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:8989967
- 负责人:
- 金额:$ 18.94万
- 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 2017-12-31
- 项目状态:已结题
- 来源:
- 关键词:AddressAffectAntibiotic ResistanceAntibioticsAreaBacterial RNABindingBinding ProteinsBiochemicalBiological AssayComplexComputer AnalysisDNADNA SequenceDNA analysisDNA-Directed RNA PolymeraseDataDatabasesDependenceDevelopmentDissociationEnzymesEscherichia coliEssential GenesFluorescenceGene ExpressionGenerationsGenetic TranscriptionGoalsHealthHealthcareIn VitroKineticsLaboratoriesLearningLengthLinkMeasurementMessenger RNAMolecularMonitorNucleic AcidsOrganismOutcomePatternPlayPolymeraseProcessPropertyProteinsProtocols documentationRNARNA chemical synthesisReactionReadingResource DevelopmentResourcesRoleSamplingSequence AnalysisSeriesSorting - Cell MovementSpecific qualifier valueSystemTestingTimeVariantWorkbasefollow-upin vivoinsightinterestmeltingnext generation sequencingnovelpromoterresearch 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 DNA 模板并对其进行操作的蛋白质的活性中起着重要作用。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细胞机制的理解,并且对于开发用于治疗目的的控制或抑制这些蛋白质活性的方法也很重要。尽管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),它是许多功能依赖于 DNA 序列的蛋白质的杰出例子。 RNA 聚合酶执行 DNA 模板指导的 mRNA 合成。这种多步反应中的每一步都可能以多种(有时是复杂的)方式依赖于 DNA 模板的序列,而这些方式很难理清。我们提出,通过允许对大量 DNA 模板序列变体进行高度并行分析的实验方法,可以极大地增强和加速对 RNAP 功能(更一般地说,任何在 DNA 模板上运行的蛋白质)的 DNA 模板依赖性的理解。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 RNAP(初始转录序列;ITS)转录的前约 20 bp DNA 模板在控制启动子逃逸中的作用,这是 RNAP 聚合酶离开启动子以开始 RNA 产物持续延伸的过程。 ITS 对基因表达的结果具有显着影响,但将 ITS 序列与其对转录的影响联系起来的机制仍然是启动子 DNA 功能中最不为人所知的方面之一。我们的初步数据表明,通过我们提出的方法,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收集详尽的实验数据集,将 ITS 序列与 RNAP 活性相关联,这对启动子逃逸可能很重要。将采用计算分析和后续实验来深入了解 ITS 的作用。该项目的影响将是三重的。首先,我们将获得数据,以填补了解控制 RNA 聚合酶活性和决定基因表达的因素方面的重要空白。其次,我们将开发一个实验方法模板,可以在涉及 DNA 模板上操作的蛋白质的其他系统的研究中进行复制。第三,将建立一个详尽的实验结果数据库,将 DNA 模板序列与 RNAP 活性相关联,并可用作假设检查和假设生成的资源。
项目成果
期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Real-Time Observation of Backtracking by Bacterial RNA Polymerase.
细菌 RNA 聚合酶回溯的实时观察。
- DOI:10.1021/acs.biochem.5b01184
- 发表时间:2016
- 期刊:
- 影响因子:2.9
- 作者:Lass-Napiorkowska,Agnieszka;Heyduk,Tomasz
- 通讯作者:Heyduk,Tomasz
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TOMASZ HEYDUK其他文献
TOMASZ HEYDUK的其他文献
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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:
8891815 - 财政年份:2015
- 资助金额:
$ 18.94万 - 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:
$ 18.94万 - 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:
$ 18.94万 - 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:
$ 18.94万 - 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:
$ 18.94万 - 项目类别:
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