Promoter Recognition in a T7 RNA Polymerase Model System

该提案的具体目的是测试和进一步完善启动子识别的新兴结构模型，并扩展该模型以包括有关转录早期阶段的机制细节。 在蛋白质中掺入功能基团取代允许详细探测启动子的3个区域中的关键蛋白质-DNA相互作用：上游提出的双链体识别结构域，起始位点附近的单链区域，以及之间知之甚少的过渡区。 各种方法将被应用到这个系统，以补充完善的动力学研究，并提供更好地了解特定的相互作用的化学性质。 滴定微量热法将被开发用于更详细地探测蛋白质- DNA结合相互作用。 将非干扰性的光活化核苷酸特异性地掺入到启动子DNA中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。 RNA聚合酶T7家族是研究序列特异性大分子相互作用和聚合酶基本机制的理想模型。 DNA和蛋白质中简单化学修饰的影响将被确定，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. ****