Nuclear receptors are ligand-inducible transcription regulator factors that reside within the cell nucleus and activate transcription of other proteins in response to specific lipophylic ligands such as: steroid, thyroid hormones, retinoids, vitamine D, prostaglandines, fatty acids and other ligands. All nuclear receptors have a modular structure which consists of three major domains: the N-terminal domain (AB region), the centrally located DNA binding domain (DBD), and the C-terminal ligand binding domain (LBD). In this work, we investigate the binding of retinoic acid X receptors (RXRa) to different hormone responsive elements (HRE): DR1, DR4, F2 and PAL. The RXRa construct used in our studies contained both DBD and LBD domains (RXRaDAB). The binding of protein to HRE was investigated in different buffer salt concentration and temperature using HRE DNA oligonucleotides covalently labeled with fluorescein in the 5' end. Tritations of RXRaDAB on a buffer containing 1nM FITC-HREs, accompanied by an increase in fluorescence anisotropy, were used to evaluate the protein affinity to the HREs. Tritrations were carried out in buffers containing NaCl in concentrations of 50mM, 100mM, 200mM and 400mM. The protein binding to DR1-HRE was also investigated at different temperatures (10°C, 15°C and 20°C) in a 50mM NaCl buffer.
Our results show that interactions between RXRaDAB and HREs negatively depend on the salt concentration.  No effective binding were observed for 200mM and 400mM concentrations of NaCl at the protein concentrations used in our experiments. Dissociation constants were calculated for DR1, DR4 and F2, and are, respectively 10nM, 40nM and 50nM. RXRaDAB didn't bind to PAL in none of the observed conditions up to the 200nM concentration of RXRaDAB. Small variations in the binding constants between DR1, DR4 and F2 HREs might indicate the role of AB region in binding and DNA recognition of HREs. No temperature dependence was observed for DR1-HRE in a 50mM NaCl buffer, which is characteristic of a significant entropic contribution in RXRa association mechanism to this response element.