Administrative Supplement for Flow Quench Instrument

点击翻译按钮获取中文摘要

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.