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 该提案的目的是阐明真核发散转录的分子机制 双向启动子。发散的转录，定义为从A 普通启动子提供了一种附加的转录调节方式。已经观察到这种现象 几乎无处不在，对于细胞存活所必需。链特异性测序方法，例如全局运行 在测序和新生的伸长转录测序上，估计有70％以上的活动 从酵母到人类的启动子表现出不同的转录。这可能是低估的 转录对已被证明是受调节的，因此似乎是单向的。另外，发散 成绩单对，非编码反义转录本的敲低导致肺，心脏和大脑不当 发展。当前的发散转录模型是主动启动子生成转录 允许的状态单个Med-PIC组装，释放一个单个POL II，该pol II产生一种感觉或 反义转录本，然后分解，从而清除组装另一个图片以重复的空间 过程。使用缩短DNA的Med-PIC的先前结构研究支持了该模型 模板缺乏上游激活序列（UAS）的完整和内源补体 没有激活蛋白。在我的最初研究中，我调查了图片在自然双向上的组装 通过利用包含UAS（S）的整个无核体启动子区域（NFR）的DNA模板（NFR）来启动子 和核心启动子。重要的是，该启动子体系结构通常是不同的转录 发生。有趣的是，我观察到两张图片通过共激活因子介体在体外进行了斑点，以下称为 DMED-PIC以激活蛋白依赖性方式。在AIM 1中，我将解决结构上全面的观点 使用冷冻电子显微镜单个粒子分析的新型和杂种方法（Cryo-EM）的DMED-PIC SPA）和冷冻电子层析成像（Cryo-ET）。我将确定分歧的组装途径 转录，从而通过求解DMED-PIC的结构来揭示可能的调节机制。目标 2，我将通过酵母全细胞提取物转录测定法研究DMED-PIC在体外进行转录。这些 拟议的实验将使用技术创新的方法来提高我们对新颖的理解 DMED-PIC及其在发散转录中的功能。