title: Characterization of RNA aptamer binding by the Wilms' tumor suppressor protein WT1.
abstract: The interaction of the zinc finger protein WT1 with RNA aptamers has been investigated using a quantitative binding assay, and the results have been compared to those from a previous study of the DNA binding properties of this protein. A recombinant peptide containing the four zinc fingers of WT1 (WT1-ZFP) binds to representatives of three specific families of RNA aptamers with apparent dissociation constants ranging from 13.8 +/- 1.1 to 87.4 +/- 10.4 nM, somewhat higher than the dissociation constant of 4.12 +/- 0.4 nM for binding to DNA. An isoform that contains an insertion of three amino acids between the third and fourth zinc fingers (WT1[+KTS]-ZFP) also binds to these RNAs with slightly reduced affinity (the apparent dissociation constants ranging from 22.8 to 69.8 nM) but does not bind to DNA. The equilibrium binding of WT1-ZFP to the highest-affinity RNA molecule was compared to the equilibrium binding to a consensus DNA molecule as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1-ZFP with both nucleic acids is an entropy-driven process. Binding of WT1-ZFP to RNA has a pH optimum that is narrower than that observed for binding to DNA. Binding of WT1-ZFP to DNA is optimal at 5 mM MgCl(2), while the highest affinity for RNA was observed in the absence of MgCl(2). Binding of WT1 to both nucleic acid ligands is sensitive to increasing monovalent salt concentration, with a greater effect observed for DNA than for RNA. Point mutations in the zinc fingers associated with Denys-Drash syndrome have dramatically different effects on the interaction of WT1-ZFP with DNA, but a consistent and modest effect on the interaction with RNA. The role of RNA sequence and secondary structure in the binding of WT1-ZFP was probed by site-directed mutagenesis. Results indicate that a hairpin loop is a critical structural feature required for protein binding, and that some consensus nucleotides can be substituted provided proper base pairing of the stem of the hairpin loop is maintained.
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055, Victoria, British Columbia, Canada V8W 3P6.
authors.forename: G
authors.initials: G
authors.lastname: Zhai
authors.forename: M
authors.initials: M
authors.lastname: Iskandar
authors.forename: K
authors.initials: K
authors.lastname: Barilla
authors.forename: P J
authors.initials: PJ
authors.lastname: Romaniuk
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002414:Cations, Monovalent / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016162:Genes, Wilms Tumor; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007680:Kidney Neoplasms / Q000235:genetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011188:Potassium / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025721:WT1 Proteins; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D002414:Cations, Monovalent; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D025721:WT1 Proteins; D012313:RNA; D004247:DNA; D008274:Magnesium; D011188:Potassium