J Biol Chem 1989 Dec 05;26434:20394-402.

Cooperative DNA binding by Xenopus transcription factor IIIA. Use of a 66-base pair DNA fragment containing the intragenic control region of the 5 S RNA gene to study specific and nonspecific interactions.

???displayArticle.abstract???
The interaction of Xenopus transcription factor IIIA (TFIIIA) with a synthetic 66-base pair DNA fragment containing the intragenic control region (ICR) of the 5 S RNA gene was investigated by mobility shift gel electrophoresis and DNase I protection experiments. Specific EDTA-sensitive protein-DNA complexes were observed only in the presence of high concentrations of either the ICR-containing fragment or an excess of unlabeled nonspecific DNA. This predominant complex was determined to be composed of equal molar ratios of TFIIIA and the 66-base pair ICR-containing fragment, although complexes containing two TFIIIA molecules/ICR and three TFIIIA molecules/ICR were also found. In the absence of excess unlabeled specific or nonspecific DNA, TFIIIA was observed to interact with the labeled 66-mer as an aggregate that remained at the top of the gel matrix. Although the binding affinities of TFIIIA for the 66-base pair specific DNA fragment and the 74-base pair nonspecific DNA fragment were comparable as observed by direct measurement with mobility shift gel electrophoresis, a greater than 500-fold difference was observed by competition experiments at high DNA concentrations. Furthermore, a similar difference was also detected when proteolytically cleaved TFIIIA was used in the binding reaction, which substantially reduced the protein-protein interactions. DNase I digestion patterns of the noncoding strand of the 66-mer in the absence or presence of TFIIIA demonstrated that this region of DNA is structurally equivalent to the ICR contained within the 5 S RNA gene. In addition, the mode of binding observed in the mobility shift gels is identical to that observed by direct footprint analysis. Protein-protein interactions appear, therefore, to be an intrinsic and necessary activity of TFIIIA.

???displayArticle.pubmedLink??? 2584222
???displayArticle.link??? J Biol Chem
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: gtf3a