RUI: Defining the thermodynamics of MarR family transcription factor interactions with DNA

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MarR family proteins are found in most bacterial species. These proteins bind very tightly to DNA and regulate the expression of genes that allow the bacterium to respond to various forms of environmental stress, including noxious chemicals and antibiotics. MarR proteins bind DNA with a common structural motif, yet each is specific for a particular sequence. The energetic basis for the affinity of these proteins for their target DNA sequences is not well understand. This project seeks to determine the structural components of MarR proteins that drive association with DNA. These studies will provide a detailed understanding of how MarR proteins interact with DNA, and are also expected to provide deeper insight into the energetics of sequence-specific protein-DNA interactions in general. In addition, these investigations will provide the first description of the roles of MarR proteins in regulating genes in the pathogenic bacterium Clostridium difficile and offer insight into this organism’s response to changing environmental conditions and stresses, including its resistance to antibiotics and antimicrobials. The project will be conducted at a primarily undergraduate institution and provide substantive training opportunities for undergraduate students who will participate in all phases of the project. This diverse group of students will conduct independent research using modern techniques in biochemistry, which will prepare them for advanced studies and careers in the STEM workforce. MarR proteins associate with DNA through a winged-helix-turn-helix (wHTH) motif, with nearly all interactions involving the wHTH “recognition helix” and “wing” which form base-specific contacts with the DNA major and minor grooves, respectively. However, the thermodynamics of these high-affinity associations is poorly understood. While DNA binding proteins have traditionally been classified as either major groove binders or minor groove binders, with each class being associated with a distinct thermodynamic signature, MarR proteins do not fall neatly into either category since they associate with DNA through extensive base-specific contacts in both grooves. The first objective of this project is to determine if there is a thermodynamic signature that characterizes MarR family binding interactions with DNA and to parse the energetic contributions to binding that stems from protein interactions with the major and minor grooves. More broadly, these findings will test the prevailing view that such interactions each have a characteristic thermodynamic signature. These objectives will be met through a systematic analysis of DNA binding thermodynamics involving six MarR homologs, including two which were previously structurally-defined, as well as four previously uncharacterized MarR homologs from the bacterium, Clostridium difficile.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.