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dc.contributor.authorWong, Emily Ven
dc.contributor.authorCao, Wenxiangen
dc.contributor.authorVörös, Juditen
dc.contributor.authorMerchant, Moniqueen
dc.contributor.authorModis, Yorgoen
dc.contributor.authorHackney, David Den
dc.contributor.authorMontpetit, Benen
dc.contributor.authorDe La Cruz, Enrique Men
dc.date.accessioned2016-03-03T12:35:07Z
dc.date.available2016-03-03T12:35:07Z
dc.date.issued2016-01-29en
dc.identifier.citationWong et al. Journal of Molecular Biology (2015) Vol. 428, Issue 2, Part B,pp. 492–508. doi: 10.1016/j.jmb.2015.12.018en
dc.identifier.issn0022-2836
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/254132
dc.description.abstractmRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. Although Dbp5 has measurable ATPase activity alone, several regulatory factors (e.g., RNA, nucleoporin proteins, and the endogenous small molecule InsP6) modulate catalytic activity in vitro and in vivo to facilitate mRNA export. An analysis of the intrinsic and regulator-activated Dbp5 ATPase cycle is necessary to define how these factors control Dbp5 and mRNA export. Here, we report a kinetic and equilibrium analysis of the Saccharomyces cerevisiae Dbp5 ATPase cycle, including the influence of RNA on Dbp5 activity. These data show that ATP binds Dbp5 weakly in rapid equilibrium with a binding affinity (KT~4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (KD~0.4 mM). The overall intrinsic steady-state cycling rate constant (kcat) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RNA increases kcat and rate-limiting Pi release 20-fold, although Pi release continues to limit steady-state cycling in the presence of RNA, in conjunction with RNA binding. Together, this work identifies RNA binding and Pi release as important biochemical transitions within the Dbp5 ATPase cycle and provides a framework for investigating the means by which Dbp5 and mRNA export is modulated by regulatory factors.
dc.description.sponsorshipE.V.W. is supported by National Science Foundation Graduate Research Fellowship No. DGE-1122492 and J.V. is supported by an Alberta Innovates Health Solutions Postdoctoral Fellowship. M.M. and Y.M. were supported by a Senior Research Fellowship from the Wellcome Trust (101908/Z/13/Z) and by National Institutes of Health grant R01 GM102869. Research support for B.M. was provided by the Natural Sciences and Engineering Research Council of Canada (RGPIN 435380), Canada Foundation for Innovation (31271), Government of Alberta Research Capacity Program, and Canada Research Chairs program.
dc.languageengen
dc.language.isoenen
dc.publisherElsevier Inc.
dc.rightsAttribution 4.0 International*
dc.rightsAttribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/*
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectRNA helicaseen
dc.subjectkineticsen
dc.subjectmRNA exporten
dc.subjectmantATPen
dc.subjectthermodynamicsen
dc.subjectAdenosine Triphosphatasesen
dc.subjectDEAD-box RNA Helicasesen
dc.subjectKineticsen
dc.subjectNucleocytoplasmic Transport Proteinsen
dc.subjectPhosphatesen
dc.subjectRNAen
dc.subjectSaccharomyces cerevisiaeen
dc.subjectSaccharomyces cerevisiae Proteinsen
dc.titleP(I) Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5).en
dc.typeArticle
prism.endingPage508
prism.issueIdentifier2 Pt Ben
prism.publicationDate2016en
prism.publicationNameJournal of Molecular Biologyen
prism.startingPage492
prism.volume428en
dc.rioxxterms.funderNSF
dc.rioxxterms.funderWellcome Trust
dc.rioxxterms.funderNIH
dc.rioxxterms.projectidDGE-1122492
dc.rioxxterms.projectid101908/Z/13/Z
dc.rioxxterms.projectidR01 GM102869
dcterms.dateAccepted2015-12-22en
rioxxterms.versionofrecord10.1016/j.jmb.2015.12.018en
rioxxterms.versionVoR
rioxxterms.licenseref.urihttp://creativecommons.org/licenses/by/4.0/en
rioxxterms.licenseref.startdate2016-01-29en
dc.contributor.orcidMerchant, Monique [0000-0002-6783-7193]
dc.contributor.orcidModis, Yorgo [0000-0002-6084-0429]
dc.identifier.eissn1089-8638
rioxxterms.typeJournal Article/Reviewen
pubs.funder-project-idWellcome Trust (101908/Z/13/Z)
pubs.funder-project-idNational Institute of General Medical Sciences (NIGMS) (R01GM102869)
cam.issuedOnline2015-12-28en
dc.identifier.urlhttps://www.sciencedirect.com/science/article/pii/S0022283615007093?via%3Dihub#!en
cam.orpheus.successThu Jan 30 12:55:02 GMT 2020 - The item has an open VoR version.*
rioxxterms.freetoread.startdate2100-01-01


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International