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

综合生化和x射线晶体学分析将用于确定大肠杆菌巴氏操纵子转录激活的结构和能量基础。pap操纵子由编码细菌菌毛附属物蛋白质的基因组成。先前的工作已经建立了一种称为相位变化控制机制的调节模型，其中两个GATC序列的甲基化状态决定了二聚体亮氨酸响应调节蛋白的结合位点。上游gtac - i的甲基化导致Lrp与启动子近端位点结合，RNA聚合酶的位阻和转录抑制。如果GATC-I被未甲基化，Lrp二聚体会转移到由共调节蛋白PapI介导的上游位点，GATC-II被甲基化，转录被激活。本课题的实验将为进一步评价和探索该相位变化模型提供结构和定量基础。热力学解离常数将通过凝胶位移分析来测量Lrp与野生型和选定突变位点的结合，并探索GATC甲基化和Pap I结合的后果。x射线晶体学将确定无配体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射线晶体学数据将被分析并用于开发DNA控制区结构的模型。这是一个power奖，将允许PI，谁目前是一个研究助理，建立一个独立的研究项目。

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.