Structural Mechanism of RNA Polymerase II Early Elongation

RNA聚合酶II早期延伸的结构机制

• 批准号: 8656136
• 负责人: JIANHUA FU
• 金额: $ 29万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8656136
• 关键词: 3-Dimensional; Affect; Affinity; Belief; Binding; Biochemical; Biological Assay; C-terminal; Cell Cycle Progression; Cell Differentiation process; Cell Nucleus; Cell physiology; Cells; Characteristics; Complex; Coupled; Coupling; Crystallization; Crystallography; DNA; DNA Polymerase I; DNA Polymerase III; Data; Development; Disease; Elongation Factor; Enzymes; Eukaryota; Gel Chromatography; Gene Expression; Genes; Genetic; Genetic Programming; Genetic Transcription; Goals; Heart; Hormones; Individual; Left; Link; Mediating; Molecular; Multienzyme Complexes; Normal Cell; Nuclear; Oncogene Proteins; Pathway interactions; Phase; Phosphorylation; Phosphotransferases; Polymerase; Positive Transcriptional Elongation Factor B; Preparation; Process; Property; Proteins; Protocols documentation; RNA; RNA Caps; RNA Polymerase II; RNA Processing; Reading; Recruitment Activity; Regulation; Repression; Research; Robotics; Signal Transduction; Staging; Stimulus; Structure; Surface; System; Techniques; Terminal Repeat Sequences; Testing; Trans-Activators; Transcription Process; Transcriptional Regulation; Work; Yeasts; base; cancer cell; chromatin modification; detection of nutrient; histone modification; in vivo; negative elongation factor; programs; promoter; reconstitution; research study; response; scale up; tool; tumorigenesis

DESCRIPTION (provided by applicant): We aim to understand the physical basis for the regulation of RNA polymerase II (Pol II) machinery and the crosstalk between the transcription and RNA processing pathways. The machinery is exceedingly large, consisting of Pol II at its heart and layers of trans-acting regulatory factors that modulate the Pol II activities. The advent of the crystal structure of Pol II core enzyme (513 kDa) has made it feasible to unravel the apparatus in more elaborative fashions. We have developed a structural paradigm in which functional Pol II complexes containing trans-acting regulators can be determined and mechanistic hypotheses emerging from the structures tested biochemically and genetically. Transcription is the first step in gene expression. Signals from many cellular processes such as nutrient sensing, catabolite repression, hormone stimulation, cell differentiation and responses to environmental stimuli ultimately reach the level of transcription to exert their effects. Aberration of transcriptional control is one of the mechanisms underlying tumorigenesis. The main characteristic of cancer cells is that they are highly proliferative due to their abilities to evade the programmed lifetime limit on normal cells. This means disruption of normal genetic programs, or more precisely, malfunction in the expression of genetic information stored in the DNA. As such, oncogene products are frequently found to be key players in the control networks of signal transduction, cell cycle progression and transcription regulation. The early elongation process of Pol II has been recognized as a major rate-limiting step in transcription regulation. The process is closely linked with cotranscriptional RNA processing, histone modification, and RNA export by mechanisms that are not yet clear. We propose to elucidate structures of Pol II early-elongation complexes to help understand the molecular underpinning of the central regulatory transition that occurs during the early phase of transcription. Specific aim are: (1) Determination and verification of the crystal structures of Spt5-Spt4-Pol II elongation complexes; (2) Defining the mechanism of cotranscriptional capping within the early elongation complex; and (3) Crystallization of Spt5/4-Pol IIO-CE, the complete early elongation/capping complex. Results will illuminate how interactions from trans-acting regulator (e.g., Spt5-Spt4) bound at the surface of Pol II control DNA/RNA transactions at the interior of the polymerase - whether there is a conformational relay system among the cis-acting Pol II subunits. The work will also determine the mechanistic parameters underlying cotranscriptional RNA 5' capping and assess the hypothesis of expanded Pol II spatial organization induced by the recruitment of processing factors. The proposed research will finally integrate the functional players of early elongation to allow for structural elucidation of the holo-complex that mediates the RNA processing-coupled transcription in the nucleus.

描述（由申请人提供）：我们旨在了解RNA聚合酶II（POL II）机械调节的物理基础以及转录和RNA处理途径之间的串扰。该机械非常大，由Pol II的心脏和调节Pol II活动的跨作用调节因素组成。出现 在Pol II核心酶（513 kDa）的晶体结构中，可以在更精明的时尚中揭开设备的可行性。我们已经开发了一个结构范式，其中可以确定含有跨作用调节剂的功能性POL II复合物，并从生化和遗传学上测试的结构中出现机械假设。 转录是基因表达的第一步。来自许多细胞过程的信号，例如营养感应，分解代谢物抑制，激素刺激，细胞分化以及对环境刺激的反应最终达到转录水平以发挥其作用。转录控制的像差是肿瘤发生的基础机制之一。癌细胞的主要特征是它们由于能力而高度增殖 逃避正常细胞的编程寿命限制。这意味着破坏正常遗传程序，或更确切地说是在DNA中存储的遗传信息表达中的故障。因此，在信号转导，细胞周期进程和转录调控的控制网络中，经常发现癌基产品是关键参与者。 POL II的早期伸长过程已被认为是转录调控的主要限制步骤。该过程与尚不清楚的机制密切相关的RNA处理，组蛋白修饰和RNA导出。 我们建议阐明Pol II早期延长复合物的结构，以帮助理解转录早期阶段发生的中央调节过渡的分子基础。具体目的是：（1）确定和验证SPT5-SPT4-POL II延伸复合物的晶体结构； （2）定义早期伸长络合物中共交集封盖的机制； （3）SPT5/4-POL IIO-CE的结晶，完整的早期伸长/封端复合物。 结果将阐明在聚合酶内部的Pol II控制DNA/RNA交易表面结合的反式调节剂（例如SPT5-SPT4）的相互作用 - 是否存在构象继电器系统之间的构型继电器系统。这项工作还将确定共转录RNA 5'限额的机械参数，并评估由募集处理因子引起的扩展的Pol II空间组织的假设。拟议的研究最终将整合早期伸长的功能参与者，以允许阐明整体复合物，从而介导核中RNA加工耦合的转录。