P(I) Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5).
Authors
Wong, Emily V
Cao, Wenxiang
Vörös, Judit
Hackney, David D
Montpetit, Ben
De La Cruz, Enrique M
Publication Date
2016-01-29Journal Title
Journal of Molecular Biology
ISSN
0022-2836
Publisher
Elsevier Inc.
Volume
428
Issue
2 Pt B
Pages
492-508
Language
English
Type
Article
This Version
VoR
Metadata
Show full item recordCitation
Wong, E. V., Cao, W., Vörös, J., Merchant, M., Modis, Y., Hackney, D. D., Montpetit, B., & et al. (2016). P(I) Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5).. Journal of Molecular Biology, 428 (2 Pt B), 492-508. https://doi.org/10.1016/j.jmb.2015.12.018
Abstract
mRNA 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.
Keywords
RNA helicase, kinetics, mRNA export, mantATP, thermodynamics, Adenosine Triphosphatases, DEAD-box RNA Helicases, Kinetics, Nucleocytoplasmic Transport Proteins, Phosphates, RNA, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Sponsorship
E.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.
Funder references
Wellcome Trust (101908/Z/13/Z)
National Institute of General Medical Sciences (NIGMS) (R01GM102869)
Embargo Lift Date
2100-01-01
Identifiers
External DOI: https://doi.org/10.1016/j.jmb.2015.12.018
This record's URL: https://www.repository.cam.ac.uk/handle/1810/254132
Rights
Attribution 4.0 International, Attribution 4.0 International
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