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)：在基因转录过程中，RNA聚合酶的延长可能会受到与DNA结合的许多蛋白质的阻碍(路障)。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)可能失去对启动子的控制。RNA聚合酶在不影响其调节功能的情况下穿过障碍的机制尚不清楚。以前的机械性单分子研究主要集中在RNA聚合酶破坏核小体上。然而，核小体只存在于真核生物中，与DNA非特异性相互作用，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，来自所有王国的生物体的许多转录因子识别DNA上的特定位置来塑造基因组和调节转录，并且不会经历复杂途径调节的化学修饰。取而代之的是，它们对环境线索做出反应，例如DNA超级卷曲、浓度，以及它们以不同的亲和力和合作方式与其结合的多个操作符的存在。这些可调节的、合作的相互作用决定了DNA的结构修饰，如DNA弯曲、包装和环化，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。利用磁钳和原子力显微镜成像技术，比较了三种模型转录因子，即乳胶抑制因子、L抑制因子和186噬菌体CI抑制因子对核糖核酸聚合酶转录延长的影响。这些互补技术提供了对作用于单个DNA分子(MT)上的活性复合体的动态测量，以及吸附在表面(AFM)上的核蛋白复合体的详细静态图像，是阐明RNAP延伸超过Tf的最直接的大分子分析。这项研究的结果将有助于我们(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)指示操纵基因和构建合成转录调控电路的新方法。因此，总的目标是 这项建议是为了了解蛋白质-蛋白质的协同性和蛋白质介导的远程相互作用，如DNA环可能如何影响路障的强度，以及DNA张力和转录产生的DNA超级卷曲是否可能通过这些TF促进RNAP的延伸。目的1将重点研究Tf结合亲和力、环化、DNA张力和DNA超螺旋对LacI障碍的影响。目的2将重点研究Tf结合亲和力、寡聚化、环化、DNA超螺旋的张力和惯用手对弱L CI路障的影响。目的3将重点研究交替包裹或环、DNA张力和DNA超螺旋对186CI抑制物的影响。