MECHANISMS OF EUKARYOTIC TRANSCRIPTION INITIATION

项目总结/摘要 转录起始在细胞表型的建立和调节中起着关键作用， 所有的生物体，但在真核生物中的分子机制的启动仍然不清楚。该过程涉及 RNA聚合酶（RNAP）的募集、DNA解链、转录起始位点扫描、NTP的启动 通过RNAP水解，并过渡到进行性转录延伸。在真核生物中，转录 由最少由TATA盒结合蛋白（TBP）组成的前起始复合物（PIC）引发， TFIIB、IIE、IIF、IIH和RNAP II。由于这种复合物无可争议的生物学意义， 已经收集了关于PIC的生物化学和结构数据，目的是理解机制。 这些研究已经确定了艾德并描述了PIC成分，并提供了 复杂的，是非常有用的发展机制假说。然而，由于结构 快照，它们代表静态图像，缺乏关于PIC因子动态的信息， DNA模板在启动过程中。最终，了解复杂的工作原理需要了解 PIC是如何在沿着启动的路径在定义的结构状态之间转换的。我们已经开始 使用单分子测定法成功地观察到中间状态之间的动态转换， 适合于转录起始之后。我们的长期目标是确定转录背后的机制- 启动子序列、转录因子和转录激活因子的调控。 本研究旨在确定PIC活性诱导的DNA模板的结构转变 通过应用单分子生物物理分析纯化的艾德转录因子， evisiae和智人。我们的磁镊和光镊研究的结合将使 让我们对一个复杂而不同步的过程有不同的见解。使用酵母和人类 系统将独特地允许我们比较这些高度同源的复合物之间的起始机制 它们表现出不同的活动，与它们扫描起始位点的倾向和它们被激活的能力有关， 由超螺旋DNA组成。我们将测试竞争模型的启动站点扫描机制，直接测量 最近证实的Ssl 2在以下情况下的dsDNA移位酶活性的速率和持续合成能力： TFIIH，并将测试我们在酵母的初步工作中观察到的5 bp开放复合物的假设， PIC是真核生物启动的保守特征，通过人PIC监测DNA打开。 这项研究是创新的，因为它使用高分辨率的单分子技术， 直接测定PIC依赖的启动子DNA构象变化和dsDNA转位活性 TFIIH实时该项目的结果将产生很大的影响，因为它们将回答基本问题， 关于导致真核生物转录起始的动态过程的机制问题。

Project Summary/Abstract Transcription initiation plays a pivotal role in the establishment and regulation of cellular phenotypes in all organisms, yet the molecular mechanism of initiation in eukaryotes remains unclear. The process involves the recruitment of RNA polymerase (RNAP), DNA melting, transcription start-site scanning, the initiation of NTP hydrolysis by RNAP, and the transition into processive transcription elongation. In Eukaryotes, transcription is initiated by the pre-initiation complex (PIC) which minimally consists of of TATA-box binding protein (TBP), TFIIB, IIE, IIF, IIH, and RNAP II. Due to the indisputable biological significance of this complex, a plethora of biochemical and structural data have been collected on PICs with the goal of understanding mechanism. These studies have identified and described PIC components and have provided illuminating images of the complex that are exceptionally useful for developing mechanistic hypotheses. However, as structures are snapshots, they represent static images and lack information regarding the dynamics of the PIC factors and the DNA template during initiation. Ultimately, knowledge of how the complex works requires understanding how the PIC transitions between defined structural states along the pathway to initiation. We have begun to successfully observe the dynamic transitions between intermediate states using single-molecule assays well- suited to following transcription initiation. Our long-term goal is to determine the mechanisms behind transcrip- tion initiation and its regulation by promoter sequence, transcription factors, and transcriptional activators. This proposal aims to determine the structural transitions of the DNA template induced by PIC activity by applying single-molecule biophysical assays to purified transcription factors from both Saccharomyces cer- evisiae and Homo sapiens. The combination of our magnetic tweezers and optical tweezers studies will allow us to make distinct insights into a complex and unsynchronized process. The use of both yeast and human systems will uniquely allow us to compare initiation mechanisms between these highly homologous complexes that exhibit distinct activities in relation to their propensity to scan for start-sites and their ability to be activated by superhelical DNA. We will test competing models for the mechanism of start-site scanning, directly measure the rate and processivity of the recently demonstrated dsDNA translocase activity of Ssl2 in the context of TFIIH, and will test the hypothesis that the 5 bp open complex we have observed in preliminary work on yeast PICs is a conserved feature of eukaryotic initiation by monitoring DNA opening by human PICs. The proposed research is innovative because it uses high-resolution single-molecule techniques to di- rectly measure PIC-dependent conformational changes of promoter DNA and the dsDNA translocation activity of TFIIH in real-time. The results from the project will have a large impact as they will answer fundamental mechanistic questions regarding the dynamic processes that lead to transcription initiation in Eukaryotes.