Mechanisms of Transcription-Coupled DNA Supercoiling

描述(由申请人提供)：在理解移位RNA聚合酶如何转录调节DNA拓扑以及转录偶联DNA超螺旋(TCDS)如何激活基因表达方面存在根本差距。例如，序列特异性DNA结合蛋白在TCDS中的作用仍不完全清楚。这项拟议研究的长期目标是了解转录如何影响DNA拓扑、染色体结构以及耦合的DNA交易，如DNA复制和基因表达。本应用的目的是确定某些序列特异的DNA结合蛋白，如噬菌体DNA复制启动子O蛋白和乳糖抑制因子，在体外和大肠杆菌中如何调节TCDS，并确定TCDS激活基因表达的机制。中心假设是“双超螺旋结构域”模型是TCDS的机制，在该模型中，核蛋白复合体，特别是那些包含由紧密包裹在特定序列DNA结合蛋白周围的DNA组装而成的稳定环状超螺旋的核蛋白复合体，可以形成拓扑障碍，阻碍独立的染色体超螺旋结构域的扩散和合并。在这种情况下，“受限的”局域DNA超螺旋可能激活或抑制耦合的DNA交易。这一假说是基于我们实验室提供的强大的初步数据提出的，并将通过以下四个具体目标进行验证：1)确定某些序列特定的DNA结合蛋白在已定义的蛋白质系统中有效刺激TCDS的机制；2)研究序列特定的DNA结合蛋白对大肠杆菌中TCDS的影响；3)开发一种新的系统，以线性噬菌体N15为基础，研究TCDS对鼠伤寒沙门氏菌leu-500启动子的激活作用；4)在佛罗里达国际大学建立一个具有国家竞争力的研究项目(PI的发展目标)。这一应用将为理解TCDS的机制及其在基因表达中的作用提供重要的知识。它还将为PI提供必要的资源，使其在四年资助期内过渡到非记分支助。 公共卫生相关性：这项研究的意义源于它可能为更好地理解一个基本的生物学过程提供基础：基因转录和表达。它也为进一步了解DNA拓扑结构提供了基础，DNA拓扑结构在基因组稳定性和某些人类遗传性疾病(如脆性X综合征和亨廷顿病)中发挥着重要作用。

DESCRIPTION (provided by applicant): A fundamental gap exists in understanding how transcription by a translocating RNA polymerase modulates DNA topology and how transcription-coupled DNA supercoiling (TCDS) activates gene expression. For instance, the roles of sequence-specific DNA-binding proteins in TCDS are still not fully understood. The long- term goal of the proposed research is to understand how transcription affects DNA topology, chromosome structure, and the coupled DNA transactions, such as DNA replication and gene expression. The objectives of this application are to determine how certain sequence-specific DNA-binding proteins, such as bacteriophage lambda DNA replication initiator O protein and lactose repressor, regulate TCDS in vitro and in E. coli and to determine the mechanism by which TCDS activates gene expression. The central hypothesis is that the "twin- supercoiled-domain" model is the mechanism responsible for TCDS in which nucleoprotein complexes, especially those containing stable toroidal supercoils assembled from tightly-wrapping DNA around certain sequence-specific DNA-binding proteins, can form topological barriers that impede the diffusion and merger of independent chromosomal supercoil domains. In this case, the "confined" localized DNA supercoils may activate or inhibit the coupled DNA transactions. This hypothesis has been formulated on the basis of strong preliminary data produced in our laboratory and will be tested by pursuing four specific aims: 1) to determine the mechanisms by which certain sequence-specific DNA-binding proteins potently stimulate TCDS in the defined protein systems; 2) to study effects of the sequence-specific DNA-binding proteins on TCDS in E. coli; 3) to develop a novel system, based on a linear coliphage N15, to study activation of the Salmonella typhimurium leu-500 promoter by TCDS; 4) to establish a nationally competitive research program at Florida International University (the PI's development objective). This application will provide important knowledge for understanding the mechanism of TCDS and its roles in gene expression. It will also provide the necessary resources for the PI to transit to non-SCORE support within a four-year funding period. Public Health Relevance: The significance of this research stems from its potential to provide a basis for better understanding of an essential biological process: gene transcription and expression. It also provides a foundation for further understanding DNA topology, which plays an important role in genome stability and certain human hereditary diseases, such as fragile X syndrome and Huntington's disease.