Structural Studies of Eukaryotic Transcription

描述(申请人提供)：转录调控主要集中在启动过程，涉及基本转录机制的组成部分(RNA聚合酶II和一般转录因子)和介体，一个大的(25个多肽，分子量1丙二醛)多亚基复合体，在真核生物中保守，是转录反应调控信息所必需的。所有这些组分聚集在一起形成一个巨大的预起始复合体(超过60个不同的多肽，MW~2.5 MDA)，负责转录起始。在这一应用中，我们建议应用最先进的大分子冷冻电子显微镜(Cryo-EM)和图像分析技术来确定和分析关键转录复合体的结构，并研究Mediator对转录的调控机制。冷冻-EM图像分析是唯一适合提供关于这些大型和结构动态复合体的信息，我们的EM研究(以及其他许多人的工作)为目前对转录调控的理解做出了重大贡献。我们现在建议扩大这项工作，以获得揭示监管机制的结构性信息。在目标1中，我们将利用我们最近在Mediator纯化方面的突破，获得具有足够高分辨率的Mediator的低温EM结构，以识别与单个蛋白质亚基相对应的密度。3D差异图谱和分子标记技术将被用来定位特定的亚基，并揭示介体复合体的整体组织。在目标2中，我们将确定激活剂和转录因子如何影响介体构象，从而通过影响介体与RNA聚合酶II和一般转录因子的相互作用来实现调节。在目标3中，我们将确定和分析一个最小的预引发复合体(由RNAPII、TBP、TFIIB、TFIIF和启动子DNA组成的催化核心)的3D结构，并与头部复合体结合，该介体模块直接负责将调控信息传递到预引发复合体。这一结构将揭示Mediator如何通过对预引发复合体的组装、稳定性和结构的影响来实现调节。这些目的的完成将为揭示转录调控机制提供关键的结构信息。公共卫生相关性：本申请中提出的研究的总体目标是获得关于Mediator的结构和机制信息，Mediator是调节所有真核生物中转录启动的大分子组装。转录调控控制基因的表达，是细胞分化和发育的基础，对维持细胞内环境的稳定至关重要。转录调控问题与许多疾病(特别是癌症)之间的因果关系解释了这些研究对公共健康的意义。

DESCRIPTION (provided by applicant): Regulation of transcription is largely focused on the initiation process that involves components of the basal transcription machinery (RNA polymerase II and the general transcription factors) and Mediator, a large (25 polypeptides, MW ~1 MDa) multi-subunit complex conserved throughout eukaryotes that is essential for transcriptional response to regulatory information. All of these components come together to form a giant preinitiation complex (over 60 different polypeptides, MW ~2.5 MDa) responsible for transcription initiation. In this application we propose to apply the most advanced macromolecular cryo-electron microscopy (cryo-EM) and image analysis techniques to determine and analyze the structures of key transcription complexes and investigate the mechanism of transcription regulation by Mediator. Cryo-EM image analysis is uniquely suited to provide information about these large and structurally dynamic complexes and our EM studies (along with the work of many others) have contributed significantly to the current understanding of transcription regulation. We now propose to extend this work to obtain structural information that will reveal the mechanism of regulation. In Aim 1 we will take advantage of our recent breakthrough in Mediator purification to obtain a cryo-EM structure of Mediator with high enough resolution to identify density corresponding to individual protein subunits. 3D difference mapping and molecular labeling techniques will be used to localize specific subunits and reveal the overall organization of the Mediator complex. In Aim 2 we will determine how activators and transcription factors influence Mediator conformation in a way that might enable regulation by affecting the interaction of Mediator with RNA polymerase II and the general transcription factors. In Aim 3 we will determine and analyze the 3D structure of a minimal preinitiation complex (a catalytic core comprising RNAPII, TBP, TFIIB, TFIIF, and promoter DNA) in combination with the Head complex, the Mediator module directly responsible for transducing regulatory information to the preinitiation complex. This structure will reveal how Mediator might enable regulation through effects on the assembly, stability, and structure of the preinitiation complex. Completion of these aims will provide structural information critical for unraveling the mechanism of transcription regulation. PUBLIC HEALTH RELEVANCE: The overall goal of the research proposed in this application is to obtain structural and mechanistic information about Mediator, the large macromolecular assembly that regulates transcription initiation in all eukaryotes. Transcription regulation controls gene expression, underlies cellular differentiation and development, and is critical for maintaining cellular homeostasis. The causal relationship between problems with transcription regulation and many diseases (notably cancer) explains the significance of these studies to public health.