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）：基因转录过程中的RNA聚合酶伸长可能会受到与DNA结合的许多蛋白质（障碍）的阻碍。另外，RNA聚合酶的位移可以抑制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聚合酶在不损害其调节功能的情况下通过障碍延伸的机制知之甚少。以前的机械，单分子研究集中在RNA聚合酶破坏核小体上。但是，仅在真核生物中发现的核小体与DNA非特异性相互作用，并且是翻译后修饰的底物，可调节DNA的染色质重塑和转录。相比之下，跨越所有王国的生物体的许多TF都识别DNA上的特定位点以塑造基因组和调节转录，并且不经过由复杂途径调节的化学修饰。取而代之的是，他们响应环境线索，例如DNA超串联，集中度以及与不同亲和力和合作息息相关的多个操作员的存在。这些可调的，合作的相互作用决定了建筑DNA的修饰，例如DNA弯曲，包装和循环，其作用尚未在较早的体内或体外的转录障碍研究中解决。将使用磁镊子（MT）和AFM成像进行比较，将比较三个模型TFS，LAC抑制剂（LACI），L抑制剂和186个噬菌体CI抑制器对RNA聚合酶（RNAP）的转录伸长的影响。这些互补技术提供了在单个DNA分子（MT）上运行的活性复合物的动态测量，以及吸附在表面上（AFM）上的核蛋白复合物的详细静态图像，并且是最直接的大分子分析分析，用于省略了RNAP的细节，而RNAP则是rnap的详细信息。这项研究的结果将有助于我们（i）了解基因组环境中转录因子（TFS）的复杂反应如何，（ii）指出了操纵基因并构建转录的合成调节回路的新方法。因此，总体目标 该建议是了解蛋白质 - 蛋白质的合作性和蛋白质介导的远距离相互作用（例如DNA循环）如何影响障碍的强度，以及如果DNA张力和转录生成的DNA超螺旋可能会促进通过这些TF促进RNAP的延长。 AIM 1将重点关注TF结合亲和力，环循环，DNA张力和DNA超螺旋在强LACI障碍上的影响。 AIM 2将重点关注TF结合亲和力，低聚，循环，DNA张力和DNA超螺旋在弱LCI障碍上的影响。 AIM 3将集中于替代包装或循环，DNA张力以及DNA超串制对186 CI阻遏物的影响。