Mechanisms of Transcription-Coupled DNA Supercoiling

描述（由申请人提供）：在理解易位RNA聚合酶的转录如何调节DNA拓扑结构以及转录偶联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中的作用仍然没有完全理解。拟议研究的长期目标是了解转录如何影响DNA拓扑结构、染色体结构以及耦合的DNA事务，如DNA复制和基因表达。本申请的目的是确定某些序列特异性DNA结合蛋白，如噬菌体λ DNA复制起始子O蛋白和乳糖阻遏子，如何在体外和在E.大肠杆菌，并确定TCDS激活基因表达的机制。核心假设是“双超螺旋结构域”模型是负责TCDS的机制，其中核蛋白复合物，特别是那些含有由紧密包裹DNA围绕某些序列特异性DNA结合蛋白组装的稳定环形超螺旋的核蛋白复合物，可以形成阻碍独立染色体超螺旋结构域扩散和合并的拓扑屏障。在这种情况下，“受限”的局部DNA超螺旋可以激活或抑制耦合的DNA交易。这一假设是在我们实验室的初步研究基础上提出的，并将通过以下四个具体目标进行验证：1）确定某些序列特异性DNA结合蛋白在特定蛋白质系统中有效刺激TCDS的机制; 2）研究序列特异性DNA结合蛋白对E.大肠杆菌; 3）开发一种基于线性大肠杆菌噬菌体N15的新系统，以研究TCDS对鼠伤寒沙门氏菌leu-500启动子的激活; 4）在佛罗里达国际大学建立一个具有全国竞争力的研究计划（PI的开发目标）。这一应用将为理解TCDS的作用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，以便在四年的资助期内过渡到非SCORE支持。 公共卫生相关性：这项研究的意义在于它有可能为更好地理解一个基本生物过程：基因转录和表达提供基础。它还为进一步理解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中起着重要作用，如脆性X综合征和亨廷顿病。

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.