Stuctural basis of polymerase recruitment to promoters

在原核生物和真核生物中基因表达的起始需要与RNA聚合酶相互作用以将聚合酶募集到启动子的特异性因子。 原核生物中的募集是通过σ因子完成的，σ因子识别特定的DNA序列并将聚合酶递送到转录起始位点附近。 Sigma因子已经抵抗了超过25年的详细结构/功能分析，并且关于Sigma因子/DNA识别事件的确切性质几乎没有明确的信息。 初步数据表明，E。大肠杆菌，sigma 70，现在就在手边。通过从两种嗜热生物中克隆出密切相关的因子，将从分子上详细地确定sigma 70的蛋白质/DNA相互作用。突变和足迹分析将补充结构阐明，以提供一个全面的观点到sigma 70/DNA相互作用。 此外，σ 70的C-末端区域4的相互作用将进一步表征为噬菌体T4的转录抑制物/抗σ因子AsiA，作为区域4如何作为原核基因的激活子和抑制子的通信位点的实例。 在这方面，一个意想不到的化学计量的区域4/AsiA复合物已被确定，导致有趣的假设，即AsiA作为一个DNA模拟取代sigma 70的DNA。 描述了解决蛋白质、蛋白质/DNA和蛋白质/蛋白质复合物的新方法。 真核生物的聚合酶募集因子的“类似物”也将在本提案中进行研究。 真核蛋白是Rap 30，TFIIF的一个亚基，与sigma 70具有序列和功能同源性。 Rap 30 DNA结合域已被确定，其三维结构和与DNA接触的蛋白质残基的鉴定都已被确定为初步数据的一部分。 与sigma 70不同，Rap 30不具有序列偏好性，并且不以高亲和力结合DNA。为了接近其与DNA结合的三维结构的解决方案，已经开发了用于多核NMR光谱的新的标记方案，从而允许非特异性蛋白质/DNA相互作用复杂问题的一般解决方案。 在这方面，最感兴趣的是一个强大的DNA标记方案，它允许以新的方式分析DNA结构的发展，是必不可少的复杂结构的解决方案。由此产生的结构将提供新的见解Rap 30在DNA包装的真核前起始复合物的作用。 一个真核preinitiation复合物的初步模型。

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.