Supplemental request for MAX-TL Ultracentrifuge and rotor

MAX-TL 超速离心机和转子的补充请求

• 批准号: 10386257
• 负责人: Dylan J Taatjes
• 金额: $ 4.92万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10386257
• 关键词: Address; Area; Biochemical; Biological Assay; Biology; Cells; Chemicals; Code; Communication; Complex; DNA Binding; DNA Polymerase II; DNA-Directed RNA Polymerase; Development; Disease; Enhancers; Enzymes; Gene Expression; Genes; Genetic Transcription; Goals; Growth and Development function; Heart Diseases; Human; Human Genome; In Vitro; Liquid substance; Location; Malignant Neoplasms; Mediator of activation protein; Methods; Modeling; Molecular; Nucleic Acids; Oncogenic; Phase; Phosphotransferases; Population; Positive Transcriptional Elongation Factor B; Proteins; RNA; RNA Processing; Regulation; Research; Signal Transduction; System; Testing; Transcription Elongation; Transcription Factor TFIIA; Transcription Initiation; Transcriptional Regulation; Untranslated RNA; Work; base; experimental study; follow-up; genome editing; genome-wide; in vitro Assay; insight; nervous system disorder; novel strategies; programs; promoter; public health relevance; reconstitution; success; therapeutic target; transcription factor; transcription factor TFIIH; transcriptomics

SUMMARY/ABSTRACT (for R35 GM139550) Our research program combines detailed biochemical reconstitution experiments with powerful cell-based assays, with a goal of gaining fundamental mechanistic insights about RNA polymerase II (pol II) function and its regulation. The 12-subunit human pol II enzyme transcribes all protein-coding and many non-coding RNAs in the human genome. Pol II transcription initiation is regulated by the 4.0 MDa Pre-Initiation Complex (PIC), which contains TFIIA, IIB, IID, IIE, IIF, IIH, pol II, and Mediator. Together with sequence-specific, DNA-binding transcription factors (TFs), the PIC helps direct the timing, location, and direction of pol II transcription, genome- wide. How TFs and the PIC work together during different stages of pol II transcription (e.g. initiation, pausing, elongation) remain incompletely understood; moreover, new insights over the past 5+ years have transformed our understanding of transcription. For instance, enhancer RNA (eRNA) transcription and enhancer-promoter communication appear to drive lineage- or signal-specific (or oncogenic) gene expression programs, and liquid phase separated molecular condensates correlate with pol II activity in cells. Although new mechanistic models have emerged, such models cannot be reliably tested using only cell-based methods, in part because of the enormous complexity of cellular systems. For instance, the identity and concentration of the proteins, nucleic acids, and biochemicals that are present at any given gene in a population of cells cannot possibly be defined. In the next 5 years, we propose to leverage our unique expertise in biochemical reconstitution with cutting- edge cellular methods to address the following high-impact areas: 1) Liquid phase-separated molecular condensates and pol II function. We seek to define how (or whether) molecular condensates regulate transcription, including whether distinct compositions help control different stages of pol II transcription (e.g. initiation vs. elongation). 2) Regulation of pol II initiation, pausing, and elongation by the transcriptional kinases CDK7 (TFIIH subunit), CDK8 (Mediator-associated kinase), and CDK9 (P-TEFb kinase). We will assess what each kinase, alone and in combination with the others, contributes to the regulation of pol II activity. This will include potential “downstream” impacts on elongation rates or RNA processing. 3) Enhancer RNA (eRNA) transcription and super-enhancer function. We will dissect the mechanistic requirements for bidirectional eRNA transcription, to determine whether they are distinct from typical protein-coding genes. Furthermore, we seek to reconstitute super-enhancer function in vitro, which would serve as a framework for understanding the “rules” by which super-enhancers drive high-level transcription in human cells. (Although this aspect is ambitious, we note our recent success with reconstitution of pol II promoter-proximal pausing, which the field long considered difficult if not impossible.) Finally, we emphasize that an equally important aspect of our research plan is to rigorously test the models that emerge from our detailed and systematic in vitro assays through targeted, follow-up cell- based assays, which will implement genome-editing, chemical biology, transcriptomics, and other approaches.

摘要/摘要（R35 GM139550） 我们的研究计划将详细的生化重构实验与强大的基于细胞的实验相结合 测定，目的是获得有关RNA聚合酶II（POL II）功能的基本机理见解和 它的监管。 12个亚基人Pol II酶转录所有蛋白质编码和许多非编码RNA POL II转录启动受4.0 MDA发射络合物（PIC）的调节， 其中包含TFIIA，IIB，IID，IIE，IIF，IIH，POL II和调解人。以及序列特异性的DNA结合 转录因子（TFS），PIC有助于指导POL II转录的时间，位置和方向，基因组 - 宽的。在Pol II转录的不同阶段，TF和PIC如何一起工作（例如，暂停，暂停， 伸长）仍然不完全理解；此外，过去5年以上的新见解发生了变化 我们对转录的理解。例如，增强剂RNA（ERNA）转录和增强器启动器 沟通外观以驱动谱系 - 特异性或信号特异性（或致癌）基因表达程序和液体 相分离的分子冷凝物与细胞中的POL II活性相关。虽然新机械模型 出现了，不能仅使用基于细胞的方法可靠地测试此类模型，部分原因是 细胞系统的巨大复杂性。例如，蛋白质的身份和浓度，核 在任何给定基因中存在的酸和生化物质都无法定义。 在接下来的5年中，我们建议利用我们在生化重构方面的独特专业知识， 边缘细胞方法以解决以下高影响区域：1）液相分离的分子 冷凝水和POL II功能。我们寻求定义分子冷凝物如何调节（或）如何调节 转录，包括不同的组成是否有助于控制Pol II转录的不同阶段（例如 启动与伸长率）。 2）调节Pol II计划，暂停和伸长的转录激酶伸长率 CDK7（TFIIH亚基），CDK8（介体相关激酶）和CDK9（P-TEFB激酶）。我们将评估什么 每种激酶单独并与其他激酶结合在一起，都有助于调节Pol II活性。这会 包括潜在的“下游”对伸长率或RNA处理的影响。 3）增强子RNA（ERNA） 转录和超增强功能。我们将剖析双向ERNA的机械要求 转录，以确定它们是否与典型的蛋白质编码基因不同。此外，我们寻求 重建超级能量的体外功能，该功能将作为理解“规则”的框架 超级增强剂驱动人类细胞中的高级转录。 （尽管这方面是雄心勃勃的，但我们注意到 我们最近在重组Pol II启动子暂停的成功中取得了成功，该领域长期认为这很困难 最后，我们强调，我们的研究计划同样重要的方面是严格 测试通过针对性的随访细胞从我们详细且系统的体外测定中出现的模型 基于基于基因组编辑，化学生物学，转录组学和其他方法的测定法。