Transcriptional Control of the E. coli Pap Operon

描述（由申请人提供）：一项多学科研究，旨在 揭示转录激活的结构和能量基础， 急诊coli pap操纵子。遗传、生化和X射线 晶体学实验将被用来测试一个优雅的模型， 调节称为相位变化控制机制。该模型调用 两个上游GATC序列的DNA甲基化状态作为关键因素 确定六个可能的结合位点中的哪一个被 亮氨酸反应调节蛋白（Lrp）。Lrp二聚体的易位 沿着DNA，在由辅激活蛋白PapI介导的过程中， 对于RINA聚合酶结合和随后的转录是必需的。严格 生物化学实验将提供一套全面的结合自由能 描述Lrp蛋白固有DNA序列偏好的参数 对于各个站点，以及如何通过 生理相关效应物。几种体内遗传选择将 也可以应用于Lrp，以确定参与Lrp的特定氨基酸。 对DNA甲基化和PapI结合的反应性。最后，X光 晶体学将被用来确定高分辨率的原子结构 未配体的Lrp，二元Lrp-DNA复合物与几个不同的 单位点，和三元Lrp-PapI-DNA复合物。分析表明， 对甲基化作用的基础有相当多的一般性了解， 蛋白质-DNA相互作用，以及蛋白质诱导和 高级核蛋白形成中的内在A束弯曲 结构由该操纵子调控的皮利蛋白对于 将细菌靶向至人膀胱上皮细胞的表面 泌尿系统因此，关于pap监管的详细信息应有助于 设计抑制宿主定殖和致病性的策略。

DESCRIPTION (provided by applicant): A multidisciplinary study aimed at uncovering the structural and energetic basis for transcriptional activation in the E. coli pap operon is proposed. Genetic, biochemical and X-ray crystallographic experiments will be employed to test an elegant model for regulation known as the phase variation control mechanism. This model invokes the DNA methylation state of two upstream GATC sequences as a key factor determining which of six possible binding sites are occupied by the leucine-responsive regulatory protein (Lrp). The translocation of Lrp dimers along the DNA, in a process mediated by the coactivator protein PapI, is essential for RINA polymerase binding and subsequent transcription. Rigorous biochemical experiments will provide a comprehensive set of binding free-energy parameters describing the inherent DNA sequence preference of the Lrp protein for individual sites, together with how this is modulated by the physiologically relevant effectors. Several in vivo genetic selections will also be applied to Lrp, to identify specific amino acids involved in the responsiveness to DNA methylation and to PapI binding. Finally, X-ray crystallography will be used to determine the high-resolution atomic structure of unliganded Lrp, of the binary Lrp-DNA complexes with several different single-sites, and of the ternary Lrp-PapI-DNA complex. The analyses promise considerable general insight into the basis of methylation effects on protein-DNA interactions, as well as the interplay of protein-induced and intrinsic A-tract bending in the formation of higher-order nucleoprotein structure. The pili proteins regulated by this operon are essential for targeting of bacteria to the surface of uroepithelial cells lining the human urinary tract. Detailed information on pap regulation should thus be helpful in designing strategies to inhibit host colonization and pathogenicity.