Abstract
A long-standing goal of biomedical research has been to determine the quantitative mechanisms responsible for gene regulation and transcriptional activation. These events occur through numerous protein–protein and protein–DNA interactions, many of which are allosterically coupled. For systems where highly purified protein is available, analytical ultracentrifugation provides a means to study these linked reactions, allosteric or otherwise. Sedimentation velocity is an ultracentrifugation technique that provides rigorous insight into protein self-association, homogeneity, and gross structure. Because self-association is often in dynamic equilibrium with other reactions such as DNA binding, an explicit and independent analysis of each interaction is critical to revealing mechanism. This chapter details a protocol for using sedimentation velocity to dissect the linkage between transcription factor self-association and site-specific DNA binding.
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Acknowledgments
The authors would like to thank Michael T. Miura and Keith D. Connaghan. This work was supported by NIH grant DK061933 and the Avon Foundation for Women.
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Moody, A.D., Robblee, J.P., Bain, D.L. (2012). Dissecting the Linkage Between Transcription Factor Self-Assembly and Site-Specific DNA Binding: The Role of the Analytical Ultracentrifuge. In: Fenton, A. (eds) Allostery. Methods in Molecular Biology, vol 796. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-334-9_10
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DOI: https://doi.org/10.1007/978-1-61779-334-9_10
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