MODULATION OF DNA-BINDING SPECIFICITY WITHIN THE NUCLEAR RECEPTOR FAMILY BY SUBSTITUTIONS AT A SINGLE AMINO-ACID POSITION

Regulation of gene expression involves a large. number of transcriptio n factors with unique DNA-binding properties, Many transcription facto rs belong to families of related proteins that bind to similar but dis tinct sequences. In this study we have analyzed how amino acid substit utions at a single position in the DNA-binding domain modulate the DNA -binding specificity within the nuclear receptor family of transcripti on factors. All possible amino acids were introduced at the first posi tion in the DNA recognition helix, and the specificities of the mutant s were analyzed using response elements containing all combinations of bases at two variable base pair positions. All mutant proteins were f unctional in DNA binding, and could be divided into classes of mutants with different response element specificities. By combining functiona l data with analysis of the structural effects of the mutations by mol ecular modeling, we could identify both prohibitive steric interaction s as well as positive interactions, such as hydrogen bonds, that funct ion as important determinants for specificity. Only the residues found naturally in the glucocorticoid and estrogen receptors, glycine and g lutamate, produce unique binding specificities. The specificities of t he other mutants overlap with each other somewhat but the substitution s clearly have potential to contribute to diversity within the nuclear receptor family. (C) 1995 Wiley-Liss, Inc.