The effects of P-box substitutions in thyroid hormone receptor on DNA binding specificity.
Three "P-box" amino acids within the DNA recognition alpha-helix of members of the steroid hormone and thyroid hormone families of nuclear receptors are known to determine the identity of two of the six base pairs within the half-sites of cognate DNA elements. We introduced P-box substitutions derived from different members of the thyroid hormone/estrogen receptor (T3R/ER) family into the beta-isoform of human thyroid hormone receptor (hT3R beta) and tested the DNA binding and transactivation activities of these mutants using thyroid hormone response elements (TREs) with half-sites composed of different sequences and arranged in different orientations. Different P-box sequences derived from the T3R/ER family resulted in distinct DNA binding specificities determined by the fourth base pair of the half-site. Thyroid hormone receptor mutants containing EGA, EAA, EGS substitutions for the wild type EGG P-box bound with wild type affinity to consensus AGGTCA half-sites, regardless of orientation. TREs composed of AGGACA half-sites bound hT3R beta s with an EGG or EAA P-box sequence, but not those with EGA or EGS P-box sequence. A reversal of this specificity was observed on a direct repeat TRE with AGGGCA half-sites. Additionally, an ESG P-box substitution in hT3R beta prevented the receptor from binding to a direct repeat as a homodimer, but this mutant could bind as a heterodimer with retinoid X receptor or to the everted repeat TRE from the chicken lysozyme promoter.
Nelson
C C
CC
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Hendy
S C
SC
Faris
J S
JS
Romaniuk
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011817:Rabbits; D011960:Receptors, Estrogen / Q000378:metabolism; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*; D014158:Transcription, Genetic
D011960:Receptors, Estrogen; D011988:Receptors, Thyroid Hormone; D004247:DNA