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

T. J. Daly, Cheng-Wen Wu*

*此作品的通信作者

研究成果: Article同行評審

5 引文 斯高帕斯(Scopus)

摘要

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 in 66-base pair ICR-containing fragment, although complexes containing two TFIIIA molecules/ICR and three TFIIA 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.

原文English
頁(從 - 到)20394-20402
頁數9
期刊Journal of Biological Chemistry
264
發行號34
DOIs
出版狀態Published - 20 12月 1989

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