title: An investigation of the affinities, specificity and kinetics involved in the interaction between the Yin Yang 1 transcription factor and DNA.
abstract: Human transcription factor Yin Yang 1 (YY1) is a four zinc-finger protein that regulates a large number of genes with various biological functions in processes such as development, carcinogenesis and B-cell maturation. The natural binding sites of YY1 are relatively unconserved and have a short core sequence (CCAT). We were interested in determining how YY1 recognizes its binding sites and achieves the necessary sequence selectivity in the cell. Using fluorescence anisotropy, we determined the equilibrium dissociation constants for selected naturally occurring YY1 binding sites that have various levels of similarity to the consensus sequence. We found that recombinant YY1 interacts with its specific binding sites with relatively low affinities from the high nanomolar to the low micromolar range. Using a fluorescence anisotropy competition assay, we determined the affinity of YY1 for non-specific DNA to be between 30 and 40 μm, which results in low specificity ratios of between 3 and 220. Additionally, surface plasmon resonance measurements showed rapid association and dissociation rates, suggesting that the binding strength is regulated through changes in both k(a) and k(d). In conclusion, we propose that, in the cell, YY1 may achieve higher specificity by associating with co-regulators or as a part of multi-subunit complexes.
authors: Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
authors: Filip M
authors: FM
authors: Golebiowski
authors: Andrzej
authors: A
authors: Górecki
authors: Piotr
authors: P
authors: Bonarek
authors: Maria
authors: M
authors: Rapala-Kozik
authors: Andrzej
authors: A
authors: Kozik
authors: Marta
authors: M
authors: Dziedzicka-Wasylewska
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D050986:YY1 Transcription Factor / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D050986:YY1 Transcription Factor; C494698:YY1 protein, human; D004247:DNA