Stuctural basis of polymerase recruitment to promoters

在原核生物和真核生物中启动基因表达需要特定的因子，这些因子与RNA聚合酶相互作用，将聚合酶招募到启动子。在原核生物中的募集是由西格玛因子完成的，它识别特定的DNA序列，并将聚合酶运送到转录起始点附近。Sigma因子拒绝详细的结构/功能分析已经超过25年，关于Sigma因子/DNA识别事件的确切性质的确切信息很少被阐明。这项提案中的初步数据表明，解决大肠杆菌中主要西格玛因子的三维结构，即西格玛70，现在已经近在咫尺。通过从两个嗜热生物中克隆密切相关的因子，将从分子细节上定义sigma70的蛋白质/DNA相互作用。突变和足迹分析将补充结构阐明，提供对sigma70/DNA相互作用的全面看法。此外，Sigma70的C-末端区域4的相互作用将进一步表征噬菌体T4的转录抑制因子/抗Sigma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中心的例子。在这方面，已经发现了区域4/亚洲复合体的一个意想不到的化学计量比，导致了一个有趣的假设，即亚洲作为DNA模拟物从DNA中取代了sigma70。描述了解决蛋白质、蛋白质/DNA和蛋白质/蛋白质复合体的新方法。在这项提案中，还将研究真核生物中的聚合酶招募因子的“模拟”。该真核蛋白为Rap30，是TFIIF的一个亚基，与Sigma70具有序列和功能上的同源性。Rap30DNA结合域已经确定，其三维结构和与DNA接触的蛋白质残基的鉴定都是初步数据的一部分。与Sigma70不同，Rap30不具有序列偏好，也不以高亲和力结合DNA。为了探索其与DNA结合的三维结构的解决方案，人们开发了新的多核核磁共振谱标记方案，从而为非特异性蛋白质/DNA相互作用复杂问题提供了一个通用的解决方案。在这方面最感兴趣的是开发一种强大的DNA标记方案，该方案允许以新的方式分析DNA结构，并且对于解决复杂结构是必不可少的。所得到的结构将为RAP30在真核预起始复合体的DNA包装中的作用提供新的见解。提出了一个真核生物预引发复合体的初步模型。

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.