mRNA Capping Enzyme

描述（由申请人提供）：该项目的长期目标是了解RNA聚合酶II （RNApII）的转录如何耦合到RNA加工和终止。该项目的早期资助期产生了一个模型，其中RNApII亚基Rpb1的c端结构域（CTD）在转录周期的不同阶段显示出特有的磷酸化模式，以促进与适当因子的结合，进行共转录RNA加工。该项目产生的基础知识为CTD磷酸化周期如何影响医学上重要的过程提供了重要的见解，例如通过病毒Tat蛋白刺激HIV转录和RNApII在胚胎干细胞中发育调节基因的“暂停”。这个项目对于更好地了解介导CTD磷酸化变化的酶（激酶、磷酸酶等）以及识别这些模式的蛋白质是必要的。在母赠款的当前供资期间，正在实现四个具体目标。首先是了解两种已知的终止途径（polyA和非polyA转录本）以及RNApII如何在它们之间进行选择。第二个目的是研究在许多RNApII启动子上由发散转录产生的短的不稳定转录物的生物发生。第三个目的是使用NET-Seq来探测CTD磷酸化在RNA延伸、暂停和终止中的作用。第四个目标利用我们的发现，Rpb1 CTD可以转移到不同的RNApII亚基上，询问不同的CTD修饰和功能是否需要在相同的CTD上“顺式”发生，或者可以在两个CTD之间分裂。该补充将增加一个新的特定目标，试图回答CTD重复序列在不同位置之间是否存在功能差异。在第一个子目标中，将设计一个CTD，添加几个赖氨酸和非一致重复序列，以便质谱可以用来区分近端重复序列和远端重复序列。这些CTD片段的体内磷酸化将在野生型细胞和各种CTD修饰酶的突变体中进行分析。在第二个子目标中，将构建一系列包含非天然光反应氨基酸的CTD突变体。体内交联和相关蛋白分析将显示ctd相关因子是否优先与近端或远端重复序列相关。总之，这些实验的结果将显示CTD磷酸化模式和相关蛋白在CTD的不同区域是不同的还是相同的。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand how transcription by RNA polymerase II (RNApII) is coupled to RNA processing and termination. Earlier funding periods of this project produced 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 the "pausing" of RNApII at developmentally regulated genes in embryonic stem cells. 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 current funding period of the parent grant, four specific aims are being pursued. The first is to understand the two known termination pathways (for polyA and non-polyA transcripts) and how RNApII chooses between them. A second aim studies the biogenesis of short unstable transcripts produced by divergent transcription at many RNApII promoters. The third aim uses NET-Seq to probe the role of CTD phosphorylation in RNA elongation, pausing, and termination. The fourth aim exploits our discovery that the Rpb1 CTD can be transferred onto a different RNApII subunit, asking whether the different CTD modifications and functions need to occur in "cis" on the same CTD or can be split between two CTDs. This supplement will add one new specific aim attempting to answer whether there are functional differences between CTD repeats at different positions. In the first subaim, a CTD will be engineered that adds several lysines and non-consensus repeats so that mass spectrometry can be used to distinguish proximal and distal repeats. The in vivo phosphorylations on these CTD fragments will be analyzed in wild-type cells and mutants of various CTD modifying enzymes. In the second subaim, a series of CTD mutants will be constructed that incorporate a non-native, photoreactive amino acid in vivo. In vivo crosslinking and analysis of associated proteins will show whether CTD-associated factors preferentially associate with proximal or distal repeats. Together, the results of these experiments will show whether the CTD phosphorylation patterns and associated proteins are distinct or identical in different regions of the CTD.