Overlapping but separable determinants of DNA binding and nuclear localization map to the C-terminal end of the Caenorhabditis elegans DAF-12 DNA binding domain.
Proteins are commonly viewed as modular assemblies of functional domains. We analyzed a loss-of-function mutation in the Caenorhabditis elegans intracellular receptor DAF-12, a conservative substitution of an arginine to a lysine at position 197 (R197K). Arg(197) resides in region similar to a nuclear localization signal, just downstream of the receptor minimal zinc finger DNA binding domain (DBD) core. We found that the R197K, but not mutations of neighboring arginine or lysine residues, dramatically reduced DAF-12 transcriptional regulatory activity in a yeast reporter assay. This reduction in regulatory activity correlated with greatly decreased DNA binding affinity in vitro, suggesting a role for the DAF-12 DBD C-terminal region (dbdC), and specifically for Arg(197), in DNA binding. Remarkably, three basic residues immediately contiguous with Arg(197) played little role in DNA binding and rather affected nuclear localization; in contrast, Arg(197) itself was dispensable for nuclear localization. Thus, DAF-12 dbdC harbors overlapping but separable determinants of DNA binding and nuclear localization in a single small region.
Shostak
Yuriy
Y
Program in Biochemistry and Molecular Biology and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-2280.
Yamamoto
Keith R
KR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D004247:DNA / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D015533:Transcriptional Activation
D029742:Caenorhabditis elegans Proteins; C410905:DAF-12 protein, C elegans; D019913:Nuclear Localization Signals; D018160:Receptors, Cytoplasmic and Nuclear; D004247:DNA