Mechanistic Studies of Mediator and the p53 Tumor Suppressor

描述（由申请人提供）：p53转录因子是这项工作的重点，是一种有效的肿瘤抑制因子，通过激活控制关键细胞过程（如生长停滞和凋亡）的基因表达起作用。为了激活它的靶基因，p53与一种称为中介体的蛋白质复合物相互作用。调解帮助调节几乎所有的蛋白质编码基因的表达；因此，它位于转录调控的核心。与大多数转录因子不同，p53通过2个不同的结构域与Mediator相互作用。值得注意的是，使用电子显微镜（EM）和单粒子重建技术，我们已经表明，每个不同的p53结构域在与复合物结合时诱导了截然不同的中介构象状态。这项工作的目的是明确每种p53- mediator相互作用的机制作用，以及它们如何共同调节p53活性。Aim 1中描述的实验将使用重组的人类体外转录系统来检查p53诱导的结构变化如何影响Mediator的生化功能。在目标2中，我们将使用固定化模板分析来探索p53-Mediator结构变化是否会改变转录机制（即预起始复合物）的组装、组成或稳定性。在Aim 3中，我们将结合冷冻电镜研究和p53晶体结构对接，揭示p53四聚体中的不同结构域如何协同工作以协调Mediator中如此巨大的结构变化。

DESCRIPTION (provided by applicant): The p53 transcription factor, which is the focus of this work, is a potent tumor suppressor that works by activating expression of genes that control key cellular processes such as growth arrest and apoptosis. In order to activate its target genes, p53 interacts with a protein complex known as Mediator. Mediator helps regulate expression of nearly all protein-coding genes; thus, it lies at the heart of transcriptional regulation. Unlike most transcription factors, p53 interacts with Mediator via 2 different domains. Notably, using electron microscopy (EM) and single-particle reconstruction techniques, we have shown that each different p53 domain induces a dramatically different Mediator conformational state upon binding to the complex. The goal of this work is to clearly define the mechanistic role of each p53-Mediator interaction and how they may work together to regulate p53 activity. Experiments described in Aim 1 will use a reconstituted, human in vitro transcription system to examine how p53-induced structural changes affect the biochemical function of Mediator. In Aim 2 we will use immobilized template assays to explore whether p53-Mediator structural changes alter the assembly, composition, or stability of the transcriptional machinery (a.k.a. the Pre- Initiation Complex ). In Aim 3, we will combine cryo-EM studies with p53 crystal structure docking to reveal how different domains within the p53 tetramer work together to orchestrate such dramatic structural changes in Mediator. Taken together, these biochemical and structural studies will provide much-needed insight into the molecular mechanisms by which Mediator and p53 work together to control expression of key tumor suppressor genes.p53 is one of the most commonly mutated genes in human cancer. In order to properly function in preventing tumor formation, p53 must interact with a protein complex known as Mediator. Our work will define the fundamental molecular mechanisms by which p53 and Mediator work together to control expression of key anti-cancer genes. This will enhance our understanding of how p53 works to safeguard human cells against cancer; moreover, we anticipate that the information accumulated by our efforts will identify new strategies for controlling the anti-cancer activity of p53.