Mechanism of RNA polymerase II elongation factors

RNA聚合酶II延伸因子的机制

基本信息

批准号：
9353415
负责人：
JOSEPH C REESE
金额：
$ 39.78万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-01-01 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9353415
关键词：
Address Affect Amino Acids Binding Biochemical Biochemistry Biological Assay Cardiovascular system Cell Proliferation Cell physiology Cells Chromatin Complex DNA DNA Binding DNA Damage Data Defect Development Dimerization Disease Elongation Factor Employee Strikes Enzymes Eukaryotic Cell Event Excision Exercise Failure Gene Expression Gene Expression Regulation Genes Genetic Genetic Transcription Genome Genome Mappings Goals Health Human Impairment Jaw Kinetics Knowledge Link Macromolecular Complexes Mass Spectrum Analysis Messenger RNA Modeling Modification Molecular Biology Movement Mutation Nature Nucleic Acids Nucleosomes Pathway interactions Phenotype Physiological Play Polymerase Positioning Attribute Process Production Proteomics RNA Polymerase II Recombinants Recruitment Activity Resistance Role Site Stable Isotope Labeling Structural Models Surface Syndrome Testing Time Transcription Elongation Ubiquitination Work Yeasts base chromatin modification complement system dimer genome integrity human disease novel prevent protein complex reconstitution scaffold tool transcription factor ubiquitin ligase ubiquitin-protein ligase

项目摘要

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.

项目摘要基因调节是细胞中的一个基本过程，基因表达的改变已与人类中的许多疾病状态。基因在多个层面上受到调节，并确定这些事件如何协调对于理解细胞生理学至关重要。在此过程中的中心酶是RNA 聚合酶II（RNAPII），合成mRNA。 RNAPII在转录，被称为伸长因子（EFS）的蛋白质复合物克服。两个形成 RNAPII的障碍是核小体和DNA损伤，它会减慢或阻止完成跨基因的mRNA。该提议的目的是了解RNAPII如何与这两个障碍抗衡转录。我们已经确定了两个保守的EFS，SPT4/5（Metazoans中的DSIF）和CCR4-不播放允许RNAPII与这些障碍抗衡的重要作用。我们有一种发达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来表征机制伸长控制。 SPT4/5和CCR4不在整个真核王国中保存；因此，酵母是适当研究这两个EF的功能。其中描述的研究的第一个目标建议是研究SPT4/5复合物如何通过核小体辅助RNAPII通过此功能所需的大型亚基SPT5的域。第二个目标是表征 CCR4不合适地通过受损的DNA模板调节RNAPII的转录。我们将确定CCR4是否直接修改了RNAPII，RPB1和/或用作招募的大型亚基从DNA损伤部位去除RNAPII的因素。第三，我们使用公正的质谱法（MS）基于氨基酸（SILAC）中稳定同位素标记的蛋白质组学筛选以识别和验证 CCR4的新靶标不参与控制转录伸长率。该项目的长期目标是了解伸长因素如何帮助RNAPII转录基因并保持完整性真核细胞的基因组。