ABSTRACT
Basic-region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a leucine zipper dimerization domain and a basic region that directly contacts DNA. In all naturally occurring bZip proteins, the basic region is positioned N-terminal to the leucine zipper. We have designed a series of model bZip peptides in which the basic region of the yeast transcriptional activator GCN4 is placed C-terminal to its leucine zipper. DNA-binding studies demonstrate that the optimal reverse GCN4 (rGCN4) peptide is able to bind specifically and with wild-type affinity to DNA despite this unnatural arrangement of the two subdomains. These results suggest that a thermodynamic basis for the observed N-terminal positioning of the basic region relative to the dimerization domain is unlikely.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Leucine Zippers/genetics , Mutagenesis, Site-Directed , Protein Kinases/genetics , Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins , Transcription Factors/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Basic-Leucine Zipper Transcription Factors , Conserved Sequence/genetics , DNA-Binding Proteins/chemical synthesis , Dimerization , Escherichia coli/genetics , Fungal Proteins/chemical synthesis , G-Box Binding Factors , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinases/chemical synthesis , Protein Structure, Secondary/genetics , Protein Structure, Tertiary/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemical synthesis , Repetitive Sequences, Amino Acid/genetics , Thermodynamics , Transcription Factors/chemical synthesisABSTRACT
bZip transcription factors contain two regions that are required for DNA binding: a leucine zipper dimerization domain and a highly charged basic region that directly contacts DNA. The spacing between these subdomains is strictly conserved, and changes in this spacing result in a loss of function. Using an in vitro selection strategy, we have investigated the ability of a bZip protein with incorrect spacing between these two regions to bind specifically to DNA. Surprisingly, we find that although such a protein does not bind to its predicted site, it is possible to isolate a pool of DNAs that bind with very similar affinity to that of GCN4 for its optimum DNA site.
