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.

项目概要 真核基因表达的进展提供了与转录相关的详细而全面的列表 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 结合配合的修饰和核小体重塑酶 转录因子，揭示基因的转录平衡染色质结构和预起始复合物 启动子和增强子。尽管取得了这些进展，染色质整合仍面临重大挑战 通过转录酶学的复杂编排来重塑和修改事件 传统上是在裸DNA模板上定义的，并且缺乏对时间尺度和动力学的了解 表观遗传和转录蛋白在染色质底物上起作用。 该提案将通过开发体外 RNA 聚合酶 II (Pol II) 转录来应对这些挑战 基于纯化的酵母微型染色体的系统，其携带天然的染色质结构集和 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 引入染色质的明确改变并通过生化方法操纵转录的特定成分 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 规避集合反应的混杂异步性将允许检测短暂的反应 中间事件，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调节的假设 机制，回答超出当前方法范围的问题，并与活细胞成像协同作用 转录和染色质重塑。