Stuctural basis of polymerase recruitment to promoters

在原核生物和真核生物中，基因表达的启动需要特定的因子与RNA聚合酶相互作用，将聚合酶招募到启动子上。原核生物的招募是由识别特定DNA序列并将聚合酶传递到转录起始位点附近的sigma因子完成的。超过25年来，Sigma因子一直抵制详细的结构/功能分析，并且很少有关于Sigma因子/DNA识别事件的确切性质的明确信息被阐明。本提案中的初步数据表明，大肠杆菌中主要sigma因子sigma70的三维结构的解决方案现在已经触手可及。通过克隆两种嗜热生物中密切相关的因子，将从分子细节上定义sigma70蛋白/DNA相互作用。诱变和足迹分析将补充结构阐明，以提供对sigma70/DNA相互作用的全面看法。此外，sigma70的c端4区将进一步表征T4噬菌体的转录抑制因子/抗sigma因子AsiA的相互作用，作为4区如何作为原核基因激活因子和抑制因子的通讯位点的一个例子。在这方面，一个意想不到的区域4/AsiA复合体的化学计量学已经被确定，导致了一个有趣的假设，即AsiA作为DNA模拟物取代了DNA中的sigma70。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。来自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。该真核蛋白是TFIIF的一个亚基Rap30，与sigma70具有相同的序列和功能同源性。Rap30 DNA结合结构域已经被鉴定，其三维结构和与DNA接触的蛋白残基的鉴定都已经确定，作为初步数据的一部分。与sigma70不同，Rap30不具有序列偏好，也不具有高亲和力结合DNA。为了解决其与DNA结合的三维结构，已经开发了新的多核磁共振波谱标记方案，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。在这方面最令人感兴趣的是一种强大的DNA标记方案的发展，该方案允许以新的方式分析DNA结构，对复杂结构的解决至关重要。所得到的结构将为Rap30在真核生物起始前复合物的DNA包裹中的作用提供新的见解。提出了真核生物起始前复合物的初步模型。

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.