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转录的方式 聚合酶II（RNAPII）与RNA处理和终止耦合。这个项目以前开发了 RNAPII亚基RPB1的C末端域（CTD）显示特征的模型 在转录周期的不同阶段的磷酸化模式，以促进适当的结合 共转录RNA处理的因素。该项目产生的基本知识提供了 关于CTD磷酸化周期如何影响医学上重要的过程，例如 在发育调节的 基因。该项目是必要的，以更好地了解介导CTD变化的酶 磷酸化（激酶，磷酸酶等）以及识别这些模式的蛋白质。 在下一个资金期间，将追求三个具体目标，重点是衡量动态 转录期间的事件。第一个目标继续我们的工作直接分析CTD磷酸化位点 质谱法避免了与大多数CTD研究中使用的单克隆抗体相关的陷阱。一个 修改后的CTD（MSCTD）经过设计，以区分质量的个体近端和远端重复序列。 肽色谱法和计算分析的最新改进将提高我们的准确性和 吞吐量。体内CTD磷酸化将在各种CTD修饰酶的细胞中进行分析 迅速灭活或耗尽。与特定CTD结合蛋白或CTD相关的RNAPII分析 还将执行抗体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循环是随时间而不是伸长距离的函数进行的。这个实现 允许我们创建一个体外系统，该系统重现CTD磷酸化和 相关因素伸长复合物，促进动力学的实时分析。这固定了 模板系统将与AIM 1的MSCTD结合使用，以产生CTD的高分辨率时间过程 磷酸化，探测单个激酶和磷酸酶的贡献。第三个目标适应 固定的模板测定到单分子显微镜。我们可以可视化单个转录事件 最多三个荧光标记的转录因子，仅次于毫秒的时间分辨率 约束动力学。我们将衡量石化的图表，结合顺序和合作相互作用 多个CTD结合和伸长复杂因子。总之，这个项目将为 我们通过提供补充事件的时间分辨图片来理解基因表达 固有的静态技术，例如结构研究或基因组学。