Organization of transcriptional machinery by weak multivalent interactions

项目摘要 已经提出了由蛋白质固有无序区(IDR)介导的弱多价相互作用 在空间上将转录机制组织成多组分的集群，但我们对如何 这些IDR与特定的合作伙伴互动，以实现职能成果。因为这些相互作用是高度 传统的鉴定蛋白质-蛋白质的策略忽视了它们的动态和依赖于簇的特性 互动。我们的初步数据支持我们的主要假设，即在更高阶星系团的背景下 弱多价相互作用能够产生高度特异性的异型相互作用，从而导致功能性 原子核的组织。我们的长期目标是了解弱多价相互作用是如何组织的 转录机制的特定组成部分，以实现基因激活。这样做的目的是 GRANT是为了研究MED1IDR簇介导的相互作用的机制和功能， 介体共激活物复合体的最大亚基。Mediator是一个连接DNA结合的百万级复合体 转录因子，激活过程的下游步骤需要它动态地与 监管机制的许多组成部分。虽然最近的结构研究揭示了 这种复杂的、负责多价相互作用的结构域是动态的IDR，并且仍然未被解析。在……里面 特别是，MED1亚单位含有先前发表的600个氨基酸的c-末端IDR(MED1-IDR) 研究表明，这与星系团的形成有关。我们的初步数据显示，MED1-IDR星团选择性地划分 正性转录调节因子，排除负性调节因子或功能无关但丰富的核 蛋白质。这些数据让我们假设，IDR介导的选择性划分是一种机制 来调节转录。为了验证这一假设，我们将确认和验证这些星系团的特异性- 中介相互作用(目标1)，描述这些相互作用特有的分子特征 (目标2)，并研究这些相互作用在各种基因激活模式中的作用(目标3)。vt.在.的基础上 完成这些建议的研究，我们将了解弱多价相互作用在 转录机器的特定组件的组织中的MED1-IDR。这一贡献是巨大的 因为这将导致对普遍但经常被忽视的IDR在基因激活中的作用的新的认识。 虽然我们在这里关注的是MED1-IDR，但开发的工具和方法以及在这里学到的原则可以是 用于其他涉及基因调控的弱多价相互作用或日益增长的生物化学 由调节器的动态聚类来调节的过程。

Project Summary Weak multivalent interactions mediated by intrinsically disordered regions (IDRs) of proteins have been proposed to spatially organize the transcriptional machinery into multi-component clusters, yet we know little about how these IDRs interact with specific partners to enable functional outcomes. As these interactions are highly dynamic and cluster-dependent they have been overlooked by conventional strategies to identify protein-protein interactions. Our preliminary data support our overarching hypothesis that in the context of higher-order clusters weak multivalent interactions are capable of highly specific hetero-typic interactions leading to functional organization of the nucleus. Our long-term objective is to understand how weak multivalent interactions organize specific components of the transcriptional machinery in order to enable gene activation. The objective of this grant is to investigate the mechanism and function of cluster-mediated interactions of the IDR of MED1, the largest subunit of the mediator coactivator complex. Mediator is a megadalton complex that bridges DNA-binding transcription factors with downstream steps of the activation process requiring it to engage dynamically with many components of the regulatory machinery. While recent structural studies have revealed the architecture of this complex, the domains responsible for multivalent interactions are dynamic IDRs and remain unresolved. In particular the MED1 subunit contains a >600 amino acid c-terminal IDR (MED1-IDR) which previously published studies implicate in cluster formation. Our preliminary data show that MED1-IDR clusters selectively partition positive regulators of transcription and exclude negative regulators or functionally unrelated yet abundant nuclear proteins. These data lead us to hypothesize that IDR-mediated selective compartmentalization is a mechanism to regulate transcription. To test this hypothesis we will confirm and validate the specificity of these cluster- mediated interactions (Aim 1), characterize the molecular features underlying specificity of these interactions (Aim 2), and investigate the function of these interactions in various models of gene activation (Aim 3). Upon completion of these proposed studies, we will understand the role of weak multivalent interactions mediated by MED1-IDR in organization of specific components of the transcriptional machinery. This contribution is significant as it will lead to a new appreciation for the function of the prevalent yet often overlooked IDRs in gene activation. While we focus here on MED1-IDR, the tools and methods developed and the principles learned here can be applied to other weak multivalent interactions involved in gene regulation or the growing list of biochemical process regulated by dynamic clustering of regulators.