The landscape of NFκB transcription dynamics

摘要/摘要 转录因子在特定DNA上组装活性转录复合物的机制 序列似乎并不遵循简单的识别代码。直接读出，其中， 转录因子通过直接相互作用“读取”特定的DNA序列， 这是因为将DNA看作刚性分子的观点过于简单化。然而，微妙的，不那么微妙的，结构 当DNA与转录因子结合时就会发生变化。此外，转录的DNA结合结构域 因子表现出大范围的灵活性，并且通常包含固有的无序区域。的这些元件 灵活性赋予了转录因子-DNA分子识别的问题许多特征， 蛋白质折叠问题我们的总体假设是，转录因子-DNA结合反而会更好 通过与蛋白质折叠问题所阐明的原理类似的原理来描述。在这里，我们将重点介绍 应激反应转录因子，核因子κB（NFκB），激活数百个相关基因 在生长调节和免疫反应中的作用。我们将联合收割机严格的理论与分子生物物理学相结合 实验研究NFκB转录体复合物的组装动力学。我们将研究耦合 DNA和NFκB之间，因为它涉及串联κB位点、核小体DNA和DNA结合辅激活剂， RPS3。我们预测，NFκB和其他核蛋白组装成特定的NFκB转录体， κB-DNA位点通过协同组装过程。我们将以下列目标来检验这一假设：目标1 确定DNA环境在NFκB结合中的作用。我们将检验DNA环境在 通过研究NFκB的结合，在确定哪些NF κ B结合事件导致转录激活中的作用 一系列真正的NFκB启动子和增强子序列的理论和实验。目的2 探讨NFκB如何与核小体DNA相互作用，并可侵入或解旋核小体DNA。我们将测试 NFκB能够以依赖于NFκB的方式破坏核小体稳定性的假设 在一些实施方案中，所述方法包括测定核小体包裹的DNA的浓度和序列，从而暴露DNA以用于核小体包裹。 转录的起始。原子力显微镜和计算模拟NFκB B与 将追踪核小体。目的3研究DNA、NFκB B和NFκB之间的相互作用， 转录辅激活因子，RPS 3形式。NFκB辅激活因子RPS 3与下列细胞亚群相关并激活这些亚群： 形成高级NFκ B转录体复合物的NF κ B转录激活位点。我们将使用 AWSEM-Suite代码来预测这些较大的蛋白质复合物的结构，并将验证预测结果。 通过NMR顺磁弛豫、SAXS和HDX-MS研究蛋白质和结构域之间的长程接触 实验

Summary/Abstract The mechanism by which transcription factors assemble active transcription complexes on specific DNA sequences does not appear to follow a simple recognition code. Direct readout, wherein specific residues in the transcription factor “read” the specific DNA sequence through direct interactions is most often assumed to apply due to an oversimplified view of DNA as a rigid molecule. However, subtle, and not-so-subtle, structural changes occur when DNA binds to transcription factors. In addition, the DNA binding domains of transcription factors exhibit a large range of flexibility and often contain intrinsically disordered regions. These elements of flexibility endow the problem of transcription factor-DNA molecular recognition with many of the features of the protein folding problem. Our overall hypothesis is that transcription factor-DNA binding would instead be better described by similar principles as have been elucidated for the protein folding problem. Here, we will focus on the stress-response transcription factor, nuclear factor κB (NFκB), which activates hundreds of genes involved in growth regulation and the immune response. We will combine rigorous theory with molecular biophysical experiments to study the assembly kinetics of NFκB transcriptosome complexes. We will investigate coupling between DNA and NFκB as it relates to tandem κB sites, nucleosomal DNA, and the DNA-binding co-activator, RPS3. We predict that NFκB and additional nuclear proteins assemble into specific NFκB transcriptosomes on κB-DNA sites via a cooperative assembly process. We will test this hypothesis with the following aims: Aim 1 Determine the role of DNA context in NFκB binding. We will test the hypothesis that DNA context plays a key role in determining which NFκB binding events result in transcription activation by studying the binding of NFκB to a series of bona fide NFκB promoter and enhancer sequences both theoretically and experimentally. Aim 2 Explore how NFκB interacts with nucleosomal DNA and can invade or unwind nucleosomal DNA. We will test the hypothesis that NFκB is capable of disrupting nucleosome stability in a manner dependent on NFκB concentration and the sequence of DNA that is wrapped by the nucleosome, thereby exposing DNA for the initiation of transcription. Atomic force microscopy and computational modeling of the NFκB interaction with nucleosomes will be pursued. Aim 3 Determine how the ternary interaction between DNA, NFκB and the transcription co-activator, RPS3 forms. The NFκB coactivator, RPS3, associates with, and activates, subsets of NFκB transcription activation sites forming higher-order NFκB transcriptosome complexes. We will use the AWSEM-Suite code to predict the structures of these larger protein complexes and will verify the predicted long-range contacts between proteins and domains by NMR paramagnetic relaxation, SAXS, and HDX-MS experiments.