Mechanisms of Transcription-Coupled DNA Supercoiling

转录偶联 DNA 超螺旋机制

基本信息

批准号：
7813784
负责人：
Fenfei Leng
金额：
$ 24.65万
依托单位：
FLORIDA INTERNATIONAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-01 至 2013-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7813784
关键词：
Affect Antineoplastic Agents Bacteriophage lambda Bacteriophages Binding Sites Biological Process Camptothecin Cells Chromosome Structures Coliphages Complex Coupled DNA DNA Sequence DNA Topoisomerases DNA biosynthesis DNA-Binding Proteins DNA-Directed RNA Polymerase Data Development Diffusion Doxorubicin Enzymes Escherichia coli Florida Foundations Fragile X Syndrome Funding Gene Expression Genes Genetic Recombination Genetic Transcription Genome Stability Goals Grant Health Hereditary Disease Human Huntington Disease In Vitro International Knowledge Laboratories Lactose Malignant Neoplasms Modeling Molecular Nucleoproteins Plant Roots Play Point Mutation Process Processed Genes Progress Reports Proteins Research Resources Role Salmonella typhimurium Sequence-Specific DNA Binding Protein Superhelical DNA System Testing Transcription Initiation Twin Multiple Birth Universities Work base career in vivo innovation novel plasmid DNA programs promoter public health relevance stem

项目摘要

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.

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性的差距。例如，序列特异性 DNA 结合蛋白在 TCDS 中的作用仍未完全了解。本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 事务，例如 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（例如噬菌体 lambda DNA 复制起始蛋白 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 激活基因的机制表达。核心假设是“双超螺旋结构域”模型是 TCDS 的机制，其中核蛋白复合物，特别是那些含有由紧密包裹的 DNA 围绕某些序列特异性 DNA 结合蛋白组装而成的稳定环形超螺旋的核蛋白复合物，可以形成拓扑结构。阻碍独立染色体超螺旋结构域扩散和合并的障碍。在这种情况下，“受限”的局部 DNA 超螺旋可能会激活或抑制耦合的 DNA 事务。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，并将通过追求四个具体目标进行测试：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结合蛋白对大肠杆菌TCDS的影响； 3) 开发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具有全国竞争力的研究项目（PI的发展目标）。该应用将为理解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 在四年资助期内过渡到非 SCORE 支持提供必要的资源。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基因转录和表达这一重要的生物过程提供基础。它还为进一步了解 DNA 拓扑结构奠定了基础，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 综合征和亨廷顿舞蹈病）中发挥着重要作用。