Structural basis of TraD and sbmA recognition by TraM of F-like plasmids

  • Author / Creator
    Wong, Joyce JW
  • Bacterial conjugation is the process of plasmid DNA transfer from a donor cell to a recipient cell. This process is mediated in F-like plasmids by proteins expressed by the tra operon. The relaxosome forms at oriT (origin of transfer), where the nicking and unwinding of a single-stranded copy of the plasmid begins, and the transferosome forms a transmembrane pore through which the DNA is transferred.
    TraM is a tetrameric relaxosomal protein which binds to 3 sites at oriT –sbmA, sbmB, and sbmC. TraD is an inner membrane protein of the transferosome that is homologous to FtsK/SpoIIIE hexameric ATPases. The interaction between the C-terminal tail of TraM and TraD is essential for high conjugation efficiency. The structural basis of this interaction is revealed by the crystal structure of F TraM58-127 in complex with TraD711-717. Electrostatic complementarity is a key feature of TraM-TraD interaction, which includes the TraM K99-TraD D715 and TraM R110-TraD F717 C-terminal carboxylate interactions. An additional feature is the fit of the phenyl side chain of F717 into a hydrophobic pocket. The importance of the TraD C-terminal tail for binding to TraM was tested with a pulldown assay comparing TraD constructs with and without a C-terminal truncation of 8 residues. In vivo assays confirmed the role of the C-terminal tail and its individual residues in conjugation.
    TraM interacts with sbmA in a highly plasmid-specific manner. The basis of this is revealed by the crystal structure of pED208 TraM in complex with sbmA. The structure shows that the N-terminal domain of TraM is a dimeric ribbon-helix-helix fold which recognizes the DNA bases which make up the binding motif. Two tetramers are bound to sbmA on opposite faces of the DNA without protein-protein contact, confirming the TraM-sbmA binding stoichiometry obtained from various biophysical methods. The cooperative nature of TraM binding to sbmA is therefore entirely through DNA distortions observed in the crystal structure, which include underwinding and kinking. Efforts to determine the structural basis of F TraM-sbmA interaction were undertaken but no diffraction-quality crystals were obtained.

  • Subjects / Keywords
  • Graduation date
    Fall 2012
  • Type of Item
  • Degree
    Doctor of Philosophy
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    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
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  • Institution
    University of Alberta
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  • Department
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Frost, Laura .S. (Biological Sciences)
    • Hazes, Bart (Biochemistry and Medical Microbiology and Immunology)
    • Lemieux, M. Joanne (Biochemistry)
    • Schumacher, Maria (Biochemistry)