The roles of TFIIB and TFIIF in transcription by DNA-directed RNA Polymerase II

 描述(申请人提供)：DNA指导的RNA聚合酶II(RNAPII)在真核生物中高度保守，在细胞生活中发挥着重要作用，特别是基因转录。在转录过程中，RNAPII将基因转录成信使RNA，这一过程受到通用转录因子TBP、TFIIA、TFIIE、TFIIH、TFIIB和TFIIF的辅助。有意义的生化实验表明，转录启动是一个高度动态的事件，可能有几个离散的阶段，在这个阶段，一般的转录因子识别、融化和加载RNAPII活性位点内的核酸支架(NAS)。这个过程对于所有真核生物来说都是普遍的，是基因调控的核心；因此，了解它的分子细节将提供基本的线索，可能导致潜在的药物操纵基因表达。既然目前的技术使我们能够结晶和解决大型多组分复合体的高分辨结构，我们就可以开始在原子水平上研究转录机制，并解决这个特定的问题：在转录泡泡加载和启动子逃逸过程中，一般转录因子TFIIB和TFIIF在DNA稳定中扮演什么角色？这项提议的目的是使用化学交联法、X射线结晶学和核磁共振等新方法来回答这个问题。在两种倾向的方法中，我们将首先建立在初步数据的基础上，这些数据表明有可能重建和结晶化学计量比的RNAPII-TFIIF络合物，以及获得到2.9？的各向异性衍射。改善结晶条件 我们将恳求一种新的化学交联方法，它将有助于防止结晶学试验中的复杂解离。其次，我们将展示重构由TFIIB、TFIIF、NAS和RNAPII组成的早期转录中间体的能力，并使用荧光探针研究个体因素如何影响NAS的开启/关闭末端。我们还将使用核磁共振表征TFIIF-亚基与NAS和TFIIB的相互作用，这将为为什么该亚单位中的某些突变促进转录缺陷提供结构洞察。我们强大的初步结果，对完整的核酸支架的独特理解，以及与多蛋白质复合体的合作记录，使我们处于从事这一研究的独特地位。一旦实现这些目标，将加深我们对一般转录因子与RNAPII协同作用启动转录的复杂机制的理解，并为研究与疾病有关的新型多蛋白复合体提供蓝图。

 DESCRIPTION (provided by applicant): DNA-directed RNA Polymerase II (RNAPII) is highly conserved among eukaryotic organisms and plays a fundamental role in cellular life, specifically gene transcription. During transcription, RNAPII transcribes genes into messenger RNA, a process aided by the general transcription factors: TBP, TFIIA, TFIIE, TFIIH, TFIIB and TFIIF. Meaningful biochemical experiments have demonstrated that transcription initiation is a highly dynamic event, with the possibility of discrete stages where the general transcription factors recognize, melt and load a nucleic acid scaffold (NAS) inside RNAPII's active site. This process is universal, for all eukaryotic species, and is at the core of gene regulation; therefore, understanding its molecular details will provide essential clues that could potentially lead to pharmacological manipulation of gene expression. Now that present technology enables us to crystallize and solve high-resolution structures of large multicomponent complexes, we can begin to examine the transcriptional machinery at the atomic level and address this specific question: What are the roles of the general transcription factors, TFIIB and TFIIF, in DNA stabilization during transcription bubble loading and promoter escape? The intention of this proposal is to answer this question using novel methodologies in chemical cross-linking, X-ray crystallography, and NMR. In a two prone approach we will first build on preliminary data which has shown that it is possible to reconstitute and crystallize a stoichiometric RNAPII - TFIIF complex, as well as obtain anisotropic diffraction to 2.9 Å. To improve crystallization conditions we will implore a novel chemical-crosslinking methodology, which will help prevent complex dissociation during crystallography trials. Secondly, we will demonstrate the ability to reconstitute early transcribing intermediates comprised of TFIIB, TFIIF, the NAS, and RNAPII, and investigate how the individual factors affect the opening/closing ends of the NAS using fluorescent probes. We will also characterize the interactions of the TFIIF -subunit with the NAS and TFIIB using NMR, which will provide structural insight into why certain mutants within this subunit promote transcription defects. Our strong preliminary results, distinctive understanding of the complete nucleic acid scaffold, and track record working with multi-protein complexes, places us in a unique position to pursue this line of research. Once accomplished, these goals will enhance our understanding of the intricate mechanisms by which general transcription factors cooperatively interact with RNAPII to initiate transcription, and provide a blueprint for studying novel multi-protein complexes implicated in disease.