UV Resonance Raman and Fluorescence Spectroscopic Investigations of DNA Structure and Protein-DNA Complexes

许多涉及核酸的过程，如复制和转录，都涉及复杂结构的形成，其中DNA由于不同蛋白质的结合而被迫采用环状或弯曲的形状。该项目重点研究形成环状DNA结构所需的蛋白质与DNA的相互作用，以确定形成环状结构所需的重要接触和形成环状结构所需的能量。本研究的一个核心问题是DNA结构是否在这些复杂的组装体中发挥蛋白质结合和识别的作用。两种不同的方法用于研究相互作用：荧光光谱和激光技术，紫外共振拉曼光谱。使用荧光法，将检查配合物的性质和配合物的整体结构。这是通过两种不同类型的荧光实验来完成的：测量局部DNA柔韧性和结合亲和力的各向异性；和共振能量转移来测量蛋白质和DNA上特定位点之间的相对距离。拉曼技术监测分子的内部振动，这是对分子结构和键合的敏感测量。使用这种技术，可以选择性地检查蛋白质或DNA的不同部分，从而可以直接调查重要区域和接触点。这些实验探讨了调节蛋白和基因之间形成的复合物，有助于当前对细胞中基因组过程如何被调节的理解。更广泛的影响：本科生和研究生的教学和培训是这项工作产生的主要教育影响。该项目将营造一个促进包括课堂在内的各级研究和教育一体化的环境。受资助机构的几门课程将直接借鉴该项目的研究。另一个影响来自代表性不足的群体参与研究和致力于提高妇女在科学领域的地位。这项工作加强了卫斯理大学的研究基础设施，其中包括建立用于荧光寿命研究的多用户时间相关单光子计数仪器。卫斯理大学的这些仪器将供当地科学家使用。

Many processes involving nucleic acids, such as replication and transcription, involve the formation of complex structures, wherein the DNA is forced to adopt a looped or bent shape because of the binding of different proteins. This project focuses on the interaction of proteins, required for forming looped DNA structures, with DNA in order to determine the contacts that are important for forming the looped structures and the energy needed to form the loop. A central question of this research is whether DNA structure plays a role in protein binding and recognition in these complex assemblies. Two different methods are used to study the interactions: fluorescence spectroscopy and a laser technique, UV resonance Raman spectroscopy. Using the fluorescence method, the nature of the complexes and the overall architecture of the complexes will be examined. This is accomplished by two different types of fluorescence experiments: anisotropy to measure local DNA flexibility and binding affinity; and resonance energy transfer to measure the relative distance between specific sites on the proteins and the DNA. The Raman technique monitors the internal vibrations of molecules, which are a sensitive measure of the structure and bonding of a molecule. Using this technique, different parts of the protein or the DNA can be selectively examined, which allows direct investigation of the important regions and contact points. These experiments probe the complexes formed between regulatory proteins and genes and contribute towards current understanding of how genomic processes are regulated in the cell. Broader impacts: Teaching and training of undergraduate and graduate students are the main educational impacts that result from this work. The project will foster an environment that promotes the integration of research and education at all levels, including the classroom. Several of the courses at the grantee institution will draw directly from research in this project. An additional impact stems from the participation of underrepresented groups in the research and from a commitment to the advancement of women in science. The research infrastructure of Wesleyan University is enhanced by this work, which involves the building of a multi-user time-correlated single photon counting instrument for fluorescence lifetime studies. This instrumentation housed at Wesleyan will be available to local scientists.