Mechanism of RNA polymerase II elongation factors

项目摘要 基因调控是细胞中的一个基本过程，基因表达的变化与 人类的多种疾病状态。基因在多个水平上受到调控，并决定这些事件如何 是相互协调的，这对理解细胞生理学至关重要。这一过程中的中心酶是RNA。 聚合酶II(RNAPII)，合成mRNAs。RNAPII在以下方面遇到了许多类型的障碍 转录，这被称为延伸因子(EFS)的蛋白质复合体所克服。两个可成形的 RNAPII的障碍是核小体和DNA损伤，它们减缓或阻止RNAPII的完成 跨基因的mRNAs。本提案的目标是了解RNAPII如何应对这两个障碍 转到抄写。我们已经确定了两个保守的EFS，Spt4/5(后生动物中的DSIF)和CCR4-不起作用 在允许RNAPII应对这些障碍方面发挥了重要作用。我们已经开发了一种方法， 充分利用酵母菌的生物化学、强大的遗传学和分子生物学来表征其机制 延伸率控制。Spt4/5和CCR4-NOT在真核生物王国中是保守的；因此，酵母是一种 合适的模型来研究这两种EFS的功能。其中描述的研究的第一个目标是 建议研究Spt4/5复合体如何帮助RNAPII通过核小体，并确定 该功能所需的其大亚基Spt5的结构域。第二个目标是描述 CCR4-非复合体通过损伤的DNA模板调节RNAPII的转录。我们会 确定CCR4-Not是否直接修饰RNAPII、Rpb1的大亚基和/或作为支架招募 将RNAPII从DNA损伤部位移除的因素。第三，我们使用无偏质谱(MS) 基于培养中氨基酸稳定同位素标记的蛋白质组学筛选(SILAC)用于鉴定和验证 CCR4的新靶点--不参与控制转录延伸。这个项目的长期目标是 了解延伸因子如何帮助RNAPII转录基因并维持基因的完整性 真核细胞的基因组。

Project Summary Gene regulation is a fundamental process in cells and alterations in gene expression have been linked to numerous disease states in humans. Genes are regulated at multiple levels and determining how these events are coordinated is critical to understanding cellular physiology. The central enzyme in this process is RNA Polymerase II (RNAPII), which syntheses mRNAs. RNAPII encounters many types of barriers during transcription, which are overcome by protein complexes called elongation factors (EFs). Two formable roadblocks for RNAPII are the nucleosome and DNA damage, which slows or prevents the completion of mRNAs across genes. The goal of this proposal is to understand how RNAPII contends with these two barriers to transcription. We have identified two conserved EFs, Spt4/5 (DSIF in metazoans) and Ccr4-Not as playing an important role in allowing RNAPII to contend with these barriers. We have a developed approaches that fully exploit the biochemistry, powerful genetics and molecular biology of yeast to characterize the mechanism of elongation control. Spt4/5 and Ccr4-Not are conserved across the eukaryotic kingdom; thus, yeast is an appropriate model to study the function of these two EFs. The first goal of the studies described in this proposal is to investigate how the Spt4/5 complex aids RNAPII passage through the nucleosome and identify the domains of its large subunit, Spt5, required for this function. The second goal is to characterize the role of the Ccr4-Not complex in regulating the transcription of RNAPII through damaged DNA templates. We will determine if Ccr4-Not directly modifies the large subunit of RNAPII, Rpb1, and/or serves as a scaffold to recruit factors that remove RNAPII from sites of DNA damage. Third, we use an unbiased mass spectrometry (MS) proteomics screen based on stable isotope labeling by amino acids in culture (SILAC) to identify and verify novel targets of Ccr4-Not involved in controlling transcription elongation. The long term goal of this project is to understand how elongation factors assist RNAPII in transcribing genes and maintaining the integrity of the genome of eukaryotic cells.