How elongating RNAP navigates protein-mediated DNA looping and wrapping

延长 RNAP 如何引导蛋白质介导的 DNA 环化和包裹

• 批准号: 8895353
• 负责人: Laura Finzi
• 金额: $ 30.86万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8895353
• 关键词: Address; Affect; Affinity; Bacteriophage Genetics; Bacteriophage lambda; Bacteriophages; Binding; Biological Assay; Bypass; Chemicals; Chromatin Loop; Communicable Diseases; Complex; Cues; Cytolysis; DNA; DNA Modification Process; DNA-Binding Proteins; DNA-Directed RNA Polymerase; Disease; Epigenetic Process; Equilibrium; Eukaryota; Genes; Genetic; Genetic Transcription; Genome; Genomics; Goals; Handedness; Histones; Human; Image; In Vitro; Investigation; Kinetics; Knowledge; Lac Repressors; Lactose Factors; Lead; Life; Lysogeny; Magnetism; Malignant Neoplasms; Measurement; Mediating; Modeling; Modification; Molecular; Nucleoproteins; Nucleosomes; Organism; Pathway interactions; Patients; Positioning Attribute; Post-Translational Protein Processing; Probability; Process; Protein Binding; Proteins; Regulation; Regulatory Element; Repressor Proteins; Research; Role; Shapes; Site; Superhelical DNA; Surface; Techniques; Testing; Therapeutic; base; chromatin remodeling; design; dimer; experience; fight against; improved; in vivo; insight; lambda repressor; promoter; public health relevance; response; single molecule; synthetic construct; tool; transcription factor

DESCRIPTION (provided by applicant): RNA polymerase elongation during gene transcription may be hindered by the many proteins bound to DNA (roadblocks). Alternatively, displacement by RNA polymerase could inhibit the activity of a DNA-bound protein, and a transcription factor (TF), for example, might lose control of a promoter. The mechanism by which RNA polymerases elongate through roadblocks without compromising their regulatory function is poorly understood. Previous mechanistic, single-molecule studies have focused on RNA polymerase disrupting nucleosomes. However, nucleosomes, which are only found in eukaryotes, interact with DNA non-specifically, and are substrates for post-translational modifications that regulate chromatin remodelling and transcription of DNA. In contrast, many TFs from organisms spanning all kingdoms recognize specific sites on DNA to shape the genome and regulate transcription, and do not undergo chemical modifications regulated by complex pathways. Instead, they respond to environmental cues such as DNA supercoiling, concentration, and the presence of multiple operators to which they bind with different affinities and cooperatively. These tunable, cooperative interactions determine architectural DNA modifications such as DNA bending, wrapping and looping, the role of which has not been addressed in earlier studies on transcription roadblocks either in vivo or in vitro. The effect of three model TFs, the lac repressr (LacI), the l repressor and the 186 bacteriophage CI repressor, on transcriptional elongation by RNA polymerase (RNAP), will be compared and contrasted using magnetic tweezers (MT) and AFM imaging. These complementary techniques provide dynamic measurements of active complexes operating on single DNA molecules (MT), and detailed static images of nucleoprotein complexes adsorbed on a surface (AFM), and are the most direct macromolecular analyses for elucidating the mechanistic details by which RNAP elongates past a TF. The results of this investigation will help us (i) understand how transcriptional factors (TFs) generat complex responses in genomic contexts, and (ii) indicate new ways in which to manipulate genes and construct synthetic regulatory circuits for transcription. Therefore, the overall goal of this proposal is to understand how protein-protein cooperativity and protein-mediated long-range interactions, such as DNA looping may affect the strength of a roadblock, and if DNA tension and transcription-generated DNA supercoiling may facilitate RNAP elongation through these TFs. Aim 1 will focus on the effects of TF binding affinity, looping, DNA tension and handedness of DNA supercoiling on the strong LacI roadblock. Aim 2 will focus on the effects of TF binding affinity, oligomerization, looping, DNA tension and handedness of DNA supercoiling on the weak l CI roadblock. Aim 3 will focus on the effects of alternate wrapping or looping, DNA tension and handedness of DNA supercoiling on the 186 CI repressor.

描述（由申请人提供）：基因转录过程中RNA聚合酶的延伸可能受到许多与DNA结合的蛋白质（路障）的阻碍。或者，RNA聚合酶的置换可以抑制DNA结合蛋白的活性，例如，转录因子（TF）可能会失去对启动子的控制。RNA聚合酶通过路障伸长而不损害其调节功能的机制知之甚少。以前的机制，单分子研究集中在RNA聚合酶破坏核小体。然而，仅在真核生物中发现的核小体与DNA非特异性地相互作用，并且是调节染色质重塑和DNA转录的翻译后修饰的底物。相反，许多来自所有生物界的TF识别DNA上的特定位点来塑造基因组并调节转录，并且不经历由复杂途径调节的化学修饰。相反，它们会对环境线索做出反应，例如DNA超螺旋、浓度以及它们以不同亲和力和合作方式结合的多个操纵基因的存在。这些可调的合作相互作用决定了DNA的结构修饰，如DNA弯曲、缠绕和成环，其作用在早期的体内或体外转录障碍研究中尚未得到解决。利用磁镊（MT）和原子力显微镜（AFM）成像，比较了三种模型转录因子（LacI、L1和186噬菌体C1）对RNA聚合酶（RNAP）转录延伸的影响。这些互补的技术提供了动态测量的活性复合物上操作的单个DNA分子（MT），和详细的静态图像的核蛋白复合物吸附在表面上（AFM），是最直接的大分子分析阐明的机制细节RNAP延长过去的TF。这项研究的结果将帮助我们（i）理解转录因子（TF）如何在基因组环境中产生复杂的反应，以及（ii）指示操纵基因和构建转录合成调控回路的新方法。因此，总体目标是 这个建议是为了了解蛋白质-蛋白质协同性和蛋白质介导的长程相互作用，如DNA成环可能会影响路障的强度，以及DNA张力和转录产生的DNA超螺旋是否可能促进RNAP通过这些TF的延伸。目的1将集中在TF结合亲和力，成环，DNA张力和DNA超螺旋的手性对强LacI路障的影响。目的2将集中于TF结合亲和力、寡聚化、成环、DNA张力和DNA超螺旋的手性对弱I CI路障的影响。目的3将着重于交替缠绕或成环、DNA张力和DNA超螺旋旋向性对186 CI阻遏物的影响。