TFIIH and Transcription Regulation

摘要 RNA聚合酶II(PolII)对转录的调控是控制细胞生长、分化和分化的关键。 发展。在全基因组转录所需的因素中，有进化上保守的， 10亚基TFIIH复合体，在转录过程的多个步骤中发挥基础作用 包括启动子开放和信使核糖核酸的加工，但其分子机制尚不清楚。这个 该项目的长期目标是阐明TFIIH调节polII的分子机制。 抄写。这个项目的基本原理是，了解这些机制将揭示分子 多种不同的发育性和躯体疾病的基础，包括神经障碍， 黄体综合征、皮肤病和癌症。这个合作项目的目标是建立一个 对TFIIH如何控制启动子等基本转录事件的结构和机制的理解 打开，Pol II暂停，以及信使核糖核酸加工。我们最近完成了本地TFIIH的结构分析 使用化学交联/质谱分析(CXMS)和3D建模技术对化合物(人和酵母)进行了分析。 结果预测了两个截然不同的、保守的蛋白质-蛋白质相互作用网络，涉及必要的 酶亚基XPB/SSL2和CDK7/Kin28。转录需要ATPase/转位酶XPB/Ssl2 启动，因为它融化启动子DNA模板，使单链DNA与PolII活性部位结合。 CDK7/Kin28激酶可磷酸化PolII大亚基的C-末端结构域(CTD)以及 其他关键底物(例如P-TEFb)，共同实现对POL II启动、暂停和 M RNA的加工。在目标1中，我们将研究XPB/Ssl2 ATPase/转位酶是如何通过一种 涉及p52/Tfb2和p8/Tfb5的相互作用网络；在目标2中，我们将研究特定残基和 CCNH/Ccl1和MAT1/Tfb3中的结构域控制CDK7/Kin28的活性。为了实现这些目标， 我们将利用酵母遗传学、紫外线响应型交联剂的定点插入、 和体内功能筛选，以在结构上精炼和功能上验证蛋白质-蛋白质界面 太好了。这些数据将指导一系列人体细胞的体外和基于细胞的分析，这些分析将严格地测试 在全基因组转录中，特定分子接口在调节TFIIH功能中的作用。我们 将采用由PIS和我们的合作者建立的方法，包括体外重组 转录、高锰酸盐足迹(体外和细胞内)、Gro-Seq、ChIP或ChIP-Seq和RNA-Seq。如果 成功后，这些实验将使我们能够将给定的蛋白质-蛋白质界面与特定的 转录相关事件，如PolII暂停、启动子打开或交替剪接。最后，强有力的 将对XPB和CDK7的特定化学抑制剂进行评估，以更清楚地定义它们在总体上的作用 和基因特异性(这里将研究P53反应)转录，同时作为关键的阳性对照 XPB或CDK7活性中断。

SUMMARY Regulation of transcription by RNA polymerase II (pol II) is essential to control cell growth, differentiation and development. Among the factors required for genome-wide pol II transcription is the evolutionarily conserved, 10-subunit TFIIH complex, which plays fundamental roles in multiple steps of the transcription process including promoter opening and mRNA processing; however, the molecular mechanisms remain unclear. The long term goal of this project is to elucidate the molecular mechanisms by which TFIIH regulates pol II transcription. The rationale for this project is that understanding these mechanisms will reveal the molecular bases for multiple and diverse developmental and somatic diseases, including neurological disorders, progeroid syndromes, skin diseases and cancer. The objectives of this collaborative project are to establish a structural and mechanistic understanding of how TFIIH controls basic transcriptional events such as promoter opening, pol II pausing, and mRNA processing. We recently completed structural analysis of native TFIIH complexes (both human and yeast) using chemical crosslinking/mass spectrometry (CXMS) and 3D modeling. The results predict two distinct, conserved protein-protein interaction networks involving the essential enzymatic subunits XPB/Ssl2 and CDK7/Kin28. The ATPase/translocase XPB/Ssl2 is required for transcription initiation, as it melts the promoter DNA template to allow single-stranded DNA to engage the pol II active site. The CDK7/Kin28 kinase phosphorylates the C-terminal domain (CTD) of the pol II large subunit as well as other key substrates (e.g. P-TEFb) that collectively enable precise regulation of pol II initiation, pausing, and mRNA processing. In Aim 1 we will investigate how the XPB/Ssl2 ATPase/translocase is regulated by an interaction network involving p52/Tfb2 and p8/Tfb5; in Aim 2 we will investigate how specific residues and domains within CCNH/Ccl1 and MAT1/Tfb3 govern CDK7/Kin28 kinase activity. To accomplish these goals, we will utilize a powerful combination of yeast genetics, site-directed insertion of UV-responsive crosslinkers, and in vivo functional screens to structurally refine and functionally validate protein-protein interfaces within TFIIH. These data will guide a series of in vitro and cell-based assays in human cells that will rigorously test the roles of specific molecular interfaces in the regulation of TFIIH function in transcription, genome-wide. We will employ methods that are well-established by the PIs and our collaborators, including reconstituted in vitro transcription, permanganate footprinting (in vitro and in cells), GRO-Seq, ChIP or ChIP-Seq, and RNA-Seq. If successful, these experiments will allow us to link a given protein-protein interface to regulation of specific transcription-associated events such as pol II pausing, promoter opening, or alternate splicing. Finally, potent and specific chemical inhibitors of XPB and CDK7 will be evaluated to more clearly define their roles in general and gene-specific (p53 response will be studied here) transcription while serving as key positive controls for disruption of XPB or CDK7 activity.