Stuctural basis of polymerase recruitment to promoters

启动子招募聚合酶的结构基础

• 批准号: 6641173
• 负责人: Milton H. Werner
• 金额: $ 26.72万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6641173
• 关键词: DNA; DNA binding protein; DNA directed RNA polymerase; DNA footprinting; Escherichia coli; bacteriophage T4; chemical structure function; computer simulation; conformation; crystallization; gene induction /repression; genetic promoter element; genetic regulation; intermolecular interaction; model design /development; nuclear magnetic resonance spectroscopy; physical model; protein folding; protein purification; site directed mutagenesis; stoichiometry; structural biology; thermophilic organism

Initiation of gene expression in prokaryotes and eukaryotes requires specific factors which interact with RNA polymerase to recruit polymerase to the promoter. Recruitment in prokaryotes is accomplished by sigma factors which recognize specific DNA sequences and deliver polymerase proximal to the start site of transcription. Sigma factors have resisted detailed structure/function analysis for more than 25 years and little definitive information has been elucidated on the precise nature of the sigma factor/DNA recognition event. Preliminary data in this proposal demonstrates that the solution of the three-dimensional structure of the primary sigma factor in E. coli, sigma70, is now at hand. Through the cloning of the closely related factors from two thermophilic organisms, the protein/DNA interaction of sigma70 will be defined in molecular detail. Mutagenic and footprinting analysis will complement structure elucidation to provide a comprehensive view into the sigma70/DNA interaction. In addition, the interaction of the C-terminal region 4 of sigma70 will be further characterized for the transcriptional repressor/anti-sigma factor AsiA of bacteriophage T4 as an example of how region 4 acts as a communication locus for activators and suppressors of prokaryotic genes. In this regard, an unexpected stoichiometry of the region 4/AsiA complex has been identified, leading to the intriguing hypothesis that AsiA acts as a DNA mimic to displace sigma70 from the DNA. Novel approaches to the solution of the protein, protein/DNA and protein/protein complexes are described. The 'analog' of a polymerase recruitment factor from eukaryotes is also to be studied in this proposal. The eukaryotic protein is Rap30, a subunit of TFIIF, which shares sequence and functional homology with sigma70. The Rap30 DNA-binding domain has been identified, its three-dimensional structure and the identification of the protein residues in contact with DNA have all be determined as a part of preliminary data. Unlike sigma70, Rap30 does not possess sequence preferences and does not bind DNA with high affinity. To approach the solution of its three-dimensional structure bound to DNA, novel labeling schemes for multinuclear NMR spectroscopy have been developed, permitting a general solution to the non- specific protein/DNA interaction complex problem. Of greatest interest in this regard is the development of a robust DNA labeling scheme which permits analysis of DNA structure in new ways and is essential to the solution of the complex structure. The resultant structure will provide new insight into the role of Rap30 in DNA wrapping for a eukaryotic pre-initiation complex. A preliminary model of a eukaryotic preinitiation complex is presented.

原核生物和真核生物中基因表达的启动需要与RNA聚合酶相互作用的特定因素，以募集聚合酶与启动子。 原核生物中的募集是通过识别特定DNA序列并为转录开始部位提供聚合酶的Sigma因子来完成的。 Sigma因子已经抵抗了25年以上的详细结构/功能分析，并且几乎没有确定的确切信息在Sigma因子/DNA识别事件的确切性质上。 该提案中的初步数据表明，在大肠杆菌（Sigma70）中，主要sigma因子的三维结构的解决方案现已介绍。通过从两种嗜热生物的紧密相关因素克隆，Sigma70的蛋白/DNA相互作用将以分子细节定义。诱变和足迹分析将补充结构阐明，以提供对Sigma70/DNA相互作用的全面视图。 此外，Sigma70的C末端区域4的相互作用将进一步为噬菌体T4的转录阻遏物/抗杀菌因子亚洲亚洲，作为区域4如何充当激活剂和降低原基因抑制因子的示例。 在这方面，已经确定了区域4/亚洲复合物的意外化学计量法，从而引起了一个有趣的假设，即亚洲充当DNA模拟于从DNA中取代Sigma70的DNA。 描述了蛋白质，蛋白质/DNA和蛋白质/蛋白质复合物溶液的新方法。 该提案还可以研究来自真核生物的聚合酶募集因子的“类似物”。 真核蛋白是TFIIF的亚基Rap30，它与Sigma70共享序列和功能同源性。 已经鉴定出Rap30 DNA结合结构域，其三维结构以及与DNA接触的蛋白质残基的鉴定都被确定为初步数据的一部分。 与Sigma70不同，RAP30不具有序列偏好，也不结合具有高亲和力的DNA。为了接近与DNA结合的三维结构的溶液，已经开发了用于多核NMR光谱的新型标记方案，从而允许对非特异性蛋白/DNA相互作用复杂问题的一般解决方案。 在这方面的最大兴趣是发展强大的DNA标记方案，该方案允许以新的方式分析DNA结构，对于复杂结构的解决方案至关重要。最终的结构将为真核前发射复合物在DNA包裹中的作用提供新的见解。 提出了真核生物前启动复合物的初步模型。