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超卷曲（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中的作用仍未完全了解。该研究的长期目标是了解转录如何影响DNA拓扑结构、染色体结构和耦合DNA交易，如DNA复制和基因表达。本应用程序的目的是确定某些序列特异性DNA结合蛋白，如噬菌体lambda DNA复制启动器O蛋白和乳糖抑制蛋白，如何在体外和大肠杆菌中调节TCDS，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”模型是导致TCDS的机制，其中核蛋白复合物，特别是那些含有稳定的环形超线圈的核蛋白复合物，由紧密包裹在特定序列的DNA结合蛋白周围的DNA组装而成，可以形成拓扑屏障，阻碍独立染色体超螺旋结构域的扩散和合并。在这种情况下，“受限”的局部DNA超级线圈可能激活或抑制耦合DNA交易。这一假设是在我们实验室产生的强有力的初步数据的基础上制定的，并将通过追求四个特定目标来进行测试：1)确定某些序列特异性dna结合蛋白在定义的蛋白质系统中有效刺激TCDS的机制；2)研究序列特异性dna结合蛋白对大肠杆菌TCDS的影响；3)建立基于线性噬菌体N15的新型系统，研究TCDS对鼠伤寒沙门菌leu-500启动子的激活作用；4)在佛罗里达国际大学建立具有全国竞争力的研究项目（PI的发展目标）。这一应用将为了解TCDS的机制及其在基因表达中的作用提供重要的知识。它还将提供必要的资源，使PI在四年供资期内转为非score支助。

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.