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

0074674荷尔蒙综合生化和X射线结晶学分析将被用来确定E.ColiPAP操纵子转录激活的结构和能量基础。纸操纵子由编码细菌菌毛附属物蛋白质的基因组成。以前的工作导致了一个被称为相位变化控制机制的调节模型，在这个模型中，两个GATC序列的甲基化状态决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与启动子近端结合，RNA聚合酶空间位阻和转录抑制。如果GATC-I被非甲基化，LRP二聚体在辅调节蛋白PAPI和GATC-II的介导下转移到上游位置，则GATC-II甲基化，转录被激活。该基金的实验将为评估和进一步探索这一阶段变化模型提供结构和定量的基础。通过凝胶位移分析LRP与野生型和选定的突变位点的结合，将测量热力学解离常数，并将探索GATC甲基化和Pap I结合的后果。X射线结晶学将确定未连接的LRP、LRP-DNA络合物和三元LRP-PAPI-DNA络合物的高分辨原子结构。这些生化和X射线结晶学数据将被分析并用于开发DNA控制区结构的模型。这是一项POWRE奖，将允许目前担任研究助理的PI建立独立的研究项目。

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.