Kinetic mechanism of transcription on native minichromosome

项目总结 真核基因表达的进展提供了与转录相关的详细和全面的清单 蛋白质及其生化活性和结构-功能关系，并揭示了组蛋白的重要性 与序列特异性DNA结合协同的修饰和核小体重塑酶 转录因子，揭示转录平衡的染色质结构和基因的预起始复合体 促进剂和增强剂。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 通过复杂的转录酶学协调来重塑和修改事件 传统上定义在裸DNA模板上，在缺乏时间尺度和动力学知识的情况下 哪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)转录来应对这些挑战 基于纯化的酵母微染色体的系统，该系统携带一套自然的染色质结构和 修饰，并对转录激活剂作出反应。有了这个微型染色体平台，我们的目标是 在染色质中引入明确的变化并生化操作转录的特定成分 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 为了避免整体反应的混杂的不同步，将允许检测到短暂的反应 中间体，以及中间事件的时间顺序的定义。因此，此方法允许直接 对多步转录过程的动力学受染色质调控的假设的检验 机制，回答当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。

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.