Mechanism and Regulation of RNA Polymerase II Elongation

RNA聚合酶II延伸的机制和调控

• 批准号: 8086247
• 负责人: Craig Kaplan
• 金额: $ 26.16万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8086247
• 关键词: Affect; Amanitins; Catalysis; Cell physiology; Cells; Chromatin; Chromatin Structure; Code; DNA; DNA Polymerase II; Data; Defect; Detection; Development; Dissection; Elongation Factor; Eukaryota; Eukaryotic Cell; Exhibits; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genome; Goals; Growth; Guanosine Triphosphate; Health; Human; Human Development; In Vitro; Lead; Link; Maintenance; Mediating; Modeling; Mutation; Nature; Nucleotides; Organism; Outcome; Phase; Phenotype; Polymerase; Predisposition; Prevention; Process; Property; Proteins; Publishing; RNA; RNA Polymerase II; RNA Processing; Regulation; Regulator Genes; Research; Resolution; Role; Running; Saccharomyces cerevisiae; Series; Site; Small Nuclear RNA; Specific qualifier value; Stimulus; System; Testing; Transcript; Transcription Initiation; Transcription Initiation Site; Work; base; chromatin modification; chromatin remodeling; disorder prevention; flexibility; genome-wide; in vivo; inhibitor/antagonist; mutant; novel; promoter; protein expression; research study; response

DESCRIPTION (provided by applicant): PROJECT SUMMARY Regulation of elongation by RNA Polymerase II (Pol II) is less well understood than initiation and has wide- ranging importance for gene expression and chromatin structure. Pol II elongation in eukaryotes has several main functions. Of primary importance, Pol II is responsible for expression of protein-coding gene and certain essential snRNA genes. Pol II transcription, including the elongation phase, has wide-ranging impacts on chromatin structure and composition due to cotranscriptional chromatin remodeling and modification. A low level of transcription is pervasive throughout eukaryotic genomes, revealing new challenges to the regulation and control of proper gene expression. A large number of factors have been linked to Pol II transcription during the elongation phase, but their roles in the maintenance and control of Pol II elongation are relatively unknown. The proposed research will identify the mechanisms and regulation of Pol II elongation in vivo using the structurally, genetically and molecularly amenable Saccharomyces cerevisiae. The research builds on the previous identification of a critical role for the highly conserved, conformationally flexible trigger loop (TL) of the Pol II large subunit, Rpb1, in substrate selection, control of elongation rate and as the direct target for the Pol II-elongation specific inhibitor, 1-amanitin. We have generated a series of Pol II mutants with alterations in just a few trigger loop residues that have differences in elongation rate over two orders of magnitude in vitro. In the current work, these mutants with a range of Pol II rates will be used to dissect mechanisms of Pol II elongation in vivo using a multipronged approach. First, it will be determined how mutations that alter Pol II elongation rate in vitro affect Pol II processivity and elongation rate in vivo using chromatin IP, detection of nascent RNA and transcription run on. Second, the consequences of alteration of Pol II activity by substrate limitation in vivo will be determined using a novel genetic system. This system will allow testing of a long standing but unproven phenotype that has been used for the putative identification of elongation factors. Third, a specific model will be tested for Pol II elongation contribution to initiation through the efficiency of promoter escape, wherein transcription begins but may be unsuccessful in reaching a mature elongation phase. This model has broad implications for how elongation factors might control transcription, and how promiscuous initiation may be normally limited. Finally we will determine at nucleotide resolution effects on Pol II pausing, distribution, transcription start site selection and transcriptome content in response to alteration of Pol II TL function. These experiments will reveal the nature of elongation defects in vivo, and how alteration of elongation contributes to gene expression. This work will establish new paradigms for the dissection of Pol II elongation factor function. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE This project aims to uncover basic mechanisms for gene expression in a model eukaryotic organism, Baker's yeast. The proteins responsible for control of gene expression in Baker's yeast are highly related to proteins with the same functions in human cells. Precise control of gene expression is required for proper human development and prevention of disease. Our studies will directly impact the understanding of gene expression in humans, and thus are relevant to human health.!

描述（由申请人提供）： RNA聚合酶II（Pol II）对延伸的调控不如起始那么好理解，对基因表达和染色质结构具有广泛的重要性。真核生物中的Pol II延伸具有几个主要功能。最重要的是，Pol II负责蛋白质编码基因和某些必需的snRNA基因的表达。Pol II转录，包括延伸阶段，由于共转录染色质重塑和修饰，对染色质结构和组成具有广泛的影响。低水平的转录在整个真核基因组中普遍存在，揭示了对适当基因表达的调控的新挑战。大量的因素已被链接到Pol II转录在延伸阶段，但它们的作用，在维护和控制Pol II延伸是相对未知的。拟议的研究将确定的机制和调节Pol II延长在体内使用的结构，遗传和分子适合酿酒酵母。该研究建立在先前鉴定的高度保守的，构象灵活的触发环（TL）的Pol II大亚基，RPB 1，在底物选择，控制延伸率和作为直接目标的Pol II延伸特异性抑制剂，1-鹅膏蕈碱的关键作用的基础上。我们已经产生了一系列Pol II突变体，其仅在几个触发环残基中具有改变，所述改变在体外具有超过两个数量级的延伸率差异。在目前的工作中，这些突变体与一系列的Pol II率将被用来解剖Pol II在体内使用多管齐下的方法伸长的机制。首先，它将确定如何在体外改变Pol II的延伸率的突变影响Pol II的持续合成能力和延伸率在体内使用染色质IP，检测新生RNA和transcriptionrunon. Second，改变Pol II的活动在体内底物限制的后果将使用一种新的遗传系统确定。该系统将允许测试长期存在但未经证实的表型，该表型已用于延伸因子的推定鉴定。第三，将测试特定模型的Pol II延伸通过启动子逃逸效率对起始的贡献，其中转录开始但可能不成功地达到成熟延伸期。这个模型对延伸因子如何控制转录，以及混杂起始通常如何受到限制具有广泛的意义。最后，我们将确定核苷酸分辨率对Pol II暂停，分布，转录起始位点选择和转录组含量的影响，以响应Pol II TL功能的改变。这些实验将揭示体内伸长缺陷的性质，以及伸长的改变如何有助于基因表达。这项工作将为Pol II延伸因子功能的研究建立新的范式。 公共卫生相关性： 本项目旨在揭示真核生物模型--面包酵母中基因表达的基本机制。在面包酵母中负责控制基因表达的蛋白质与人类细胞中具有相同功能的蛋白质高度相关。基因表达的精确控制是人类正常发育和疾病预防所必需的。我们的研究将直接影响对人类基因表达的理解，因此与人类健康有关。