Promoter Recognition in a T7 RNA Polymerase Model System

这个提议的具体目的是测试和进一步完善一个新兴的启动子识别结构模型，并扩展该模型，以包括关于转录早期阶段的机制细节。在蛋白质中加入功能基取代可以详细探测启动子3个区域的关键蛋白质- dna相互作用：上游的双链识别域，起始位点附近的单链区域，以及两者之间鲜为人知的过渡区域。各种方法将被应用于这个系统，以补充已建立的动力学研究，并提供对特定相互作用的化学性质的更好理解。滴定微热法将被发展用于更详细地探测蛋白质- DNA结合相互作用。在启动子DNA中加入非干扰的光激活核苷酸将为进一步的蛋白质诱变研究提供急需的方向。%%%%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RNA聚合体提供了一个理想的模型系统，不仅可以研究序列特异性大分子相互作用的基本方面，还可以研究聚合酶的基本机制。简单的化学修饰对DNA和蛋白质的影响将被确定，以完善我们对识别界面的理解。​

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. ****