mRNA Capping Enzyme

项目摘要。这个项目的长期目标是了解rna如何转录。 聚合酶II(RNAPII)与RNA的加工和终止有关。该项目之前开发了一个 RNAPII亚基Rpb1的C-末端结构域(CTD)显示特征的模型 转录周期不同阶段的磷酸化模式促进适当的结合 共转录RNA加工的因素。该项目产生的基础知识提供了 对CTD磷酸化循环如何影响重要的医学过程的重要见解，如 病毒Tat蛋白对HIV转录的刺激和RNAPII在发育调节中的“暂停” 基因。这个项目对于更好地了解介导CTD变化的酶是必要的 磷酸化(激酶、磷酸酶等)以及识别这些模式的蛋白质。 在下一个供资期间，将追求三个具体目标，重点是衡量 转录过程中的事件。第一个目标是继续我们的工作，通过直接分析CTD磷酸化位点 质谱仪，避免了与大多数CTD研究中使用的单抗相关的陷阱。一个 改良的CTD(MsCTD)被设计用来根据质量区分单个近端和远端重复序列。 多肽层析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修饰酶所在的细胞中进行分析 迅速失活的或耗尽的。特异性CTD结合蛋白或CTD相关RNAPII的分析 还将进行抗体检测，以确定它们的结合特异性。第二个目标是利用我们最近的 发现CTD循环是时间的函数，而不是延伸距离的函数。这一认识 使我们能够创建一个体外系统，重现CTD磷酸化和 对延伸率复合体的相关因素进行分析，便于实时动态分析。这是固定的 模板系统将与AIM 1的msCTD相结合，以产生高分辨率的CTD时间历程 磷酸化，探索单独的激酶和磷酸酶的作用。第三个目标适应了 固定化模板分析到单分子显微镜。我们可以将单个转录事件可视化 最多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、结合顺序和相互作用 多个CTD结合和延伸的复杂因素。总而言之，这个项目将为 我们通过提供时间分辨的事件图像来理解基因表达，这补充了 固有的静态技术，如结构研究或基因组学。

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.