Analysing the rearrangement of holo-TFIID with cross-linking/mass spectrometry

转录因子IID（TFIID）等共激活因子是通过与核心转录机制和调控蛋白相互作用以及通过修饰染色质来影响转录的多蛋白质复合物。最近的研究表明，TFIID通过改变其构象来处理这些信号，导致DNA结合TFIID的重排结构。TFIID的构象灵活性和其兆道尔顿的大小迄今为止阻止了整个复合物的高分辨率结构的获取。主机实验室开创了一种使用交联和质谱（CLMS）的新方法，以获得蛋白质及其复合物在溶液中的结构见解。这种方法特别适合于研究复杂环境中的结构变化。使用最先进的综合结构方法，结合联合收割机交联/质谱，冷冻电子显微镜和X射线，我将获得高分辨率的结构TFIID。该体外研究将通过TFIID复合物在其天然环境中的结构分析来补充。我将在活细胞中交联TFIID，以避免生物化学纯化过程中弱相互作用、构象重排或重组表达产物的损失。我的目标是阐明转录控制的核心步骤，并学习最前沿的技术。通过我在这里工作中实施的技术对细胞进行结构分析，有可能从根本上改变结构生物学的进行方式，使其从高度限制的体外条件迁移到更自然的环境。

Coactivators like the transcriptional factor IID (TFIID) are multi-protein complexes that impact transcription through interactions with the core transcription machinery and regulatory proteins, as well as by modifying chromatin. Recent studies suggest that TFIID processes these signals by changing its conformation, resulting in a rearranged structure of DNA bound-TFIID. The conformational flexibility of TFIID and its megadalton size have so far prevented the acquisition of high-resolution structures of the entire complex.The host lab has pioneered a novel approach using cross-linking and mass spectrometry (CLMS) to obtain structural insights of proteins and their complexes in solution. This method is especially well suited to study structural changes in complex environments. Using state-of-the-art integrative structural approaches that combine cross-linking/mass spectrometry, cryo-electron microscopy and X-ray, I will acquire high-resolution structures of TFIID. This in vitro study will be complemented by the structural analysis of TFIID complexes in their native environment. I will cross-link TFIID in living cells to avoid the loss of weak interactions, conformational rearrangements during biochemical purification or artefacts from recombinant expression. My aim is to elucidate a central step in transcription control and to learn a cutting edge technique. Structural analyses in cells by the technologies implemented in my work here have the potential to fundamentally change the way structural biology is conducted, allowing its migration from highly restrictive in vitro conditions towards more native environments.