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.

总结/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。微妙的和不那么微妙的结构变化 当DNA与转录因子结合时发生。我们的总体假设是转录因子-DNA结合 相反，将通过与针对蛋白质折叠问题所阐明的原理类似的原理来更好地描述。 我们已经发现，230个氨基酸的固有无序转录激活结构域（NATRA）， NFκB显著改变其DNA结合特异性。由于磁畴是无序的，我们需要测量它的 毫秒级的动态变化。我们所知道的最好的方法是通过流动淬火耦合到 HDX-MS。因此，我们要求购买流动淬火仪器的设备补充。这 仪器预计将揭示DNA结合到DNA上后，DNA的总体种群的微妙变化。 DNA结合域，被认为是解耦的。这对实现千年发展目标1至关重要。 亲本R 01。我们还将使用该仪器来确定在三重复合物中的细微变化 形成与DNA和RPS 3，转录共激活研究目的3。HDX-MS结果将告知 计算模拟与父母补助金中的实验相结合。