Promoter Recognition in a T7 RNA Polymerase Model System

9308670马丁，这项建议的具体目的是测试和进一步完善一个新的启动子识别结构模型，并将该模型扩展到包括关于转录早期阶段的机械细节。通过在蛋白质中引入功能基团取代，可以在启动子的三个区域详细探测关键的蛋白质-DNA相互作用：建议的上游双链识别区域，起始点附近的单链区域，以及之间鲜为人知的过渡区。将对这一系统采用各种方法，以补充已建立的动力学研究，并更好地了解具体相互作用的化学性质。滴定微量热法将被开发来更详细地探测蛋白质与DNA的结合作用。将非干扰光激活核苷酸特异地掺入启动子DNA中，将为进一步的蛋白质诱变研究提供必要的方向。%本研究的目的是了解转录的酶学，包括转录起始的化学基础。T7家族的RNA聚合体提供了一个理想的模型系统，在其中不仅可以研究序列特异性大分子相互作用的基本方面，而且还可以研究聚合酶的基本机制。为了加深我们对识别界面的理解，我们将确定DNA和蛋白质中简单的化学修饰的效果。****

9308670 Martin The specific aims of this proposal are to test and to further refine an emerging structural model for promoter recognition and to extend that model to include mechanistic detail regarding the early stages of transcription. The incorporation of functional group substitutions in the protein allow detailed probing of critical protein-DNA interactions in 3 areas of the promoter: a proposed duplex recognition domain upstream, a single stranded region near the start site, and the poorly understood transition region in between. A variety of approaches will be applied to this system to complement well-established kinetic studies and to provide a better understanding of the chemical nature of specific interactions. Titration microcalorimetry will be developed to probe protein - DNA binding interactions in more detail. Specific chemical incorporation of nonperturbing photoactivatable nucleotides into promoter DNA will provide much needed direction to further protein mutagenesis studies. %%%% The goal of this research is to understand the enzymology of transcription, including the chemical basis of transcript initiation. The T7 family of RNA polymerses present an ideal model system in which to study not only the fundamental aspects of sequence-specific macromolecular interactions, but also the basic mechanisms of polymerases. The effects of simple chemical modifications in the DNA and the protein will be determined, in order to refine our understanding of the recognition interface. ****