Journal
FEBS JOURNAL
Volume 279, Issue 17, Pages 3147-3158Publisher
WILEY-BLACKWELL
DOI: 10.1111/j.1742-4658.2012.08693.x
Keywords
fluorescence anisotropy; non-specific DNA; surface plasmon resonance (SPR); YY1; zinc fingers
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Funding
- Polish Ministry of Science and Higher Education [3128/P01/2006/31]
- European Union [POIG.02.01.00-12-064/08]
- Polish Innovation Economy Operational Programme [POIG.02.01.00-12-167/08]
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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 mu m, 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 ka and kd. 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.
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