SOLUTION STRUCTURE OF DNA BINDING PROTEINS

真核生物与DNA序列特异性蛋白质相互作用 转录因子ADR 1及其同源DNS UAS 1将被研究。 ADR 1的DNA结合域含有两个Cys 2-His 2锌指基序 其沿着短N-末端序列是必需的， 足以进行高亲和力、特异性的蛋白质-DNA相互作用， 参与酿酒酵母中AHD 2基因的调节。 这两个锌指在过去都被详细研究过 授予期，使用合成指肽和1H同源物 核磁共振（NMR）光谱。 多维异谱核磁共振波谱学的进展使其成为 可以确定大蛋白质的三维结构， 复杂的比已经接近使用homopolysis光谱。 我们建议将这些技术应用于DNA结合区域， 在E.杆菌 表达的蛋白片段保留了较高的 对UAS 1 DNA序列的亲和力。 蛋白质的同位素- 富含15 N和/或13 C和杂原子NMR光谱将被 用于确定蛋白质的结构， 不存在UAS 1 DNA序列。 此外，标记材料 将使我们能够检查蛋白质的动力学特性， DNA的存在和缺失。 一旦详细描述了野生动物的结构和特性- 型蛋白质-DNA复合物已经获得，突变体已被证明 将检查具有改变的DNA结合特性的细胞。 这些研究 将提供大量关于特定蛋白质-DNA的信息 ADR 1锌指的相互作用，特别是Cys 2-His 2 锌指结构。 这项研究的最终目标是 用新的DNA设计新的含锌指蛋白质的能力 结合特异性。

The sequence-specific protein-DNA interaction between the eukaryotic transcription factor, ADR1, and its cognate DNS, UAS1, will be studied. The DNA-binding domain of ADR1 contains two Cys2-His2 zinc finger motifs that, along with a short N-terminal sequence, are necessary and sufficient for the high-affinity, specific protein-DNA interaction that is involved in regulation of the AHD2 gene in Saccharomyces cerevisiae. These two zinc fingers have each been studied in detail over the past granting period, using synthetic finger peptides and 1H homonuclear nuclear magnetic resonance (NMR) spectroscopy. Advances in multidimensional heteronuclear NMR spectroscopy make it possible to determine three-dimensional structure of large proteins and complexes than have been approachable using homonuclear spectroscopy. We propose to apply these techniques to the DNA-binding region has been over-expressed in E. coli. The expressed protein fragment retains high affinity for the UAS1 DNA sequence. The protein will be isotopically- enriched with 15N and/or 13C and heteronuclear NMR spectroscopy will be used to determine the structure of the protein both in the presence and absence of the UAS1 DNA sequence. In addition, the labelled material will allow us to examine dynamical properties of the protein, again in the presence and absence of DNA. Once a detailed description of the structure and properties of the wild- type protein-DNA complex has been obtained, mutants that have been shown to have altered DNA-binding properties will be examined. These studies will provide a wealth of information concerning the specific protein-DNA interactions of the ADR1 zinc fingers in particular and of the Cys2-His2 zinc finger motif in general. An ultimate goal of this research is the ability to design new zinc finger-containing proteins with new DNA binding specificities.