Abstract
The DExD/H-box RNA-dependent ATPase Dbp5 plays an essential role in the nuclear export of mRNA. Dbp5 localizes to the nuclear pore complex, where its ATPase activity is stimulated by Gle1 and its coactivator inositol hexakisphosphate. Here, we present the crystal structure of the C-terminal domain of Dbp5, refined to 1.8 A. The structure reveals a RecA-like fold that contains two defining characteristics not present in other structurally characterized DExD/H-box proteins: a C-terminal alpha-helix and a loop connecting beta5 and alpha4, both of which are composed of conserved and unique elements in the Dbp5 primary sequence. Using structure-guided mutagenesis, we have identified several charged surface residues that, when mutated, weaken the binding of Gle1 and inhibit the ability of Gle1 to stimulate Dbp5's ATPase activity. In vivo analysis of the same mutations reveals that those mutants displaying the weakest ATPase stimulation in vitro are also unable to support yeast growth. Analysis of the correlation between the in vitro and in vivo data indicates that a threshold level of Dbp5 ATPase activity is required for cellular mRNA export that is not met by the unstimulated enzyme, suggesting a possible mechanism by which Dbp5's activity can be modulated to regulate mRNA export.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Adenosine Triphosphatases / metabolism*
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Amino Acid Sequence
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Binding Sites / genetics
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Catalysis
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Crystallization
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Crystallography, X-Ray
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DEAD-box RNA Helicases / chemistry
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DEAD-box RNA Helicases / genetics
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DEAD-box RNA Helicases / metabolism*
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In Situ Hybridization
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Models, Molecular
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Molecular Sequence Data
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Mutation
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Nuclear Pore / metabolism
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Nuclear Pore Complex Proteins / genetics
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Nuclear Pore Complex Proteins / metabolism*
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Nucleocytoplasmic Transport Proteins / chemistry
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Nucleocytoplasmic Transport Proteins / genetics
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Nucleocytoplasmic Transport Proteins / metabolism*
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Protein Binding
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Protein Structure, Secondary
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Protein Structure, Tertiary
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RNA Transport
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RNA, Fungal / genetics
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RNA, Fungal / metabolism
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RNA, Messenger / genetics
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RNA, Messenger / metabolism*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
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Sequence Homology, Amino Acid
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Two-Hybrid System Techniques
Substances
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GLE1 protein, S cerevisiae
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Nuclear Pore Complex Proteins
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Nucleocytoplasmic Transport Proteins
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RNA, Fungal
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RNA, Messenger
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Saccharomyces cerevisiae Proteins
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Adenosine Triphosphatases
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DBP5 protein, S cerevisiae
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DEAD-box RNA Helicases