The role of flexibility in the specific interactions of even zinc fingers to DNA.
Item
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Title
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The role of flexibility in the specific interactions of even zinc fingers to DNA.
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Identifier
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AAI9405525
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identifier
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9405525
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Creator
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Feng, Waldo C.
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Contributor
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Adviser: Harel A. Weinstein
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Date
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1993
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biophysics, Medical | Biology, Molecular | Biophysics, General
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Abstract
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The molecular basis of specificity and affinity in protein-DNA complexes was explored with the study of interactions between the canonical (CC/HH) zinc finger motifs of Transcription Factor IIIA (X.laevis) and the 5S gene. The nine zinc fingers of TFIIIA can be classified as "odd" or "even" based upon the number of intervening residues (3 or 4) between the conserved histidine ligands in the zinc cluster. The systematic arrangement of the "odd" and "even" zinc fingers in TFIIIA and other multi-zinc finger proteins suggests an underlying functional role in protein-DNA binding.;To assess the molecular basis of such a functional role, the structural and dynamic properties of the zinc fingers were explored with molecular dynamics simulations which revealed major differences in the properties of the two classes of zinc fingers. The time dependent motions of the zinc cluster were found to be correlated with the structure of the DNA-binding helix in the "even" zinc finger only, producing a degree of structural flexibility not available in the "odd" zinc finger. The binding of zinc finger proteins containing both "odd" and "even" fingers is proposed to be dependent on the observed difference in the flexibility of the two types.;This hypothesis was probed in gel retardation assays with native and mutant TFIIIA zinc fingers obtained from an in vitro transcription/translation system. A deletion mutant missing one residue between the conserved histidines of the third zinc finger of TFIIIA (i.e., making an "even" to "odd" ZF) was constructed. In gel shift experiments, TFIIIA containing the mutant finger 3 was found to have decreased DNA binding affinity, in agreement with the inferences developed from theoretical studies that the "even" zinc fingers of TFIIIA are required for positioning groups of "odd" for sequence specific recognition.;A detailed molecular model, consistent with known structural data (e.g., footprinting) of the TFIIIA-DNA complex was constructed. The model incorporates the structural implications of the observed flexibility of the "even" zinc fingers and proposes a novel mode of interaction for zinc fingers 4, 7, and 9 to DNA.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
