Department of Clinical Neuroscience, Karolinska Institutet, 17176 Stockholm, Sweden.
Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4093-8. doi: 10.1073/pnas.0914612107. Epub 2010 Feb 10.
Transcription factor-DNA interactions are life sustaining and therefore the subject of intensive research. In spite of vast effort, quantitative in vivo studies of the molecular mechanisms underlying these fundamental interactions remain challenging. In the preceding paper, we designed synthetic Sex combs reduced (Scr) peptides and validated genetically their function as transcriptional regulators. Here we present a controllable system for quantitative studies of protein-DNA interactions in live cells that enables us to "titrate" the concentration of the synthetic Scr peptides in a single cell. Using methods with single-molecule sensitivity, advanced fluorescence imaging and fluorescence correlation spectroscopy (FCS), we were able to study the kinetics of Scr-DNA interactions in live salivary gland cells, where Scr is normally expressed during development. We discerned freely moving Scr molecules, characterized the specific and nonspecific Scr peptide-DNA interactions, and estimated their corresponding dissociation constants (K(d)) in vivo. Our results suggest that the synthetic Scr transcription factors find their specific target sites primarily by multiple association/dissociation events, the rapidity of which is largely owed to electrostatic interactions. Based on these new findings, we formulate a model mechanism and emulate the kinetics of Scr homeodomain-DNA interactions in live cells using numerical simulations.
转录因子与 DNA 的相互作用对生命至关重要，因此一直是研究的热点。尽管已经付出了巨大的努力，但对这些基本相互作用的分子机制进行定量的体内研究仍然具有挑战性。在前一篇论文中，我们设计了合成的 Sex combs reduced (Scr) 肽，并通过遗传验证了它们作为转录调节剂的功能。在这里，我们提出了一种可用于定量研究活细胞中蛋白质-DNA 相互作用的可控系统，使我们能够在单个细胞中“滴定”合成 Scr 肽的浓度。我们使用具有单分子灵敏度的方法、先进的荧光成像和荧光相关光谱 (FCS)，能够在正常发育过程中表达 Scr 的唾腺细胞中研究 Scr-DNA 相互作用的动力学。我们区分了自由移动的 Scr 分子，表征了特异性和非特异性 Scr 肽-DNA 相互作用，并估计了它们在体内的相应解离常数 (K(d))。我们的结果表明，合成的 Scr 转录因子主要通过多次结合/解离事件找到其特定的靶位，其快速性在很大程度上归因于静电相互作用。基于这些新发现，我们提出了一个模型机制，并使用数值模拟模拟了 Scr 同源域-DNA 相互作用在活细胞中的动力学。
