mRNA Capping Enzyme

描述（由申请人提供）：该项目的目标是了解RNA聚合酶II的c端结构域如何用于将转录与基因表达中的几个起始后步骤偶联。这些事件包括mRNA盖帽、剪接和聚腺苷化，以及转录延伸和终止的调节。目前的数据支持CTD磷酸化模式在转录的不同阶段发生变化的模型。每种磷酸化状态都可以被一组不同的ctd相互作用蛋白识别。这允许延长和mRNA加工因子的动态交换，每一个都在转录周期的适当时间招募。本项目的实验将测试该模型，并确定CTD，其各种激酶和磷酸酶，及其相关的延伸和mRNA加工因子之间的物理和功能关系。提出了五个具体目标。第一个目标将使用染色质免疫沉淀来调查所有已知的延伸和mRNA加工因子的交联模式。一旦知道了野生型的模式，实验将在不同的突变菌株中重复进行。待检测的突变体包括CTD激酶和磷酸酶，以及延伸和加工因子本身。模式的变化将表明与转录复合体的相互依赖关系。在第二个目标中，亲和层析将用于分离与特定磷酸化形式的CTD结合的蛋白质。鉴定的蛋白质将进一步表征其在基因表达中的作用。具体目标3将是开发体外系统来复制体内观察到的一些CTD修饰变化。这些体外系统将用于测试和扩展来自目标1和目标2的因子相互作用模型。具体目标4将是鉴定和表征一种假定的CTD丝氨酸5磷酸酶。具体目标5将是破译Bur1/Bur2激酶复合物在调节转录伸长过程中发生的事件中的作用。提出的实验将大大扩展我们对基因表达的各个步骤如何整合的理解。很明显，转录后起始事件在许多系统中受到基因活性调节的调节。清楚了解基因表达的基本机制将为未来的治疗提供基础，包括基因替代疗法和直接调节细胞和病毒基因表达。

DESCRIPTION (provided by applicant): The goal of this project is to understand how the C-terminal domain of RNA polymerase II is used to couple transcription with several post-initiation steps in gene expression. These events include mRNA capping, splicing, and polyadenylation, as well as regulation of transcription elongation and termination. Current data supports a model in which the pattern of CTD phosphorylation changes at different stages of transcription. Each phosphorylation state may be recognized by a distinct set of CTD-interacting proteins. This allows a dynamic exchange of elongation and mRNA processing factors, each one recruited at the appropriate time(s) of the transcription cycle. The experiments in this project will test this model and identify physical and functional relationships between the CTD, its various kinases and phosphatases, and its associated elongation and mRNA processing factors. Five specific aims are proposed. The first aim will use chromatin immunoprecipitation to survey the crosslinking patterns of all known elongation and mRNA processing factors. Once the wild-type patterns are known, the experiments will be repeated in various mutant strains. Mutants to be assayed include CTD kinases and phosphatases, as well as the elongation and processing factors themselves. Changes in patterns will suggest an interdependence for association with transcription complexes. In the second aim, affinity chromatography will used to isolate proteins that bind to specific phosphorylated forms of the CTD. Proteins identified will be further characterized as to their roles in gene expression. Specific Aim 3 will be to develop in vitro systems for reproducing some of the CTD modification changes observed in vivo. These in vitro systems will be used to test and extend the models of factor interactions derived from Aims 1 and 2. Specific Aim 4 will be to identify and characterize a putative CTD serine 5 phosphatase. Specific Aim 5 will be to decipher the role of the Bur1/Bur2 kinase complex in regulating events occurring during transcription elongation. The experiments proposed would significantly extend our understanding of how various steps in gene expression are integrated. It is clear that post-transcription initiation events are regulated in many systems for modulation of gene activity. A clear understanding of the fundamental mechanisms of gene expression will provide the groundwork for future therapies, including gene replacement therapies and direct modulation of cellular and viral gene expression.