mRNA Capping Enzyme

mRNA加帽酶

基本信息

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
项目类别：
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

项目摘要

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。该项目的早期资助阶段产生了一个模型，其中 C 末端。 RNApII 亚基 Rpb1 的结构域 (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 处理的适当因子的结合。 CTD 磷酸化循环影响医学上的重要过程，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这些变化的两种酶是必要的。 CTD 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资助期内，将追求三个具体目标：第一个是创建一个系统。通过质谱法直接分析 CTD 磷酸化位点，避免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一致性重复序列，以便可以使用质谱法。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。还将进行与特定 CTD 结合蛋白（体内和体外）相关的 RNApII 以确定其 CTD 结合特异性。在第二个目标中，将构建一系列 CTD 突变体，其中包含非天然的光反应性氨基酸。体内交联和相关蛋白的分析将显示 CTD 相关因子是否优先与近端或远端重复序列相关。在第三个目标中，我们将分析在基因中特定位置被阻断的 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 复合物将阻断延伸。将通过 ChIP 来表征停滞的复合物，以进行 CTD 修饰和相关蛋白质。分析这些被阻断的复合物的清除，以及它们的清除是否在各种转录终止或修复偶联因子突变的细胞中停滞不前，这些实验将极大地增进我们对转录过程中发生的事件的理解。伸长，无论是在无阻碍伸长期间还是在 DNA 损伤阻断时。