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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.
AuthorsG Zhai, M Iskandar, K Barilla, P J Romaniuk
JournalBiochemistry (Biochemistry) Vol. 40 Issue 7 Pg. 2032-40 (Feb 20 2001) ISSN: 0006-2960 [Print] United States
PMID11329270 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Cations, Monovalent
  • DNA-Binding Proteins
  • Peptide Fragments
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Transcription Factors
  • WT1 Proteins
  • RNA
  • DNA
  • Magnesium
  • Potassium
Topics
  • Base Sequence
  • Binding Sites
  • Cations, Monovalent (metabolism)
  • DNA (metabolism)
  • DNA-Binding Proteins (chemistry, genetics, metabolism)
  • Genes, Wilms Tumor
  • Humans
  • Hydrogen-Ion Concentration
  • Kidney Neoplasms (genetics)
  • Magnesium (metabolism)
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Peptide Fragments (genetics, metabolism)
  • Point Mutation
  • Potassium (metabolism)
  • RNA (metabolism)
  • RNA-Binding Proteins (chemistry, genetics, metabolism)
  • Recombinant Proteins (metabolism)
  • Transcription Factors (chemistry, genetics, metabolism)
  • WT1 Proteins
  • Zinc Fingers (genetics)

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