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

0074674 Horton将使用综合的生物化学和X射线晶体学分析来确定大肠杆菌pap操纵子中转录激活的结构和能量基础。pap操纵子由编码细菌皮利附属物蛋白质的基因组成。先前的工作已经导致了一个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亮氨酸响应调节蛋白的结合位点。上游GATC-I的甲基化导致Lrp与启动子近端位点结合，RNA聚合酶的空间阻断和转录抑制。如果GATC-I变成非甲基化的Lrp二聚体，则其易位到由辅助调节蛋白PapI介导的上游位点，并且GATC-II被甲基化并且转录被激活。本基金的实验将为评估和进一步探索这种相位变化模型提供结构和定量的基础。将通过凝胶位移分析测定Lrp与野生型和选定突变位点结合的热力学解离常数，并探讨GATC甲基化和Pap I结合的后果。X射线晶体学将确定未配体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POWRE奖，将允许PI，谁是目前的研究助理，建立和独立的研究项目。

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.