Elucidating the Molecular Mechanism of Divergent Transcription

项目摘要 本研究的目的是阐明真核生物趋异转录的分子机制， 双向启动子趋异转录，定义为从一个或多个转录物中产生的有义和反义转录物。 共同启动子提供了另一种转录调控模式。这一现象已被观察到 几乎无处不在，是细胞生存所必需的。链特异性测序方法，如全局运行 在测序和新生延伸转录测序中估计，超过70%的活性 从酵母到人的启动子表现出不同的转录。这可能是一个低估的分歧， 转录本对已被证明是受调节的，因此表现为单向的。此外，在分歧 转录物对，敲低非编码反义转录物导致肺、心脏和脑 发展目前的趋异转录模型是一个活性启动子产生转录， 允许的状态，其中单个Med-PIC组装，释放单个pol II，其产生感测或 反义转录物，然后拆解，从而为另一个单个PIC的组装清除空间，以重复 过程这一模型得到了先前利用缩短的DNA的Med-PIC结构研究的支持。 模板缺乏上游激活序列（UAS）的完整和内源性互补序列，或 缺乏激活蛋白。在我最初的研究中，我研究了PIC在自然双向 通过利用包含具有UAS的完整无核小体启动子区（NFR）的DNA模板 和侧翼核心启动子。重要的是，这种启动子结构是趋异转录通常 发生。有趣的是，我观察到两个PIC在体外通过共活化剂介体二聚化，下文称为 dMed-PIC以激活蛋白依赖的方式。在目标1中，我将解决一个结构全面的视图 dMed-PIC使用低温电子显微镜单颗粒分析（cryo-EM）的新的混合方法 SPA）和冷冻电子断层扫描（cryo-ET）。我将决定通往发散的装配路径 转录，从而揭示可能的调控机制，通过解决dMed-PIC的结构。在Aim中 2、通过酵母全细胞提取物转录实验研究dMed-PIC在体外的转录情况。这些 拟议的实验将使用技术创新的方法，以促进我们对小说的理解 dMed-PIC及其在趋异转录中的作用

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.