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.
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.
Daly
T J
TJ
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794.
Wu
C W
CW
D000818:Animals; D001482:Base Composition; D004247:DNA / Q000378:metabolism*; D003850:Deoxyribonuclease I; D005796:Genes; D007438:Introns; D009710:Nucleotide Mapping; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D012335:RNA, Ribosomal / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000378:metabolism*; D014357:Trypsin; D014982:Xenopus laevis
D010446:Peptide Fragments; D012335:RNA, Ribosomal; D012341:RNA, Ribosomal, 5S; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D004247:DNA; D003850:Deoxyribonuclease I; D014357:Trypsin