Determinants of target gene specificity for ROR alpha 1: monomeric DNA binding by an orphan nuclear receptor.
The ROR alpha isoforms are orphan members of the steroid/thyroid/retinoid receptor superfamily. Previous DNA-binding studies indicated that ROR alpha isoforms bind to response elements consisting of a single copy of the core recognition sequence AGGTCA preceded by a 6-bp A/T-rich sequence and that the distinct amino-terminal domains of each isoform influence DNA-binding specificity. In this report, we have investigated in detail the protein determinants of target gene specificity for the ROR alpha 1 isoform and have now identified the minimal sequence both in its amino- and carboxy-terminal domains required for high-affinity DNA binding. High-resolution methylation and ethylation interference analyses and mixing of truncated proteins in a DNA-binding assay show that ROR alpha 1 presumably binds along one face of the DNA helix as a monomer. By analogy to previous studies of the orphan receptors NGFI-B and FTZ-F1, extensive mutational analysis of the ROR alpha 1 protein shows that a domain extending from the carboxy-terminal end of the second conserved zinc-binding motif is required for specific DNA recognition. However, point mutations and domain swap experiments between ROR alpha 1 and NGFI-B demonstrated that sequence-specific recognition dictated by the carboxy-terminal extension is determined by distinct subdomains in the two receptors. These results demonstrate that monomeric nuclear receptors utilize diverse mechanisms to achieve high-affinity and specific DNA binding and that ROR alpha 1 represents the prototype for a distinct subfamily of monomeric orphan nuclear receptors.
Giguère
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Molecular Oncology Group, Royal Victoria Hospital, Montréal, Québec, Canada.
McBroom
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LD
Flock
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D004247:DNA