Dunlap David, Zurla Chiara, Manzo Carlo, Finzi Laura
Department of Cell Biology, Emory University, Atlanta, GA, USA.
Methods Mol Biol. 2011;783:295-313. doi: 10.1007/978-1-61779-282-3_16.
Transcription factors mediate the formation of nucleoprotein complexes that are critical for efficient regulation of epigenetic switches. In these complexes, DNA is frequently bent or looped by the protein; other times, strong interactions lead the DNA to fully wrap the regulatory protein(s). The equilibrium between the bending, looping, full and partial wrapping of DNA governs the level of transcriptional regulation and is tuned by biophysical parameters. Characterization of the structure, kinetics, and thermodynamics of formation of such nucleoprotein complexes is fundamental to the understanding of the molecular mechanisms that underlie the operation of the genetic switches controlled by them. Here, we describe in detail how to perform tethered particle motion experiments aimed at understanding how protein-DNA interactions influence the formation and breakdown of these regulatory complexes.
转录因子介导核蛋白复合物的形成，这些复合物对于表观遗传开关的有效调控至关重要。在这些复合物中，DNA常被蛋白质弯曲或成环；其他时候，强烈的相互作用会使DNA完全包裹调节蛋白。DNA的弯曲、成环、完全和部分包裹之间的平衡决定了转录调控的水平，并由生物物理参数进行调节。表征此类核蛋白复合物形成的结构、动力学和热力学，对于理解由它们控制的遗传开关运作的分子机制至关重要。在这里，我们详细描述如何进行拴系粒子运动实验，旨在了解蛋白质与DNA的相互作用如何影响这些调节复合物的形成和分解。
