Probing the Effects of Highly Bent and Twisted DNA on Transcription by RNA Polymerase

细胞含有大量以复杂模式表达的基因，以确保适当的细胞功能和分化。已经确定，调节蛋白在转录控制和调节RNA聚合酶（RNAP）的活性中起核心作用。此外，与超螺旋、环状DNA拓扑结构和染色质重塑相关的各种证据表明，RNAP活性也可能通过DNA的纳米机械性质来调节。我们假设DNA模板的弯曲和扭曲直接调节RNAP的活性。为了验证这一假设，我们将产生长度约为100个碱基对的DNA微环，以在不存在阻遏物或任何其他调节蛋白的情况下将弯曲应变能和扭转引入生理相关量级的DNA模板中。使用这些小环DNA模板和基于分子信标的转录测定直接检测合成的mRNA，我们将表征T7-RNAP动力学性质的变化。 我们还将开发检测方法来跟踪单个DNA微环的转录，并直接调节DNA分子的扭转，同时记录RNAP的活性。最后，我们将利用计算杆模型来预测DNA拓扑结构和转录之间的关系。我们对DNA模板力学对RNA聚合酶活性的作用的研究对生物学、医学和物理科学具有更广泛的意义。这项工作将填补我们在DNA力学生物学基础知识方面的一个重要空白。拟议的测定将有助于详细表征DNA模板拓扑结构和力学对RNAP活性的影响。如果成功，这项研究将扩大我们对转录调控的理解，并迫使我们如何思考和处理遗传开关和基因调控的范式转变。因此，这项工作对医学科学具有重要意义，并将有助于合理理解细胞调节，细胞控制和组织工程。这里开发的多学科方法和技术也将有利于许多其他DNA结合酶的研究，基因调控的其他领域和分子马达和细胞骨架的研究。

Cells contain a large number of genes that are expressed in complex patterns to ensure proper cellular function and differentiation. It is well established that regulatory proteins play a central role in transcriptional control and regulate the activity of RNA polymerases (RNAP). In addition, various lines of evidence related to supercoiling, looped DNA topologies, and chromatin remodeling suggest that RNAP activity may also be regulated via the nano-mechanical properties of DNA. We hypothesize that bending and twisting of the DNA template directly regulate the activity of RNAPs. To test this hypothesis we will generate DNA minicircles of a length of about 100 base pairs to introduce bending strain energy and torsion into DNA templates of a physiologically relevant magnitude in the absence of repressors or any other regulatory proteins. Using these minicircle DNA templates and molecular beacon-based transcription assays to directly detect synthesized mRNA, we will characterize changes in the kinetic properties of T7-RNAP. We will also develop assays to track the transcription from single DNA minicircles and to directly modulate torsion in DNA molecules while recording the activity of RNAP. Lastly, we will utilize computational rod models to predict the relationship between DNA topology and transcription. Our research on the role of DNA template mechanics on the activity of RNA polymerases has broader implications for the biological, medical and physical sciences. The work will close an important gap in our fundamental knowledge of the biology of DNA mechanics. The proposed assays will facilitate a detailed characterization of the effects that DNA template topologies and mechanics have on the activity of RNAPs. If successful, this research expands our understanding of transcriptional regulation and compels a paradigm shift in how we think about and approach genetic switches and gene regulation. Thus, the work has important implications for the medical sciences and will contribute to a rational understanding of cell regulation, cellular control and tissue engineering. The multidisciplinary approach and techniques developed here will also benefit the study of numerous other DNA-binding enzymes, other areas of gene regulation and the study of molecular motors and the cytoskeleton.