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

J Biol Chem. 2005 Feb 25;280(8):6554-60. doi: 10.1074/jbc.M412928200. Epub 2004 Dec 15.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Binding Sites
  • Caenorhabditis elegans Proteins / chemistry*
  • Caenorhabditis elegans Proteins / genetics
  • Caenorhabditis elegans Proteins / physiology*
  • DNA / metabolism*
  • Mutagenesis, Site-Directed
  • Nuclear Localization Signals*
  • Protein Structure, Tertiary
  • Receptors, Cytoplasmic and Nuclear / chemistry*
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / physiology*
  • Transcriptional Activation

Substances

  • Caenorhabditis elegans Proteins
  • DAF-12 protein, C elegans
  • Nuclear Localization Signals
  • Receptors, Cytoplasmic and Nuclear
  • DNA