Mechanism and Regulation of RNA Polymerase II Elongation

RNA聚合酶II延伸的机制和调控

• 批准号: 8449311
• 负责人: Craig Kaplan
• 金额: $ 26.12万
• 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/8449311
• 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.

描述（由申请人提供）： 项目摘要 RNA 聚合酶 II (Pol II) 对延伸的调节不像起始那样被人们所了解，但它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 snRNA 基因的表达。由于共转录染色质重塑和修饰，Pol II 转录（包括延伸期）对染色质结构和组成具有广泛的影响。低水平转录在真核基因组中普遍存在，这给正确基因表达的调控带来了新的挑战。在延伸阶段，大量因子与 Pol II 转录有关，但它们在维持和控制 Pol II 延伸中的作用相对未知。拟议的研究将使用结构上、遗传上和分子上均可接受的酿酒酵母来确定体内 Pol II 延伸的机制和调节。该研究建立在先前对 Pol II 大亚基 Rpb1 的高度保守、构象灵活的触发环 (TL) 在底物选择、延伸率控制以及作为 Pol II 延伸特异性抑制剂 1-鹅膏蕈碱的直接靶点中的关键作用的认识的基础上。我们已经生成了一系列 Pol II 突变体，仅改变了几个触发环残基，这些突变体在体外的伸长率差异超过两个数量级。在目前的工作中，这些具有一系列 Pol II 速率的突变体将用于通过多管齐下的方法剖析体内 Pol II 伸长的机制。首先，使用染色质 IP、新生 RNA 检测和转录运行，确定体外改变 Pol II 延伸率的突变如何影响 Pol II 持续合成能力和体内延伸率。其次，通过体内底物限制改变 Pol II 活性的后果将使用新型遗传系统来确定。该系统将允许测试长期存在但未经证实的表型，该表型已用于推定鉴定延伸因子。第三，将通过启动子逃逸的效率测试特定模型的Pol II延伸对起始的贡献，其中转录开始但可能不成功到达成熟延伸阶段。该模型对于延伸因子如何控制转录以及通常如何限制混杂起始具有广泛的意义。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内容的影响，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影响基因表达。这项工作将为 Pol II 延伸因子函数的解析建立新的范例。