Administrative Supplement for Flow Quench Instrument

流动淬火仪行政补充

• 批准号: 10799448
• 负责人: ELIZABETH A. KOMIVES
• 金额: $ 4.25万
• 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/10799448
• 关键词: Administrative Supplement; Amides; Amino Acids; Code; Complex; Computer Simulation; Coupled; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; Disease; Equipment; Family; Gene Expression; Genetic Transcription; Inflammatory; Measures; Nuclear; Parents; Population; RPS3 gene; Specificity; Time; Transcription Coactivator; Transcriptional Activation Domain; Virus Diseases; Work; biological adaptation to stress; experimental study; instrument; millisecond; parent grant; protein folding; response; transcription factor

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. Subtle, and not-so-subtle, structural changes occur when DNA binds to transcription factors. 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. We have discovered that the 230 amino acid intrinsically disordered transcription activation domain (TAD) of NFκB dramatically alters its DNA binding specificity. Because the domain is disordered, we need to measure its dynamic changes on the millisecond timescale. The best way we know to do this is by Flow Quench coupled to HDX-MS. We are therefore requesting an equipment supplement to purchase the Flow Quench instrument. This instrument is expected to reveal subtle changes in the ensemble population of the TAD upon DNA binding to the DNA-binding domain that had been thought to be decoupled from the TAD. This will be critical to Aim 1 of the parent R01. We will also use the instrument to determine subtle changes in the TAD upon ternary complex formation with DNA and RPS3, the transcription co-activator studied in Aim 3. The HDX-MS results will inform the computational simulations integrated with experiments in the parent grant.

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 序列似乎不遵循简单的识别代码，或者不那么微妙的结构变化。 当 DNA 与转录因子结合时，就会发生这种情况。我们的总体假设是转录因子与 DNA 结合。 相反，可以通过与蛋白质折叠问题所阐明的类似原理来更好地描述。 我们发现，230个氨基酸的本质上无序的转录激活域（TAD） 由于 NFκB 结构域是无序的，因此我们需要测量它的 DNA 结合特异性。 我们知道实现这一点的最佳方法是结合 Flow Quench。 因此，我们请求购买 Flow Quench 仪器的设备补充。 仪器有望揭示 DNA 与 TAD 结合后 TAD 总体群体的微妙变化 DNA 结合域被认为与 TAD 分离，这对于目标 1 至关重要。 我们还将使用该仪器来确定三元复合物上 TAD 的细微变化。 DNA 和 RPS3（目标 3 中研究的转录共激活剂）的形成。HDX-MS 结果将告知 计算模拟与父资助中的实验相结合。