plot was linear over the temperature range 10-25 degrees C and the binding was found to be entropy-driven and enthalpy-favorable. The effect of electrolytes in RFX5DBD-DNA association was also studied. Molecular association between RFX5DBD and RFXANK has been observed by fluorescence resonance energy transfer (FRET) measurements, changes in the ratio of the two vibronic intensities of pyrene labeled RFX5DBD in presence of RFXANK and chemical cross-linking followed by tandem mass spectrometry. Results showed that the two proteins could interact in the absence of the third subunit RFXAP, in vitro with an apparent dissociation constant (K(d)) of 128 nM.
authors.affiliation: Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064, India.
authors.forename: Madhumita
authors.initials: M
authors.lastname: Chakraborty
authors.forename: Amitava
authors.initials: A
authors.lastname: Sengupta
authors.forename: Dipankar
authors.initials: D
authors.lastname: Bhattacharya
authors.forename: Subrata
authors.initials: S
authors.lastname: Banerjee
authors.forename: Abhijit
authors.initials: A
authors.lastname: Chakrabarti
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D000949:Histocompatibility Antigens Class II / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D000071701:Regulatory Factor X Transcription Factors; D012045:Regulatory Sequences, Nucleic Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D000949:Histocompatibility Antigens Class II; C093524:RFX5 protein, human; C117892:RFXANK protein, human; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D004247:DNA