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XB-ART-25529
J Biol Chem 1990 Oct 15;26529:17593-600.
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Characterization of the equilibrium binding of Xenopus transcription factor IIIA to the 5 S RNA gene.

Romaniuk PJ .


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A nitrocellulose filter-binding assay has been developed to study the interaction of Xenopus transcription factor IIIA (TFIIIA) with its specific binding site on the 5 S RNA gene. The protein binds to a DNA restriction fragment containing a Xenopus oocyte 5 S RNA gene (5 S DNA) with an apparent association constant of 1.90 x 10(9) M-1 in 0.1 M salt, pH 7.5, at 22 degrees C. Under these assay conditions, the protein has approximately a 100-fold lower binding affinity for DNA fragments that do not contain a 5 S RNA gene. Analysis of the temperature dependence of the binding of TFIIIA to 5 S DNA indicates that the interaction is largely enthalpy driven at temperatures above 19 degrees C, while it is largely entropy driven at lower temperatures. One molecule of TFIIIA binds per 5 S RNA gene, and this bimolecular complex dissociates with first order kinetics, having a half-life of 15.6 min. The DNA binding activity of the protein exhibits a broad pH optimum from 6.0 to 8.0, and is optimal at 5 mM MgCl2 decreasing rapidly at higher divalent ion concentrations. The specific binding of TFIIIA to 5 S DNA is insensitive to the identity of the monovalent cation present in the binding buffer. In comparison, the anion effects on DNA binding are dramatic, with a 100-fold decrease in binding affinity observed to follow the lyotropic series. This result suggests that there are several specific anion-binding sites on TFIIIA. Determination of the monovalent salt dependence of the association constant revealed that as many as 8 lysine-phosphate type ionic bonds are formed in the TFIIIA-DNA complex.

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Species referenced: Xenopus
Genes referenced: gtf3a