Recognition by the Thyroid Hormone Receptor of Canonical DNA Response Elements
journal contributionposted on 09.02.2010, 00:00 by Ana Carolina Migliorini Figueira, Luís Maurício T. R. Lima, Leonardo H. F. Lima, Americo T. Ranzani, Guilherme dos Santos Mule, Igor Polikarpov
To shed more light on the molecular requirements for recognition of thyroid response elements (TREs) by thyroid receptors (TRs), we compared the specific aspects of DNA TRE recognition by different TR constructs. Using fluorescence anisotropy, we performed a detailed and hierarchical study of TR−TRE binding. This was done by comparing the binding affinities of three different TR constructs for four different TRE DNA elements, including palindromic sequences and direct repeats (F2, PAL, DR-1, and DR-4) as well as their interactions with nonspecific DNA sequences. The effect of MgCl2 on suppressing of nonselective DNA binding to TR was also investigated. Furthermore, we determined the dissociation constants of the hTRβ DBD (DNA binding domain) and hTRβ DBD-LBD (DNA binding and ligand binding domains) for specific TREs. We found that a minimum DNA recognition peptide derived from DBD (H1TR) is sufficient for recognition and interaction with TREs, whereas scrambled DNA sequences were unrecognized. Additionally, we determined that the TR DBD binds to F2, PAL, and DR-4 with high affinity and similar Kd values. The TR DBD-LBD recognizes all the tested TREs but binds preferentially to F2, with even higher affinity. Finally, our results demonstrate the important role played by LBDs in modulating TR−DNA binding.