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: 9025936
• 负责人: Laura Finzi
• 金额: $ 11.07万
• 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/9025936
• 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聚合酶的延伸可能会受到许多与DNA结合的蛋白质（障碍）的阻碍。或者，RNA 聚合酶的置换可能会抑制 DNA 结合蛋白的活性，并且转录因子 (TF) 可能会失去对启动子的控制。 RNA聚合酶在不损害其调节功能的情况下延伸通过路障的机制尚不清楚。先前的机制、单分子研究主要集中在 RNA 聚合酶破坏核小体上。然而，仅在真核生物中发现的核小体与 DNA 非特异性相互作用，并且是调节染色质重塑和 DNA 转录的翻译后修饰的底物。相比之下，来自跨所有界的生物体的许多转录因子识别 DNA 上的特定位点以塑造基因组并调节转录，并且不会经历复杂途径调节的化学修饰。相反，它们对环境线索做出反应，例如 DNA 超螺旋、浓度以及它们以不同的亲和力和合作方式结合的多个操纵子的存在。这些可调节的、协作的相互作用决定了 DNA 结构修饰，例如 DNA 弯曲、包裹和成环，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。将使用磁镊子 (MT) 和 AFM 成像来比较和对比三种模型 TF（lac 阻遏物 (LacI)、l 阻遏物和 186 噬菌体 CI 阻遏物）对 RNA 聚合酶 (RNAP) 转录延伸的影响。这些互补技术提供了对单个 DNA 分子 (MT) 上运行的活性复合物的动态测量，以及吸附在表面 (AFM) 上的核蛋白复合物的详细静态图像，并且是阐明 RNAP 延伸通过 TF 的机制细节的最直接的大分子分析。这项研究的结果将帮助我们（i）了解转录因子（TF）如何在基因组环境中产生复杂的反应，以及（ii）指出操纵基因和构建转录合成调控电路的新方法。因此，总体目标是 该提案旨在了解蛋白质-蛋白质协同性和蛋白质介导的长程相互作用（例如 DNA 环）如何影响路障的强度，以及 DNA 张力和转录产生的 DNA 超螺旋是否可以通过这些 TF 促进 RNAP 延伸。目标 1 将重点关注 TF 结合亲和力、环化、DNA 张力和 DNA 超螺旋旋向性对强 LacI 障碍的影响。目标 2 将重点关注 TF 结合亲和力、寡聚化、成环、DNA 张力和 DNA 超螺旋旋向性对弱 l CI 路障的影响。目标 3 将重点关注交替缠绕或成环、DNA 张力和 DNA 超螺旋旋向性对 186 CI 阻遏物的影响。