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

Descriptions

Other title
Subject/Keyword
conjugation
F plasmid
bacteria
x-ray crystallography
structural biology
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Wong, Joyce JW
Supervisor and department
Glover, J.N. Mark (Biochemistry)
Examining committee member and department
Schumacher, Maria (Biochemistry)
Hazes, Bart (Biochemistry and Medical Microbiology and Immunology)
Frost, Laura .S. (Biological Sciences)
Lemieux, M. Joanne (Biochemistry)
Department
Department of Biochemistry
Specialization

Date accepted
2012-04-27T08:55:23Z
Graduation date
2012-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
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.
Language
English
DOI
doi:10.7939/R3NQ2R
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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