Role of the conserved leucines in the leucine zipper dimerization motif of yeast GCN4

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Abstract
Yeast GCN4 belongs to the class of eukaryotic transcription factors whose bZIP DNA-blndlng domains dimerize via a leucine zipper motif that structurally resembles a coiled coll. The leucine zipper contains 4 – 5 highly conserved leucine residues spaced exactly 7 residues apart that are located within the α-helical hydrophoblc interface between protein monomers. Here, we investigate the role of the four canonical leucines in the GCN4 leucine zipper by analyzing a series of mutated derivatives for their ability to activate transcription in vivo and to bind DNA in vitro . The GCN4 leucine zipper is surprisingly tolerant of mutations, with a wide variety of single substitutions at any of the four leucines Including basic and acidic amino acids behaving indlstinguishably from wild-type GCN4. Moreover, some derivatives containing two leucine substitutions display detectable though reduced function. These results indicate that other residues within the colled coil are crucial for efficient dimerization, and they suggest that some eukaryotic transcriptional regulatory proteins lacking the conserved leucine repeat will dimerize through a structurally homologous motif. Interestingly, our results differ in several respects from those obtained by analyzing mutations in the GCN4 leucine zipper in the context of a X repressor-GCN4 zipper hybrid protein. These apparent differences may reflect a functional interrelationship between the leucine zipper and basic region subdomains for DNA-blnding by bZIP proteins.