Kinetic mechanism of transcription on native minichromosome

天然微型染色体转录的动力学机制

• 批准号: 10418073
• 负责人: Carl Wu
• 金额: $ 57万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10418073
• 关键词: Address; Affinity; Architecture; Behavior; Binding; Biochemical; Biological; Biological Assay; Cell Extracts; Chromatin; Complement; Complex; DNA; DNA Binding; DNA Polymerase II; DNA Repair; DNA biosynthesis; DNA-Directed RNA Polymerase; Data; Dependence; Detection; Dissociation; Elements; Elongation Factor; Engineering; Enhancers; Enzymatic Biochemistry; Enzymes; Epigenetic Process; Event; Fluorescence Microscopy; Future; Gene Expression; Genes; Genetic Transcription; Genome; Heterogeneity; Histones; Immobilization; In Vitro; Kinetics; Knowledge; Label; Measures; Mediating; Mediator of activation protein; Methods; Minor; Modification; Molecular; Monitor; Nucleosomes; Outcome; Physiological; Process; Proteins; RNA; RNA Processing; Reaction; Repressor Proteins; Resolution; Step Tests; Structure-Activity Relationship; System; Testing; Tetanus Helper Peptide; Time; Transcription Coactivator; Transcription Elongation; Transcription Initiation; Transcription Process; Transcriptional Activation; Transcriptional Regulation; Yeasts; base; chromatin remodeling; fluorophore; histone modification; human disease; in vitro testing; insight; live cell imaging; millisecond; novel; particle; promoter; recruit; single molecule; transcription factor; transcription termination; yeast genome

PROJECT SUMMARY Advances in eukaryotic gene expression have provided a detailed and comprehensive list of transcription-related proteins, their biochemical activities and structure-function relationships, and revealed the importance of histone modifications and nucleosome remodeling enzymes that cooperate with sequence-specific DNA binding transcription factors, revealing a transcriptionally poised chromatin architecture and preinitiation complex at gene promoters and enhancers. Despite these advances, major challenges remain in the integration of chromatin remodeling and modifying events with the complex orchestration of transcription enzymology that were traditionally defined on naked DNA templates, and in the lack of knowledge of the timescales and kinetics by which epigenetic and transcription proteins operate on chromatin substrates. This proposal will address these challenges by developing an in vitro RNA Polymerase II (Pol II) transcription system based on purified yeast minichromosomes that carry the natural set of chromatin architectures and modifications and is responsive to transcription activators. With this minichromosome platform, we aim to introduce defined alterations in chromatin and biochemically manipulate specific components of the transcription apparatus to elucidate causal and temporal relationships. Extension of the analysis to the single-molecule-level to circumvent the confounding asynchrony of ensemble reactions will allow detection of short-lived reaction intermediates, and definition of the temporal order of intermediate events. Thus, this approach allows a direct test of the hypothesis that the kinetics of the multi-step transcription process is regulated by chromatin-based mechanisms, answering questions beyond the reach of current methods and synergistic with live-cell imaging of transcription and chromatin remodeling.

项目摘要 真核基因表达的进展提供了一个详细和全面的转录相关的清单， 蛋白质，其生化活性和结构功能关系，并揭示了组蛋白的重要性， 与序列特异性DNA结合协同作用的修饰和核小体重塑酶 转录因子，揭示了一个转录平衡的染色质结构和基因的前起始复合体 启动子和增强子。尽管取得了这些进展，但在染色质整合方面仍然存在重大挑战。 通过转录酶学的复杂编排， 传统上定义在裸DNA模板上，并且缺乏时间尺度和动力学知识， 这些表观遗传和转录蛋白作用于染色质底物。 该提案将通过开发体外RNA聚合酶II（Pol II）转录来解决这些挑战 基于纯化的酵母微型染色体的系统，所述酵母微型染色体携带染色质结构的天然集合， 修饰，并对转录激活剂有反应。通过这个微型染色体平台，我们的目标是 在染色质中引入确定的改变并以生物化学方式操纵转录的特定组分 阐明因果关系和时间关系的装置。将分析扩展到单分子水平 为了避免集合反应的混淆，将允许检测短寿命反应 中间体，以及中间事件的时间顺序的定义。因此，这种方法允许直接 检验多步转录过程的动力学受基于染色质的 机制，回答目前方法无法解决的问题，并与活细胞成像协同作用， 转录和染色质重塑。