The landscape of NFκB transcription dynamics

NFκB 转录动力学景观

• 批准号: 10686820
• 负责人: ELIZABETH A. KOMIVES
• 金额: $ 56.54万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-19 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10686820
• 关键词: Acceleration; Acquired Immunodeficiency Syndrome; Affinity; Apoptotic; Arthritis; Asthma; Atomic Force Microscopy; Autoimmune Diseases; Behavior; Binding; Biochemical; Biophysics; Cell Nucleus; Cells; Cessation of life; ChIP-seq; Code; Collaborations; Complex; Computer Models; Coupling; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; Data; Diabetes Mellitus; Disease; Elements; Endowment; Event; Excision; Exhibits; Family; Floods; Gene Expression; Genes; Genetic Enhancer Element; Genetic Transcription; Goals; Growth; I Kappa B-Alpha; Immune; Immune response; In Vitro; Inflammatory; Invaded; Kinetics; Laboratories; Length; Malignant Neoplasms; Measures; Mediating; Micro Array Data; Molecular; Monitor; Mutagenesis; Nuclear; Nuclear Export; Nuclear Proteins; Nucleosomes; Play; Process; Promoter Regions; Proteins; Publishing; RPS3 gene; Regulation; Relaxation; Research Personnel; Role; Series; Site; Slide; Speed; Stress; Structure; System; Systems Biology; TNF gene; TNFRSF5 gene; Testing; Therapeutic; Thermodynamics; Time; Transactivation; Transcription Coactivator; Transcription Initiation; Transcriptional Activation; Transcriptional Regulation; Viral; Virus Diseases; Work; biological adaptation to stress; biophysical techniques; cell growth; design; experimental study; flexibility; in vivo; molecular recognition; mutant; protein complex; protein folding; recruit; response; simulation; single-molecule FRET; stoichiometry; theories; transcription factor; transcriptome sequencing

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.

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 序列似乎没有遵循简单的识别代码。直接读数，其中的特定救援 经常假定，通过直接相互作用“读取”特定DNA序列的转录因子被认为 由于DNA作为刚性分子的过度简化视图而应用。但是，微妙且不那么微妙的结构 当DNA与转录因子结合时，发生变化。另外，转录的DNA结合结构域 因素表现出很大的灵活性，并且通常包含本质上无序的区域。这些要素 灵活性赋予转录因子-DNA分子识别的问题，具有许多特征 蛋白质折叠问题。我们的总体假设是转录因子-DNA结合会更好 通过类似的原理描述，已阐明了蛋白质折叠问题。在这里，我们将重点关注 应力反应转录因子核因子κB（NFκB）激活了涉及的数百个基因 在生长调节和免疫反应中。我们将将严格的理论与分子生物物理结合在一起 研究NFκB转录体复合物的组装动力学的实验。我们将调查耦合 与串联κB位点，核小体DNA和DNA结合共激活剂有关的DNA和NFκB之间 RPS3。我们预测NFκB和其他核蛋白在特定的NFκB转录体上汇集 κB-DNA位点通过合作组装过程。我们将以以下目的检验这一假设：目标1 确定DNA环境在NFκB结合中的作用。我们将测试DNA上下文扮演关键的假设 通过研究NFκB的结合来确定哪种NFκB结合事件导致转录激活 一系列真正的NFκB启动子和增强子序列的理论和实验序列。目标2 探索NFκB如何与核小体DNA相互作用，并可以侵入或解开核小体DNA。我们将测试 NFκB能够以依赖NFκB的方式破坏核小体稳定性的假设 浓度和由核小体包裹的DNA的序列，从而暴露于DNA 转录的开始。 NFκB相互作用的原子力显微镜和计算模型 将追求核小体。 AIM 3确定DNA，NFκB和 转录共激活器，RPS3形式。 NFκB共激活因子RPS3与并激活的子集相关联 NFκB转录激活位点形成高阶NFκB转录体复合物。我们将使用 AWSEM-SUITE代码可预测这些较大蛋白质复合物的结构，并将验证预测的 NMR顺磁松弛，SAXS和HDX-MS之间的蛋白质与域之间的远距离接触 实验。