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.
Kim
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Department of Biochemistry, University of California, Riverside 92521.
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015972:Gene Expression Regulation, Neoplastic; D013006:Growth Hormone / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D010911:Pituitary Neoplasms / Q000473:pathology; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D014284:Triiodothyronine / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D013006:Growth Hormone