Replacement of invariant bZip residues within the basic region of the yeast transcriptional activator GCN4 can change its DNA binding specificity.
Two residues are invariant in all bZip basic regions: asparagine -18 and arginine -10 (we define the first leucine of the leucine zipper of GCN4 as +1). X-ray structures of two specific GCN4-DNA complexes (Ellenberger et al., Cell, 71, 1223-1237, 1992; König & Richmond, J. Mol. Biol., 233, 139-154, 1993) demonstrate the involvement of both residues in specific base pair recognition. We replaced either asparagine -18 or arginine -10 with all other amino acids and tested the DNA binding properties of the resulting mutant peptides by gel mobility shift assays. Peptides with histidine -18 or tyrosine -10 bind with changed specificities to variants of the ATF/CREB site 5'A4T3G2A1C0*G0'T1'C2'A3'T4'3' with symmetric exchanges in positions 2/2' or 0/0', respectively. The double mutant with histidine -18 and tyrosine -10 combines the features of the parental single mutants and binds specifically to the respective double exchange target. Furthermore, the tyrosine -10 mutant clearly prefers the palindrome 5'ATGATATCAT3' over the corresponding pseudo-palindrome 5'ATGATTCA-T3', whereas the lysine -10 mutant binds better to the pseudo-palindromic AP1 site 5'ATGACTCAT3' than to the palindromic ATF/CREB site. Thus, although invariant within natural bZip proteins, asparagine -18 or arginine -10 can be functionally replaced by other amino acids, and their replacement can lead to new DNA binding specificities.
Suckow
M
M
Institut für Genetik der Universität zu Köln, Germany.
Schwamborn
K
K
Kisters-Woike
B
B
von Wilcken-Bergmann
B
B
Müller-Hill
B
B
D000595:Amino Acid Sequence; D001120:Arginine; D001216:Asparagine; D001483:Base Sequence; D001665:Binding Sites; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine; D016350:Leucine Zippers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014443:Tyrosine; D006639:Histidine; D001216:Asparagine; D004247:DNA; D001120:Arginine; D011494:Protein Kinases