mRNA Capping Enzyme

mRNA加帽酶

• 批准号: 7071045
• 负责人: Stephen Buratowski
• 金额: $ 35.1万
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7071045
• 关键词: DNA directed RNA polymerase; SDS polyacrylamide gel electrophoresis; affinity chromatography; crosslink; gene expression; genetic transcription; immunoprecipitation; matrix assisted laser desorption ionization; messenger RNA; mutant; phosphorylation; posttranscriptional RNA processing; protein protein interaction; protein structure function; protein tyrosine phosphatase; transcription factor

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.

描述（由申请人提供）：该项目的目标是了解 RNA 聚合酶 II 的 C 末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耦合。这些事件包括 mRNA 加帽、剪接和聚腺苷酸化，以及转录延伸和终止的调节。目前的数据支持一个模型，其中 CTD 磷酸化模式在转录的不同阶段发生变化。每个磷酸化状态可以被一组不同的 CTD 相互作用蛋白识别。这允许延伸和 mRNA 加工因子的动态交换，每一个因子都在转录周期的适当时间招募。该项目中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。第一个目标是使用染色质免疫沉淀来调查所有已知延伸和 mRNA 加工因子的交联模式。一旦野生型模式已知，实验将在各种突变株中重复进行。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。模式的变化表明与转录复合物的关联存在相互依赖性。第二个目标是使用亲和层析分离与特定磷酸化形式的 CTD 结合的蛋白质。鉴定出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 是开发体外系统，用于重现体内观察到的一些 CTD 修饰变化。这些体外系统将用于测试和扩展源自目标 1 和 2 的因子相互作用模型。具体目标 4 将是鉴定和表征假定的 CTD 丝氨酸 5 磷酸酶。具体目标 5 是破译 Bur1/Bur2 激酶复合物在调节转录延伸过程中发生的事件中的作用。所提出的实验将显着扩展我们对基因表达各个步骤如何整合的理解。很明显，转录后起始事件在许多基因活性调节系统中受到调节。对基因表达基本机制的清晰了解将为未来的治疗奠定基础，包括基因替代疗法以及细胞和病毒基因表达的直接调节。