POWRE: The Structural and Energetic Basis of Transcriptional Control in the E. coli pap Operon

POWRE：大肠杆菌操纵子转录控制的结构和能量基础

• 批准号: 0074674
• 负责人: Nancy Horton
• 金额: --
• 依托单位: University of California-Santa Barbara
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2002-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0074674&HistoricalAwards=false
• 关键词: POWRE; Structural; Energetic; Basis; Transcriptional

0074674HortonAn integrated biochemical and X-ray crystallographic analysis will be used to determine the structural and energetic basis for transcriptional activation in the E.coli pap operon. The pap operon consists of genes encoding the proteins of the pili appendages of bacteria. Previous work has led to a model for regulation known as the phase variation control mechanism, in which the methylation state of two GATC sequences determines the binding sites for the dimeric leucine-responsive regulatory protein. Methylation of upstream GATC-I results in Lrp binding to promoter-proximal sites, steric blockage of RNA polymerase and transcriptional repression. If GATC-I becomes umethylated Lrp dimers translocate to upstream sites mediated by the coregulator protein PapI and GATC-II is methylated and transcription is activated. The experiments in this grant will provide a structural and quantitative foundation to evaluate and further explore this phase variation model. Thermodynamic dissociation constants will be measured by gel shift analysis for Lrp binding to wild-type and selected mutant sites, and the consequences of GATC methylation and Pap I binding will be explored. Xray crystallography will determine the high resolution atomic structure of unliganded Lrp, Lrp-DNA complexes, and ternary Lrp-PapI-DNA complexes. These biochemical and Xray crystallographic data will be analyzed and used to develop models for the structure of the DNA control region. This is a POWRE award which will allow the PI, who is currently a research associate, to establish and independent research project.

0074674Hortonan综合生化和X射线晶体学分析将用于确定大肠杆菌PAP操纵子中转录激活的结构和能量基础。子宫颈抹片操纵子由编码细菌pili附属物的蛋白质的基因组成。先前的工作导致了一种被称为相变控制机制的调节模型，其中两个GATC序列的甲基化态确定了二聚体亮氨酸反应调节蛋白的结合位点。上游GATC-I的甲基化导致LRP结合与启动子螺旋位点，RNA聚合酶的空间阻滞和转录抑制。如果GATC-I变为甲基化的LRP二聚体，转移到由Coregulator蛋白Papi介导的上游位点，而GATC-II则是甲基化的，并激活了转录。该赠款中的实验将为结构和定量基础提供评估和进一步探索该相变模型的基础。热力学解离常数将通过凝胶移位分析来测量LRP与野生型和选定的突变位点结合，并将探索GATC甲基化和PAP I结合的后果。 X射线晶体学将确定非配体LRP，LRP-DNA复合物和三元LRP-PAPI-DNA复合物的高分辨率原子结构。这些生化和X射线晶体学数据将进行分析，并用于开发DNA控制区域结构的模型。这是POWRE奖，它将允许PI（目前是研究助理）建立和独立研究项目。