Biochemical and structural analysis of Salmonella typhimurium conjugative factor TraK (2014)

Undergraduates: Erin Moore, Krystle J. McLaughlin

Faculty Advisor: Matthew Redinbo
Department: Chemistry

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The human health threat posed by multi-drug resistant bacteria necessitates an understanding of the mechanisms underlying the spread of this resistance. Bacteria can propagate genes encoding antibiotic resistance through a process known as conjugative plasmid transfer (CPT). To initiate CPT, conjugative proteins assemble into the relaxosome, a multi-protein complex necessary for sequence-specific plasmid transfer. The relaxase, a component of the relaxosome, nicks DNA at the plasmid origin of transfer (oriT) and a helicase subsequently unwinds the DNA for cell-to-cell transfer. In Salmonella typhimurium plasmid pCU1, the conjugative protein TraK is hypothesized help form the relaxosome and play a central role in binding and stabilizing DNA. TraK oligomerization and site-specific binding affinity to the pCU1 oriT were analyzed through atomic force microscopy and fluorescence-anisotropy based DNA binding assays. We found that TraK primarily exists in a tetramer-dimer equilibrium and weakly binds to multiple regions of the pCU1 oriT. Further assessments are currently underway to elucidate TraK structure as well as its specificity and mechanism of DNA binding essential for CPT.