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

University Of California-Santa Barbara, Santa Barbara CA

Investigators

Abstract

0074674 Horton An 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.