title: Two of the five zinc fingers in the Zap1 transcription factor DNA binding domain dominate site-specific DNA binding.
abstract: The Zap1 transcriptional activator from Saccharomyces cerevisiae induces expression of a series of genes containing an 11 base pair conserved promoter element (ZRE) under conditions of zinc deficiency. This work shows that Zap1 uses four of its seven zinc finger domains to contact the ZRE and that two of these dominate the interaction by contacting the essential ACC-GGT ends. Two Zn finger domains (ZF1 and ZF2) do not contact DNA, and a third ZF3 may be more important for interfinger protein-protein interactions. Zn finger domains important for ZRE contact were identified from triple mutations in Zap1, changing three residues in the alpha helix in each finger known to be important for DNA contacts in Zn finger proteins. Replacement of -1, 3, and 6 helix residues in ZF4 and ZF7 reduced the affinity of Zap1 for the wild-type ZRE. In contrast, triple mutations within the intervening ZF5 and ZF6 domains had minimal effect. The data argue that fingers 4 and 7 contact the ACC-GGT ends while fingers 5 and 6 contact the 5 bp central ZRE sequence. This conclusion is corroborated by decreased Zap1 affinity for a ZRE DNA duplex containing mutations of the AC-GT ends of the ZRE, whereas transversion mutations within the central 5 bp of the ZRE had minimal effect on Zap1 binding affinity.
authors.affiliation: Department of Medicine, University of Utah Health Sciences Center, Salt Lake City, Utah 84132, USA.
authors.forename: M V
authors.initials: MV
authors.lastname: Evans-Galea
authors.forename: E
authors.initials: E
authors.lastname: Blankman
authors.forename: D G
authors.initials: DG
authors.lastname: Myszka
authors.forename: A J
authors.initials: AJ
authors.lastname: Bird
authors.forename: D J
authors.initials: DJ
authors.lastname: Eide
authors.forename: D R
authors.initials: DR
authors.lastname: Winge
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011817:Rabbits; D020218:Response Elements / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C103264:ZAP1 protein, S cerevisiae