Folding transition in the DMA-binding domain of GCN4 on specific binding to DNA

Abstract

PROTEIN-DNA recognition is often mediated by a small domain containing a recognizable structural motif, such as the helix–turn–helix¹ or the zinc-finger². These motifs are compact structures that dock against the DNA double helix. Another DNA recognition motif, found in a highly conserved family of eukaryotic transcription factors including C/EPB, Fos, Jun and CREB, consists of a coiled-coil dimerization element—the leucine-zipper—and an adjoining basic region which mediates DNA binding³. Here we describe circular dichroism and ¹NMR spectroscopic studies of another family member, the yeast transcriptional activator GCN4^4,5. The 58-residue DNA-binding domain of GCN4, GCN4-p, exhibits a concentration-dependent α-helical transition, in accord with previous studies of the dimerization properties of an isolated leucine-zipper peptide⁶. The GCN4-p dimer is ∼70% helical at 25 °C, implying that the basic region adjacent to the leucine zipper is largely unstructured in the absence of DNA. Strikingly, addition of DNA containing a GCN4 binding site (AP-1 site) increases the α-helix content of GNC4-p to at least 95%. Thus, the basic region acquires substantial α-helical structure when it binds to DNA. A similar folding transition is observed on GCN4-p binding to the related ATF/CREB site, which contains an additional central base pair. The accommodation of DNA target sites of different lengths clearly requires some flexibility in the GCN4 binding domain, despite its high α-helix content. Our results indicate that the GCN4 basic region is significantly unfolded at 25 °C and that its folded, α-helical conformation is stabilized by binding to DNA.