title: Substitution of DNA-contacting amino acids with functional variants in the Gata-1 zinc finger: a structurally and phylogenetically guided mutagenesis.
abstract: DNA-binding functionality among transcription factor proteins is afforded by a number of structural motifs, such as the helix-turn-helix, helix-loop-helix, and zinc finger domains. The common thread among these diverse structures is their sequence-specific binding to essential promoter or other genetic regulatory sequences with high selectivity and affinity. One such motif, present in a wide range of organisms from bacteria to vertebrates, is the Gata-type zinc finger. This family of DNA-binding proteins is characterized by the presence of one or two (Cys)(4) metal binding sites which recognize the protein's eponymous binding site, GATA. Unlike other conserved DNA-binding domains, Gata proteins appear to be restricted to binding consensus GATA sequences, or near variations, in DNA. Since the architecture of the Gata finger seems built around recognizing this particular sequence, we set out to define the allowable range of amino acid substitutions along the DNA-binding surface of a Gata finger that could continue to support sequence-specific DNA-binding activity. Accordingly, we set up a one-hybrid screen in yeast based on the chicken Gata-1 C-terminal zinc finger. Mutant libraries were generated at five amino acids identified in the Gata-DNA structure as likely to mediate sequence-specific contacts between the Gata finger and DNA. These libraries were designed to give as exhaustive amino acid coverage as possible such that almost all alternative amino acids were screened at each of the five probed positions. Screening and characterization of these libraries revealed several functional amino acid substitutions at two leucines which contact the DNA at the 3' and 5' flanks of the GATA binding site, but no functional substituents for amino acids near the core of the binding site. This pattern is consistent with amino acid sequences of known DNA-binding Gata fingers.
authors.affiliation: Department of Chemistry, Hope College, 35 East 12th Street, Holland, MI 49423, USA.
authors.forename: Tyson R
authors.initials: TR
authors.lastname: Vonderfecht
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Schroyer
authors.forename: Brandy L
authors.initials: BL
authors.lastname: Schenck
authors.forename: Virginia M
authors.initials: VM
authors.lastname: McDonough
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Pikaart
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D050982:GATA1 Transcription Factor / Q000737:chemistry* / Q000145:classification / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010802:Phylogeny; D011487:Protein Conformation; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D050982:GATA1 Transcription Factor; D004247:DNA