Changing Fos oncoprotein to a Jun-independent DNA-binding protein with GCN4 dimerization specificity by swapping "leucine zippers"

Abstract

A STRUCTURAL motif for DNA-binding proteins, the "leucine zipper", has been proposed for the jun,fos and myc gene products, the yeast transcriptional activator GCN4, and the C/EBP enhancer-binding protein¹. These proteins all contain a region with four or five leucine residues spaced exactly seven amino acid residues apart whose sequence is consistent with the formation of an amphipathic α-helix. It has been proposed that the leucine zipper consists of two interdigitated α-helices, one from each monomer, that constitute the dimerization function necessary for high-affinity binding to DNA; an adjacent region of basic residues is thought to be responsible for specific protein-DNA contacts¹. In support of this model, substitution of the leucine residues within the motif can abolish dimerization and DNA-binding^2–6, and a synthetic peptide corresponding to the GCN4 leucine zipper forms a-helical dimers⁷. Despite the conserved leucine residues, however, each protein has a distinct dimerization specificity. Specifically, GCN4 homodimer, Jun homodimer and Fos-Jun heterodimer proteins bind to the same DNA site, whereas Fos is unable to form homodimers, bind DNA, or interact with GCN4 (refs 8–14). Here, we alter the dimerization specificity of Fos by precisely replacing its leucine zipper with that from GCN4. This Fos-GCN4 chimaeric protein is able to bind to the target site in the absence of Jun, and can form DNA-binding heterodimers with GCN4 but not with Jun. These results indicate that the leucine zipper is sufficient to confer dimerization specificity and strongly suggest that Fos contacts DNA directly.