STRUCTURAL STUDIES OF THE LACTOSE REPRESSOR OF E COLI

转录基因调控是一个非常热门的研究领域 在生物学和医学上。大肠杆菌的乳胶操纵子是 转录基因调控。它的关键成分，乳胶抑制物 (LACR)，是遗传学上研究最深入的蛋白质之一 从生化角度来看。非络合物的最新晶体结构 四聚体LACR，LACR与诱导剂IPTG结合，LACR与 操作员DNA提供了一个框架来理解LACR如何 起到了基因开关的作用。这项提案中的研究将 扩展我们目前对LACR的结构理解。至高无上的 重要的是扩展LACR界的结构的分辨率 致DNA操作员。因为只确定了4.8埃的结构 单位分辨率，当前模型为运算符界，被抑制 LACR的构象不包括侧链。的一个主要前提 这一提议是LACR的二聚体版本，当复合到 操作者DNA，将生长出衍射到更高分辨率的晶体 比到目前为止LACR的四聚体所达到的水平更高。二聚体LACR 还将用于实现：(1)LACR界限的结构比较 对于野型和对称算子序列，(2)一种结构 LACR的天然诱导剂--别乳糖的作用基础 邻硝基苯岩藻糖苷，LACR的抗诱导剂，和(3)结构 LACR单点突变的基础，导致100倍 提高了对运营商的亲和力。

Transcriptional gene regulation is an intense field of research in biology and in medicine. The lac operon of E. coli is the paradigm for transcriptional gene regulation. Its key component, the lac repressor (lacR), is one of the most intensely studied proteins both genetically and biochemically. Recent crystal structures of the uncomplexed tetrameric lacR, lacR bound to the inducer IPTG, and lacR bound to operator DNA have provided a framework for understanding how lacR functions as a genetic switch. The research in this proposal will extend our current structural understanding of lacR. Of paramount importance is extending the resolution of the structure of lacR bound to operator DNA. Since the structure was determined at only 4.8Angstrom units resolution, the current model for the operator-bound, repressed conformation of lacR does not include side chains. A major premise of this proposal is that a dimeric version of lacR, when complexed to operator DNA, will grow crystals which diffract to higher resolution than has so far been achieved for the tetramer of lacR. Dimeric lacR will also be used to achieve: (1) a structural comparison of lacR bound to both wild-type and symmetric operator sequences, (2) a structural basis for the action of allolactose, the natural inducer of lacR, and orthonitrophenylfucoside, an anti-inducer of lacR, and (3) a structural basis for single point mutations in lacR which lead to 100-fold increased affinity for operator.