Elucidating the Molecular Mechanism of Divergent Transcription

阐明趋异转录的分子机制

• 批准号: 10679336
• 负责人: Leon Palao
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-09-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679336
• 关键词: 3-Dimensional; Architecture; Binding; Biological Assay; Biological Models; Brain; Cell Extracts; Cell Survival; Cells; Chromatin Modeling; Code; Complement; Complex; Cryo-electron tomography; Cryoelectron Microscopy; DNA; Data; Development; Dimerization; Dissection; Eukaryota; Exhibits; Galectin 1; Gene Expression; Genetic Transcription; Genome; Goals; Heart; Human; Hybrids; Image; In Vitro; Lung; Mass Spectrum Analysis; Mediator; Methods; Modeling; Molecular; Mutation; Nucleosomes; Pathway interactions; Polymerase; Prevalence; Process; Promoter Regions; Proteins; RNA Polymerase II; Resolution; Role; Structure; Tail; Transcript; Transcription Elongation; Transcriptional Regulation; Untranslated RNA; Yeasts; crosslink; dimer; experimental study; global run on sequencing; innovation; knock-down; novel; particle; preservation; promoter; reconstitution; recruit; single molecule; transcription factor

Project Summary The goal of this proposal is to elucidate the molecular mechanism of eukaryotic divergent transcription at bidirectional promoters. Divergent transcription, defined as sense and antisense transcripts generated from a common promoter, offers an additional mode of transcriptional regulation. This phenomenon has been observed to be nearly ubiquitous and necessary for cell survival. Strand-specific sequencing methods such as global run on sequencing and nascent elongation transcription sequencing have estimated that over 70% of active promoters from yeast to humans exhibit divergent transcription. This is likely an underestimate as divergent transcript pairs have been shown to be regulated and thus appear unidirectional. Additionally, of the divergent transcript pairs, knockdown of non-coding antisense transcripts results in improper lung, heart, and brain development. The current model for divergent transcription is that an active promoter generates a transcription permissible state where a single Med-PIC assembles, releases a single pol II which produces a sense or antisense transcript and then disassembles, thereby clearing space for assembly of another single PIC to repeat the process. This model is supported by previous structural studies of Med-PIC that utilized a shortened DNA template lacking a complete and endogenous complement of upstream activating sequences (UAS) or in the absence of activator proteins. In my initial study, I investigated the assembly of the PIC on a natural bidirectional promoter by utilizing a DNA template comprising an entire nucleosome-free promoter region (NFR) with UAS(s) and flanking core promoters. Importantly, this promoter architecture is where divergent transcription typically occurs. Interestingly, I observed that two PICs dimerize in vitro via the coactivator Mediator, hereinafter called dMed-PIC in an activator protein dependent manner. In Aim 1, I will resolve a structurally comprehensive view of dMed-PIC using a novel and hybrid approach of cryo-electron microscopy single particle analysis (cryo-EM SPA) and cryo-electron tomography (cryo-ET). I will determine the assembly pathway towards divergent transcription and thereby reveal possible regulatory mechanisms by solving the structure of dMed-PIC. In Aim 2, I will investigate transcription by dMed-PIC in vitro by yeast whole cell extract transcription assays. These proposed experiments will use technically innovative approaches to advance our understanding of the novel dMed-PIC and its function in divergent transcription.

项目摘要 这项建议的目的是阐明真核生物发散转录的分子机制 双向促进剂。分化转录，定义为正义和反义转录本产生于 共同启动子，提供了一种额外的转录调控模式。这种现象已经被观察到了 几乎无处不在，是细胞生存所必需的。链特异测序方法，如全局测序 关于测序和新生伸长的转录测序已经估计超过70%的活跃 从酵母到人类的启动子表现出不同的转录。这可能被低估了，因为分歧 转录本对已经被证明是受调控的，因此看起来是单向的。此外，分歧者的 转录子对，敲除非编码反义转录子会导致肺、心和脑的不正确 发展。目前发散转录的模型是一个活性启动子产生一个转录 单个MED-PIC组装、释放单个POL II的允许状态，该POL II产生或 反义转录本，然后拆解，从而为组装另一单个PIC重复腾出空间 这一过程。这一模型得到了以前利用缩短DNA的Med-PIC结构研究的支持 缺乏完整的内源互补上游激活序列(UA)的模板或在 没有激活蛋白。在我最初的研究中，我研究了PIC在自然双向上的组装 利用含有完整的无核小体启动子区域的模板进行启动子的研究(S) 以及核心推动者的侧翼。重要的是，这种启动子结构是发散转录通常 发生。有趣的是，我观察到两个PIC在体外通过辅活化子介体(以下称为 DMed-PIC以激活蛋白依赖的方式表达。在目标1中，我将解决一个结构全面的观点 用冷冻电子显微镜单粒子分析(Cryo-EM)的一种新的混合方法对DMed-PIC进行研究 Spa)和冷冻电子断层扫描(Cryo-ET)。我将确定朝向分歧者的组装路径 转录，从而通过求解DMed-PIC的结构来揭示可能的调控机制。在AIM 2、通过酵母全细胞抽提物转录实验研究DMed-PIC的体外转录。这些 拟议的实验将使用技术创新的方法来促进我们对小说的理解 DMed-PIC及其在分化转录中的作用。