Next Generation Sequencing based analysis of RNA polymerase functions

 描述(由申请人提供)：DNA序列在启动和控制结合并作用于DNA模板的蛋白质的活动中起着重要作用。人们对确定解释DNA序列依赖于这些蛋白质活性的分子机制产生了极大的兴趣。破译这些机制将大大扩展我们对许多基本细胞机制的理解，也将在开发用于治疗目的控制或抑制这些蛋白质活性的方法方面发挥重要作用。尽管在这一领域取得了重大进展，但仍有许多东西需要学习。这个项目的重点是RNA聚合酶(RNAP)，这是一个突出的例子，它的许多功能依赖于DNA序列。RNA聚合酶执行DNA模板指导的信使核糖核酸的合成。这种多步骤反应中的每一步可能以许多(有时令人费解的)方式依赖于DNA模板的序列，而这些方式很难分类。我们认为，通过实验方法可以极大地提高和加速对RNAP功能(更广泛地说，任何作用于DNA模板的蛋白质)对DNA模板的依赖关系的理解，这将允许对大量DNA模板序列变体的高度并行分析。该项目的目标是开发基于下一代测序(NGS)的方法，允许快速积累与DNA模板序列和RNAP活性相关的实验数据。我们的重点是RNAP(初始转录序列；ITS)转录的DNA模板的第一个~20bp在控制启动子逃逸中的作用，这是一个RNAP聚合酶离开启动子开始RNA产物的过程延伸的过程。ITS对基因表达的结果有显着的影响，但ITS序列与其对转录的影响之间的联系机制仍然是启动子DNA功能最不了解的方面之一。我们的初步数据表明，使用我们提出的方法，可以并行研究数十万个序列变体的RNAP活性的ITS序列依赖性。我们将使用这种方法来收集一组详尽的实验数据，这些数据将其序列与RNAP活性联系起来，这可能在启动子逃逸中起重要作用。将采用计算分析和后续实验来获得对ITS作用的机械性见解。这个项目的影响将是三方面的。首先，我们将获得能够填补在理解控制RNA聚合酶活性和决定基因表达的因素方面的重要空白的数据。其次，我们将开发一种用于实验方法的模板，该模板可以在涉及以DNA模板操作蛋白质的其他系统的研究中复制。第三，将建立一个将DNA模板序列与RNAP活性相关联的实验结果的详尽数据库，并可用作假设检验和假设生成的资源。

 DESCRIPTION (provided by applicant): The sequence of DNA plays an essential role in initiating and controlling activities of proteins that bind and operate on DNA templates. There has been a tremendous interest in determining the molecular mechanisms that explain DNA sequence dependence of the activities of these proteins. Deciphering these mechanisms will significantly expand our understanding of many basic cellular mechanisms and will be also important in the development of means to control or inhibit the activities of these proteins for therapeutic purposes. Despite significant progress in this area, much still remains to be learned. This project is focused on RNA polymerase (RNAP), which is an outstanding example of a protein whose many functions depend on the sequence of DNA. RNA polymerase performs DNA template directed synthesis of mRNA. Each step in this multistep reaction may be dependent on the sequence of DNA template in many (sometimes convoluted) ways that are difficult to sort out. We propose that the understanding of DNA template dependence of RNAP functions (and more generally, any protein operating on DNA template) could be greatly enhanced and accelerated by experimental approaches that will allow highly parallel analysis of a large number of DNA template sequence variants. The goal of this project is to develop Next Generation Sequencing (NGS) based approach that will allow rapid accumulation of experimental data relating DNA template sequence and RNAP activity. Our focus will be on the role of the first ~20 bp of DNA template transcribed by RNAP (Initially Transcribed Sequence; ITS) in controlling promoter escape, a process where RNAP polymerase leaves the promoter to begin processive elongation of RNA product. ITS has remarkable effect on the outcomes of gene expression but the mechanisms linking the sequence of ITS with their effects on transcription remain one of the least understood aspects of promoter DNA function. Our preliminary data demonstrate that with our proposed approach, ITS sequence dependence of RNAP activity for hundreds of thousands of sequence variants could be studied in parallel. We will use this approach to collect exhaustive set of experimental data correlating ITS sequence with RNAP activities that might be important in promoter escape. Computational analysis and follow up experiments will be employed to obtain mechanistic insights into the role of ITS. The impact of this project will be threefold. First, we will obtain the data that will enable filling important gaps in understanding of the factors that control activity of RNA polymerase and determine gene expression. Second, we will develop a template for experimental approach that could be replicated in studies on other systems involving proteins operating on DNA template. Third, an exhaustive database of experimental results correlating DNA template sequence with RNAP activity will be established and could be used as a resource for hypothesis checking and hypothesis generation.