POSITIVE AND NEGATIVE THYROID-HORMONE RESPONSE ELEMENTS ARE COMPOSED OF STRONG AND WEAK HALF-SITES 10 NUCLEOTIDES IN LENGTH

The steroid-thyroid hormone receptors bind to imperfect repeats of two or more half-sites. It is generally accepted that a T3 response element (TRE) half-site consists of a six-nucleotide core motif (5'-AGGT(C/A)A-3'). It is less widely appreciated that the nucleotides flanking this core motif also have a major influence on the affinity of T3 receptor (TR) for its response element. We analyzed TR-DNA interactions under conditions in which the affinity of receptor monomers for individual TRE half-sites of the rat GH (rGH) gene was measured. These studies avoided the effects of half-site spacing and orientation on receptor binding. Variations in the nucleotides flanking the core sequence can modulate receptor binding by more than 15-fold. Systematic mutational analysis of TRE half-site structure demonstrated that at least two nucleotides flanking either side of the half-site core motif strongly influence TR binding affinity and activity, indicating that half-sites are approximately 10 nucleotides long. Thus, the half-sites of most TREs overlap, and mutations in one half-site may affect the activity of its partner. The TRE half-site sequence 5'-CTGAGGTAACG-3' was bound with highest affinity by TRs. The negatively T3-responsive promoter of the rGH gene was used to investigate the functional significance of the nucleotides flanking the core motif in vivo. A promoter consisting of only 22 rGH nucleotides, containing two functional TRE half-sites which overlap the rGH TATA box, directed T3-inhibited transcription. Mutation of nucleotides flanking the core sequence of the weaker half-site dramatically reduced the activity of the element, demonstrating that the flanking sequences of the half-sites can profoundly affect TRE activity.