mRNA Capping Enzyme

mRNA加帽酶

• 批准号: 9816287
• 负责人: Stephen Buratowski
• 金额: $ 55.94万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9816287
• 关键词: Affect; Antibodies; Binding; Biological Assay; C-terminal; Cell Differentiation process; Cells; Characteristics; Chromatin; Chromatography; Collaborations; Color; Complement; Complex; Computer Analysis; Coupled; Coupling; Defect; Development; Disease; Distal; Dyes; Elongation Factor; Engineering; Enzymes; Event; Foundations; Funding; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Genomics; Goals; HIV; Immobilization; In Vitro; Individual; Kinetics; Knowledge; Label; Location; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Medical; Messenger RNA; Methods; Microscopy; Modeling; Modification; Monitor; Monoclonal Antibodies; Nature; Nuclear Extract; Pattern; Peptides; Peptidylprolyl Isomerase; Pharmaceutical Preparations; Phospho-Specific Antibodies; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Pluripotent Stem Cells; Population; Process; Productivity; Protein Binding Domain; Proteins; RNA Polymerase II; RNA Processing; RNA analysis; Resolution; Series; Specificity; Structure; System; Techniques; Thinking; Time; Total Internal Reflection Fluorescent; Transcript; Travel; Untranslated RNA; Update; Viral; Work; Yeasts; cell type; chromatin immunoprecipitation; experimental study; improved; in vivo; insight; mRNA guanylyltransferase; millisecond; promoter; response; single molecule; stoichiometry; tat Protein; temporal measurement; therapy design; transcription factor

Project Summary. The long-term goal of this project is to understand how transcription by RNA polymerase II (RNApII) is coupled to RNA processing and termination. This project previously developed a model in which the C-terminal domain (CTD) of the RNApII subunit Rpb1 displays characteristic phosphorylation patterns at different stages of the transcription cycle to promote binding of the appropriate factors for co-transcriptional RNA processing. The fundamental knowledge generated by this project provides significant insight into how the CTD phosphorylation cycle affects medically important processes such as the stimulation of HIV transcription by the viral Tat protein and "pausing" of RNApII at developmentally regulated genes. This project is necessary to better understand both the enzymes that mediate the changes in CTD phosphorylation (kinases, phosphatases, etc.) as well as the proteins that recognize these patterns. In the next funding period, three specific aims will be pursued, with a focus on measuring dynamics of events during transcription. The first aim continues our work directly analyzing CTD phosphorylation sites by mass spectrometry, avoiding pitfalls associated with the monoclonal antibodies used in most CTD studies. A modified CTD (msCTD) was engineered to discriminate individual proximal and distal repeats by mass. Recent improvements to peptide chromatography and computational analysis will improve our accuracy and throughput. In vivo CTD phosphorylations will be analyzed in cells where various CTD modifying enzymes are rapidly inactivated or depleted. Analysis of RNApII associated with specific CTD binding proteins or CTD antibodies will also be performed to determine their binding specificities. The second aim exploits our recent discovery that CTD cycle progresses as a function of time, rather than elongation distance. This realization allowed us to create an in vitro system that reproduces the progression of CTD phosphorylations and associated factors on elongation complexes, facilitating real time analysis of dynamics. This immobilized template system will be combined with the msCTD from Aim 1 to produce a high-resolution time course of CTD phosphorylation, probing the contributions of individual kinases and phosphatases. The third aim adapts the immobilized template assay to single-molecule microscopy. We can visualize individual transcription events with up to three fluorescently-labeled transcription factors, providing second to millisecond time resolution of binding kinetics. We will measure the stoichiometries, order of binding, and cooperative interactions between multiple CTD binding and elongation complex factors. Altogether, this project will make a unique contribution to our understanding of gene expression by providing a time-resolved picture of events that complements inherently static techniques such as structural studies or genomics.

项目摘要。该项目的长期目标是了解 RNA 的转录过程 聚合酶 II (RNApII) 与 RNA 加工和终止偶联。该项目之前开发了一个 模型中，RNApII 亚基 Rpb1 的 C 端结构域 (CTD) 显示出特征 转录周期不同阶段的磷酸化模式，以促进适当的结合 共转录 RNA 加工的因素。该项目产生的基础知识提供了 深入了解 CTD 磷酸化循环如何影响医学上重要的过程，例如 病毒 Tat 蛋白刺激 HIV 转录并在发育调节过程中“暂停”RNApII 基因。该项目对于更好地了解介导 CTD 变化的两种酶是必要的 磷酸化（激酶、磷酸酶等）以及识别这些模式的蛋白质。 在下一个资助期间，将追求三个具体目标，重点是衡量 转录期间的事件。第一个目标是继续我们直接分析 CTD 磷酸化位点的工作 质谱分析法，避免了大多数 CTD 研究中使用的单克隆抗体的缺陷。一个 改良 CTD (msCTD) 旨在通过质量区分个体近端和远端重复。 最近对肽色谱和计算分析的改进将提高我们的准确性和 吞吐量。将在存在各种 CTD 修饰酶的细胞中分析体内 CTD 磷酸化 迅速失活或耗尽。与特定 CTD 结合蛋白或 CTD 相关的 RNApII 分析 还将对抗体进行测定以确定其结合特异性。第二个目标利用了我们最近的 发现 CTD 周期的进展是时间的函数，而不是伸长距离的函数。这种认识 使我们能够创建一个体外系统来重现 CTD 磷酸化的进展，并且 伸长率复合物的相关因素，有助于动态的实时分析。这不动了 模板系统将与目标 1 中的 msCTD 相结合，生成 CTD 的高分辨率时间进程 磷酸化，探索单个激酶和磷酸酶的贡献。第三个目标适应 固定化模板测定到单分子显微镜。我们可以可视化各个转录事件 具有多达三个荧光标记的转录因子，提供秒到毫秒的时间分辨率 结合动力学。我们将测量化学计量、结合顺序以及之间的合作相互作用 多种 CTD 结合和延伸复杂因素。总而言之，该项目将为 通过提供事件的时间分辨图像来补充我们对基因表达的理解 本质上是静态的技术，例如结构研究或基因组学。