Characterization of the equilibrium binding of Xenopus transcription factor IIIA to the 5 S RNA gene.
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.
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
P J
PJ
Romaniuk
D000818:Animals; D004275:DNA, Ribosomal / Q000302:isolation & purification / Q000378:metabolism*; D005796:Genes; D007700:Kinetics; D009994:Osmolar Concentration; D010957:Plasmids; D011485:Protein Binding; D012341:RNA, Ribosomal, 5S / Q000235:genetics*; D015183:Restriction Mapping; D012492:Salts; D013816:Thermodynamics; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000378:metabolism*; D014981:Xenopus
D004275:DNA, Ribosomal; D012341:RNA, Ribosomal, 5S; D012492:Salts; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors