Recognition by the thyroid hormone receptor of canonical DNA response elements.
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 MgCl(2) on suppressing of nonselective DNA binding to TR was also investigated. Furthermore, we determined the dissociation constants of the hTRbeta DBD (DNA binding domain) and hTRbeta 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 K(d) 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.
Figueira
Ana Carolina Migliorini
AC
Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Saocarlense, 400, Sao Carlos, SP, Brazil 13560-970.
Lima
Luís Maurício T R
LM
Lima
Leonardo H F
LH
Ranzani
Americo T
AT
Mule
Guilherme dos Santos
Gdos S
Polikarpov
Igor
I
D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D008024:Ligands; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011988:Receptors, Thyroid Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D020218:Response Elements / Q000235:genetics*; D013816:Thermodynamics; D014284:Triiodothyronine / Q000737:chemistry / Q000378:metabolism
D004268:DNA-Binding Proteins; D008024:Ligands; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D004247:DNA