Next Generation Sequencing based analysis of RNA polymerase functions
基于下一代测序的 RNA 聚合酶功能分析
基本信息
- 批准号:8891815
- 负责人:
- 金额:$ 22.73万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2015
- 资助国家:美国
- 起止时间:2015-01-01 至 2016-12-31
- 项目状态:已结题
- 来源:
- 关键词:AddressAffectAntibiotic ResistanceAntibioticsAreaBacterial RNABindingBiochemicalBiological AssayComplexComputer AnalysisDNADNA SequenceDNA analysisDNA-Directed RNA PolymeraseDataDatabasesDependenceDevelopmentDissociationEnzymesEscherichia coliEssential GenesFluorescenceGene ExpressionGenerationsGenetic TranscriptionGoalsHealthcareIn VitroKineticsLaboratoriesLearningLengthLinkMeasurementMessenger RNAMolecularMonitorNucleic AcidsOrganismOutcomePatternPlayPolymeraseProcessPropertyProtein BindingProteinsProtocols documentationRNARNA chemical synthesisReactionReadingRelative (related person)Resource DevelopmentResourcesRoleSamplingSequence AnalysisSeriesSorting - Cell MovementSpecific qualifier valueSystemTestingTimeVariantWorkbasefollow-upin vivoinsightinterestmeltingnext generation sequencingnovelpromoterpublic health relevanceresearch studytherapeutic proteinweb site
项目摘要
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.
描述(由申请人提供):DNA序列在启动和控制结合并作用于DNA模板的蛋白质的活性方面起着至关重要的作用。人们对确定解释这些蛋白质活性的DNA序列依赖性的分子机制产生了极大的兴趣。破译这些机制将显着扩大我们对许多基本的细胞机制的理解,也将是重要的手段,以控制或抑制这些蛋白质的治疗目的的活动的发展。尽管在这一领域取得了重大进展,但仍有许多东西有待学习。该项目的重点是RNA聚合酶(RNAP),这是一个突出的例子,蛋白质的许多功能取决于DNA的序列。RNA聚合酶进行DNA模板指导的mRNA合成。这个多步反应中的每一步都可能以许多(有时是复杂的)难以区分的方式依赖于DNA模板的序列。我们建议,DNA模板依赖RNAP功能(更一般地说,任何蛋白质的DNA模板上操作)的理解可以大大增强和加速实验方法,将允许大量的DNA模板序列变异的高度并行分析。该项目的目标是开发基于下一代测序(NGS)的方法,该方法将允许快速积累与DNA模板序列和RNAP活性相关的实验数据。我们的重点将是由RNAP(初始转录序列; ITS)转录的DNA模板的第一个~20 bp在控制启动子逃逸中的作用,RNAP聚合酶离开启动子以开始RNA产物的进行性延伸的过程。ITS对基因表达的结果有显著的影响,但是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功能中最不清楚的方面之一。我们的初步数据表明,与我们提出的方法,核糖核酸酶活性的ITS序列依赖于数十万的序列变异可以并行研究。我们将使用这种方法来收集详尽的实验数据集相关的ITS序列与RNAP活动,可能是重要的启动子逃逸。计算分析和后续实验将被用来获得ITS的作用机理的见解。该项目的影响将是三方面的。首先,我们将获得数据,这将有助于填补理解控制RNA聚合酶活性和决定基因表达的因素的重要空白。第二,我们将开发一个模板的实验方法,可以复制在其他系统的研究涉及蛋白质的DNA模板上操作。第三,将建立一个详尽的数据库,将DNA模板序列与RNAP活性相关的实验结果,并可用作假设检查和假设生成的资源。
项目成果
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{{ truncateString('TOMASZ HEYDUK', 18)}}的其他基金
Next Generation Sequencing based analysis of RNA polymerase functions
基于下一代测序的 RNA 聚合酶功能分析
- 批准号:
8989967 - 财政年份:2015
- 资助金额:
$ 22.73万 - 项目类别:
New Bioanalytical Methods Based on Next Generation Sequencing
基于下一代测序的新生物分析方法
- 批准号:
8813906 - 财政年份:2015
- 资助金额:
$ 22.73万 - 项目类别:
New Bioanalytical Methods Based on Next Generation Sequencing
基于下一代测序的新生物分析方法
- 批准号:
8988583 - 财政年份:2015
- 资助金额:
$ 22.73万 - 项目类别:
Rapid homogeneous antibody-based detection of proteins
基于均质抗体的蛋白质快速检测
- 批准号:
7933141 - 财政年份:2009
- 资助金额:
$ 22.73万 - 项目类别:
Rapid homogeneous antibody-based detection of proteins
基于均质抗体的蛋白质快速检测
- 批准号:
7220119 - 财政年份:2007
- 资助金额:
$ 22.73万 - 项目类别:
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