STRUCTURAL STUDIES OF THE LACTOSE REPRESSOR OF E COLI

转录基因调控是一个激烈的研究领域， 生物学和医学。 E.大肠杆菌是 转录基因调控 它的关键成分lac阻遏物 （lacR），是研究最深入的蛋白质之一， 和生物化学。 未络合物的最新晶体结构 四聚体lacR、与诱导物IPTG结合的lacR和与诱导物IPTG结合的lacR。 操纵基因DNA提供了一个框架， 就像一个基因开关 本提案中的研究将 扩展了我们对lacR结构的理解。 至关 重要的是扩展了lacR结合结构的分辨率 与操作员DNA的联系 由于结构仅在4.8埃处测定， 单位分辨率，目前的模式为运营商绑定，压抑 lacR的构象不包括侧链。 一个大前提是， 这个建议是，当lacR的二聚体形式与 操纵子DNA，将生长出晶体， 比迄今为止对于lacR的四聚体所实现的要多。 二聚lacR 还将用于实现：（1）lacR结合的结构比较 对于野生型和对称算子序列，（2）结构 异乳糖（lacR的天然诱导物）的作用基础，以及 邻硝基苯基岩藻糖苷，lacR的抗诱导剂，和（3）结构 lacR单点突变的基础，导致100倍 增加对操作员的亲和力。

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.