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

共激活因子如转录因子IID(TFIID)是一种多蛋白质复合体，通过与核心转录机制和调节蛋白的相互作用以及通过修改染色质来影响转录。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的结构重排。到目前为止，TFIID的构象灵活性和其百万吨的大小阻碍了获得整个络合物的高分辨率结构。主办实验室开创了一种新的方法，使用交联质谱(CLMS)来获得蛋白质及其络合物在溶液中的结构洞察力。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，结合交联质谱、低温电子显微镜和X射线，我将获得TFIID的高分辨率结构。这项体外研究将得到TFIID络合物在其自然环境中的结构分析的补充。我将在活细胞中交联化TFIID，以避免在生化纯化过程中丢失弱相互作用、构象重排或重组表达产生的人工产物。我的目的是阐明转录控制的一个核心步骤，并学习一项尖端技术。通过我在这里工作中实施的技术在细胞中进行结构分析，有可能从根本上改变结构生物学的进行方式，使其从严格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.