How elongating RNAP navigates protein-mediated DNA looping and wrapping

描述（由申请人提供）：基因转录过程中的RNA聚合酶延伸可能受到许多与DNA结合的蛋白质（路障）的阻碍。另外，RNA聚合酶的置换可能抑制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控功能的情况下通过路障延长的机制尚不清楚。以前的机制，单分子研究集中在RNA聚合酶破坏核小体上。然而，核小体仅在真核生物中发现，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，来自所有生物的许多tf识别DNA上的特定位点来形成基因组并调节转录，并且不经过复杂途径调节的化学修饰。相反，它们对环境线索做出反应，如DNA超缠绕、浓度和多个操作符的存在，它们以不同的亲和力和合作方式结合。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、包裹和环，其作用在早期的体内或体外转录障碍研究中都没有得到解决。利用磁镊子（MT）和原子力显微镜（AFM）成像技术，比较3种模型TFs， 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作用于单个DNA分子（MT）上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延长通过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）如何在基因组背景下产生复杂的反应，以及（ii）指出操纵基因和构建转录合成调控回路的新方法。因此，总体目标

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.