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，基因表达的改变与细胞的生长和分化有关。 人类的许多疾病状态。基因在多个层面上受到调控， 是理解细胞生理学的关键这个过程的中心酶是RNA 聚合酶II（RNAPII），其合成mRNA。RNAPII遇到了许多类型的障碍， 转录，这是克服了蛋白质复合物称为延伸因子（EF）。两个可成形 RNAPII的障碍是核小体和DNA损伤，这会减缓或阻止RNAPII的完成。 跨基因的mRNA。本提案的目标是了解RNAPII如何应对这两个障碍 到转录。我们已经确定了两个保守的EF，Spt 4/5（后生动物中的DSIF）和Ccr 4-Not作为玩耍因子。 在使RNAPII能够应对这些障碍方面发挥了重要作用。我们有一套成熟的方法， 充分利用酵母的生物化学、强大的遗传学和分子生物学特性， 伸长控制。Spt 4/5和Ccr 4-Not在真核生物界中是保守的;因此，酵母是一种 合适的模型来研究这两种EFs的功能。本文所述研究的第一个目标是 我们的建议是研究Spt 4/5复合物如何帮助RNAPII通过核小体，并鉴定 它的大亚基Spt 5的结构域，这一功能所需。第二个目标是描述 Ccr 4-Not复合物通过受损的DNA模板调节RNAPII的转录。我们将 确定Ccr 4-Not是否直接修饰RNAPII的大亚基，Rpb 1，和/或作为支架募集 从DNA损伤位点移除RNAPII的因子。第三，我们使用无偏质谱（MS） 蛋白质组学筛选的基础上稳定同位素标记的氨基酸在文化（SILAC），以确定和验证 Ccr 4的新靶点-不参与控制转录延伸。该项目的长期目标是 了解延伸因子如何协助RNAPII转录基因和维持细胞的完整性。 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.