Transcriptional Control of the E. coli Pap Operon

大肠杆菌巴氏操纵子的转录控制

• 批准号: 6621909
• 负责人: JOHN J. PERONA
• 金额: $ 25.32万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6621909
• 关键词: DNA methylation; Escherichia coli; X ray crystallography; bacterial genetics; bacterial proteins; binding sites; genetic screening; genetic transcription; intermolecular interaction; operon; pilus; protein structure; site directed mutagenesis; transcription factor

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.

描述(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 提出了大肠杆菌纸操纵子。遗传学、生化和X射线 结晶学实验将被用来测试一个优雅的模型 调节称为相变控制机制。此模型调用 两个上游GATC序列的DNA甲基化状态是一个关键因素 确定六个可能的结合位置中的哪些由 亮氨酸反应调节蛋白(LRP)。LRP二聚体的移位 沿着DNA，在共激活蛋白PAPI的介导下， 对Rina聚合酶结合和随后的转录是必不可少的。严谨 生化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 ，以及这是如何由 生理上相关的效应器。几个体内的遗传选择将 也可用于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 将使用结晶学来确定高分辨率的原子结构 几种不同结构的二元LRP-DNA络合物的未连接LRP LRP-PAPI-DNA三元复合体。分析表明， 对甲基化影响的基础有相当广泛的见解 蛋白质-DNA相互作用，以及蛋白质诱导的和 高阶核蛋白形成过程中的本征A束弯曲 结构。受该操纵子调控的菌毛蛋白是 细菌靶向人尿路上皮细胞表面的研究 尿路。因此，关于纸张管制详细信息应有助于 设计抑制寄主定植和致病性的策略。