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）的基因表达来发挥作用。为了激活其靶基因，p53与称为Mediator的蛋白质复合物相互作用。介体有助于调节几乎所有蛋白质编码基因的表达;因此，它是转录调控的核心。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结构域诱导一个显着不同的调解员构象状态后，结合到复杂的。这项工作的目标是明确定义每一个p53介体相互作用的机制作用，以及它们如何共同调节p53活性。目的1中描述的实验将使用重建的人体外转录系统来检查p53诱导的结构变化如何影响介体的生化功能。在目标2中，我们将使用固定化模板测定来探索p53-介导物结构变化是否改变转录机器的组装、组成或稳定性（a.k.a.前启动复合体）。在目标3中，我们将结合联合收割机cryo-EM研究与p53晶体结构对接，以揭示p53四聚体中的不同结构域如何协同工作，以协调介体中如此戏剧性的结构变化。 两者合计，这些生物化学和结构的研究将提供急需的洞察介体和p53的分子机制，共同控制表达的关键肿瘤抑制基因。p53是最常见的突变基因在人类癌症。为了在预防肿瘤形成中发挥作用，p53必须与称为介体的蛋白质复合物相互作用。我们的工作将定义p53和Mediator共同控制关键抗癌基因表达的基本分子机制。这将增强我们对p53如何保护人类细胞免受癌症侵害的理解;此外，我们预计，通过我们的努力积累的信息将确定控制p53抗癌活性的新策略。

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.