Administrative Supplement for Flow Quench Instrument

摘要/摘要 转录因子在特定DNA上组装活性转录复合体的机制 序列似乎并不遵循简单的识别码。微妙和不那么微妙的结构性变化 当DNA与转录因子结合时就会发生。我们的总体假设是转录因子与DNA的结合 相反，用类似于蛋白质折叠问题所阐明的原理来描述会更好。 我们发现230个氨基酸本质上是无序的转录激活域(TAD)。 核因子κB显著改变其与DNA结合的特异性。因为这个领域是无序的，我们需要测量它的 毫秒时间尺度上的动态变化。我们所知的最好的方法是通过流动猝灭耦合到 HDX-MS因此，我们要求补充设备，以购买流动淬火仪器。这 仪器有望揭示与DNA结合后TAD整体种群的细微变化 曾被认为与TAD解偶联的DNA结合域。这将是关键的目标之一 父R01。我们还将使用该仪器来确定TAD对三元复合体的细微变化 与DNA和目标3中研究的转录共激活剂RPS3的形成。HDX-MS结果将通知 计算模拟与母体实验相结合。

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.