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The ATPase activity of the DNA transporter TrwB is modulated by protein TrwA. Implications for a common assembly mechanism of DNA translocating motors

AuthorsTato, Irantzu; Matilla, Inmaculada ; Arechaga, Ignacio ; Zunzunegui, Sandra ; Cruz, Fernando de la ; Cabezón, Elena
Issue Date2007
PublisherAmerican Society for Biochemistry and Molecular Biology
CitationJournal of Biological Chemistry 282(35): 25569-25576 (2007)
AbstractConjugative systems contain an essential integral membrane protein involved in DNA transport called the Type IV coupling protein (T4CP). The T4CP of conjugative plasmid R388 is TrwB, a DNA-dependent ATPase. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA through its central channel by a mechanism similar to that used by F1-ATPase or ring helicases. For DNA transport, TrwB couples the relaxosome (a DNA-protein complex) to the secretion channel. In this work we show that TrwA, a tetrameric oriT DNA-binding protein and a component of the R388 relaxosome, stimulates TrwBΔN70 ATPase activity, revealing a specific interaction between the two proteins. This interaction occurs via the TrwA C-terminal domain. A 68-kDa complex between TrwBΔN70 and TrwA C-terminal domain was observed by gel filtration chromatography, consistent with a 1:1 stoichiometry. Additionally, electron microscopy revealed the formation of oligomeric TrwB complexes in the presence, but not in the absence, of TrwA protein. TrwBΔN70 ATPase activity in the presence of TrwA was further enhanced by DNA. Interestingly, maximal ATPase rates were achieved with TrwA and different types of dsDNA substrates. This is consistent with a role of TrwA in facilitating the interaction between TrwB and DNA. Our findings provide a new insight into the mechanism by which TrwB recruits the relaxosome for DNA transport. The process resembles the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB system, to be targeted to the DNA followed by hexamer assembly. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
Identifiersdoi: 10.1074/jbc.M703464200
issn: 0021-9258
Appears in Collections:(IBBTEC) Artículos
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