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dc.contributor.authorLarrea, Delfina-
dc.contributor.authorDe Paz, Héctor D.-
dc.contributor.authorMatilla, Inmaculada-
dc.contributor.authorGuzmán-Herrador, Dolores L.-
dc.contributor.authorLasso, Gorka-
dc.contributor.authorCruz, Fernando de la-
dc.contributor.authorCabezón, Elena-
dc.contributor.authorLlosa, Matxalen-
dc.identifierdoi: 10.1007/s00438-017-1331-3-
dc.identifiere-issn: 1617-4623-
dc.identifierissn: 1617-4615-
dc.identifier.citationMolecular Genetics and Genomics 292(5): 1037-1049 (2017)-
dc.description.abstractConjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening–closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA.-
dc.description.sponsorshipThis work was supported by Grants BIO2013-46414-P, BFU2016-78521-R, and BFU2014-55534-C2-1-P from the Spanish Ministry of Economy and Competitiveness to ML, EC, and FdlC, respectively. GL acknowledges funding from NIH Grants GM030518, S10OD012351, and S10OD021764. DL was a recipient of a predoctoral fellowship from CSIC (JAE-PRE). DLG-H and IM were recipients of predoctoral fellowships from the University of Cantabria.-
dc.publisherSpringer Nature-
dc.subjectBacterial conjugation-
dc.subjectDNA transport-
dc.subjectMolecular motors-
dc.subjectType IV secretion systems-
dc.titleSubstrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB-
dc.description.versionPeer Reviewed-
dc.contributor.funderMinisterio de Economía y Competitividad (España)-
dc.contributor.funderNational Institutes of Health (US)-
dc.contributor.funderConsejo Superior de Investigaciones Científicas (España)-
dc.contributor.funderUniversidad de Cantabria-
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