mRNA Capping Enzyme

mRNA加帽酶

• 批准号: 8960066
• 负责人: Stephen Buratowski
• 金额: $ 54.24万
• 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 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8960066
• 关键词: Affect; Amino Acids; Amino Acyl-tRNA Synthetases; Antibodies; Binding; C-terminal; Cells; Characteristics; Chromatin; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; Consensus Sequence; Coupled; Coupling; DNA; DNA Damage; Defect; Development; Disease; Distal; Engineering; Enzymes; Event; Excision; FLP recombinase; Formaldehyde; Foundations; Funding; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Goals; HIV; Immunoblotting; In Vitro; Individual; Knowledge; Link; Location; Lysine; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Messenger RNA; Methods; Modeling; Modification; Monoclonal Antibodies; Mutate; Mutation; Nature; Pattern; Peptides; Pharmaceutical Preparations; Phenylalanine; Phospho-Specific Antibodies; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Pluripotent Stem Cells; Polymerase; Population; Process; Productivity; Protein Binding Domain; Proteins; RNA Polymerase II; RNA Processing; Reaction; Series; Site; Specificity; Staging; System; Tandem Repeat Sequences; Techniques; Testing; Time; Topoisomerase; Transcript; Transcription Elongation; Transcription-Coupled Repair; Travel; Trypsin; Ultraviolet Rays; Untranslated RNA; Update; Validation; Variant; Viral; Yeasts; arginyllysine; blocking factor; cell type; chromatin modification; cross reactivity; crosslink; embryonic stem cell; factor C; in vivo; insight; mRNA guanylyltransferase; mutant; novel strategies; promoter; protein crosslink; protein degradation; public health relevance; recombinase; repaired; research study; tandem mass spectrometry; tat Protein; termination factor; therapy design; transcription termination

 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 next funding period, three specific aims will be being pursued. The first is to create a system for directly analyzing CTD phosphorylation sites by mass spectrometry, avoiding all the pitfalls and caveats associated with the monoclonal antibodies that have been used to date. A modified CTD will be engineered that adds several basic residues and non-consensus repeats so that mass spectrometry can be used to distinguish individual 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. Analysis of RNApII associated with specific CTD binding proteins (both in vivo and in vitro) will also be performed to determine their CTD binding specificities. In the second aim, 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. In the third aim, we will analyze RNApII elongation complexes that are blocked at specific locations in genes. Elongation will be blocked by either a mutant recombinase that covalently links to nicked DNA but cannot excise, or by non- cleaving Cas9/CRISPR complexes. Stalled complexes will be characterized by ChIP for CTD modifications and associated proteins. A time course will be used to analyze the clearance of these blocked complexes, as well as whether their removal is stalled in cells mutated in various transcription termination or repair coupling factors. Together, these experiments will greatly advance our understanding of the events that occur during transcription elongation, both during unimpeded elongation and upon blockage by DNA damage.

 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) 的转录如何与 RNA 加工和终止耦合。该项目的早期资助期产生了一个模型，其中 RNApII 亚基 Rpb1 的 C 末端结构域 (CTD) 在转录周期的不同阶段显示出特征性磷酸化模式，以促进共转录 RNA 加工的适当因子的结合。该项目产生的基础知识提供了关于 CTD 磷酸化循环如何影响医学上重要过程的重要见解，例如病毒 Tat 蛋白刺激 HIV 转录以及 RNApII 在胚胎干细胞中发育调控基因上的“暂停”。该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。 在下一个资助期内，将实现三个具体目标。第一个是创建一个通过质谱法直接分析 CTD 磷酸化位点的系统，避免与迄今为止使用的单克隆抗体相关的所有陷阱和警告。将设计改良的 CTD，添加几个基本残基和非共有重复序列，以便质谱法可用于区分各个近端和远端重复序列。将在野生型细胞和各种 CTD 修饰酶的突变体中分析这些 CTD 片段的体内磷酸化。还将对与特定 CTD 结合蛋白（体内和体外）相关的 RNApII 进行分析，以确定其 CTD 结合特异性。第二个目标是构建一系列在体内掺入非天然光反应性氨基酸的 CTD 突变体。相关蛋白的体内交联和分析将显示 CTD 相关因子是否优先与近端重复或远端重复相关。第三个目标是，我们将分析在基因特定位置被阻断的 RNApII 延伸复合物。与带切口的 DNA 共价连接但不能切除的突变重组酶或非切割 Cas9/CRISPR 复合物都会阻止延伸。停滞的复合物将通过 ChIP 进行 CTD 修饰和相关蛋白的表征。将使用时间过程来分析这些被阻断的复合物的清除，以及它们的清除在各种转录终止或修复偶联因子突变的细胞中是否停滞。总之，这些实验将极大地增进我们对转录延伸过程中发生的事件的理解，无论是在无阻碍的延伸过程中还是在 DNA 损伤阻断时。