The DEAD Box Helicase YxiN Maintains a Closed Conformation during ATP Hydrolysis

Abstract DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecA-like domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysis- and unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

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ATP hydrolysis activity of the DEAD box protein Rok1p is required for in vivo ROK1 function

Nucleic Acids Research ◽

10.1093/nar/27.13.2753 ◽

1999 ◽

Vol 27 (13) ◽

pp. 2753-2759 ◽

Cited By ~ 17

Author(s):

J.-Y. Oh ◽

J. Kim

Keyword(s):

Atp Hydrolysis ◽

Dead Box ◽

The Dead ◽

Hydrolysis Activity

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ATPase activity of the DEAD-box protein Dhh1 controls processing body formation

eLife ◽

10.7554/elife.18746 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 50

Author(s):

Christopher Frederick Mugler ◽

Maria Hondele ◽

Stephanie Heinrich ◽

Ruchika Sachdev ◽

Pascal Vallotton ◽

...

Keyword(s):

Atpase Activity ◽

Atp Hydrolysis ◽

Liquid Droplets ◽

Processing Bodies ◽

Dead Box ◽

Posttranscriptional Gene Regulation ◽

The Dead ◽

Processing Body

Translational repression and mRNA degradation are critical mechanisms of posttranscriptional gene regulation that help cells respond to internal and external cues. In response to certain stress conditions, many mRNA decay factors are enriched in processing bodies (PBs), cellular structures involved in degradation and/or storage of mRNAs. Yet, how cells regulate assembly and disassembly of PBs remains poorly understood. Here, we show that in budding yeast, mutations in the DEAD-box ATPase Dhh1 that prevent ATP hydrolysis, or that affect the interaction between Dhh1 and Not1, the central scaffold of the CCR4-NOT complex and an activator of the Dhh1 ATPase, prevent PB disassembly in vivo. Intriguingly, this process can be recapitulated in vitro, since recombinant Dhh1 and RNA, in the presence of ATP, phase-separate into liquid droplets that rapidly dissolve upon addition of Not1. Our results identify the ATPase activity of Dhh1 as a critical regulator of PB formation.

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Structural and biochemical analyses of the DEAD-box ATPase Sub2 in association with THO or Yra1

eLife ◽

10.7554/elife.20070 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 16

Author(s):

Yi Ren ◽

Philip Schmiege ◽

Günter Blobel

Keyword(s):

Reaction Mechanisms ◽

Nuclear Pore ◽

Closed Conformation ◽

Dead Box ◽

Messenger Ribonucleoprotein ◽

Mrna Binding Protein ◽

The Dead ◽

Biochemical Analyses ◽

Mrna Binding ◽

Open Configuration

mRNA is cotranscrptionally processed and packaged into messenger ribonucleoprotein particles (mRNPs) in the nucleus. Prior to export through the nuclear pore, mRNPs undergo several obligatory remodeling reactions. In yeast, one of these reactions involves loading of the mRNA-binding protein Yra1 by the DEAD-box ATPase Sub2 as assisted by the hetero-pentameric THO complex. To obtain molecular insights into reaction mechanisms, we determined crystal structures of two relevant complexes: a THO hetero-pentamer bound to Sub2 at 6.0 Å resolution; and Sub2 associated with an ATP analogue, RNA, and a C-terminal fragment of Yra1 (Yra1-C) at 2.6 Å resolution. We found that the 25 nm long THO clamps Sub2 in a half-open configuration; in contrast, when bound to the ATP analogue, RNA and Yra1-C, Sub2 assumes a closed conformation. Both THO and Yra1-C stimulated Sub2’s intrinsic ATPase activity. We propose that THO surveys common landmarks in each nuclear mRNP to localize Sub2 for targeted loading of Yra1.

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When core competence is not enough: functional interplay of the DEAD-box helicase core with ancillary domains and auxiliary factors in RNA binding and unwinding

Biological Chemistry ◽

10.1515/hsz-2014-0277 ◽

2015 ◽

Vol 396 (8) ◽

pp. 849-865 ◽

Cited By ~ 23

Author(s):

Markus G. Rudolph ◽

Dagmar Klostermeier

Keyword(s):

Binding Site ◽

Rna Binding ◽

Atp Hydrolysis ◽

Current Knowledge ◽

Mechanistic Model ◽

The Core ◽

Dead Box ◽

Interaction Partners ◽

Rna Backbone ◽

The Dead

Abstract DEAD-box helicases catalyze RNA duplex unwinding in an ATP-dependent reaction. Members of the DEAD-box helicase family consist of a common helicase core formed by two RecA-like domains. According to the current mechanistic model for DEAD-box mediated RNA unwinding, binding of RNA and ATP triggers a conformational change of the helicase core, and leads to formation of a compact, closed state. In the closed conformation, the two parts of the active site for ATP hydrolysis and of the RNA binding site, residing on the two RecA domains, become aligned. Closing of the helicase core is coupled to a deformation of the RNA backbone and destabilization of the RNA duplex, allowing for dissociation of one of the strands. The second strand remains bound to the helicase core until ATP hydrolysis and product release lead to re-opening of the core. The concomitant disruption of the RNA binding site causes dissociation of the second strand. The activity of the helicase core can be modulated by interaction partners, and by flanking N- and C-terminal domains. A number of C-terminal flanking regions have been implicated in RNA binding: RNA recognition motifs (RRM) typically mediate sequence-specific RNA binding, whereas positively charged, unstructured regions provide binding sites for structured RNA, without sequence-specificity. Interaction partners modulate RNA binding to the core, or bind to RNA regions emanating from the core. The functional interplay of the helicase core and ancillary domains or interaction partners in RNA binding and unwinding is not entirely understood. This review summarizes our current knowledge on RNA binding to the DEAD-box helicase core and the roles of ancillary domains and interaction partners in RNA binding and unwinding by DEAD-box proteins.

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Mutational Analysis of the Escherichia coli DEAD Box Protein CsdA

Journal of Bacteriology ◽

10.1128/jb.01509-06 ◽

2007 ◽

Vol 189 (7) ◽

pp. 2769-2776 ◽

Cited By ~ 31

Author(s):

Anne-Marie W. Turner ◽

Cheraton F. Love ◽

Rebecca W. Alexander ◽

Pamela G. Jones

Keyword(s):

Escherichia Coli ◽

Atp Hydrolysis ◽

Mutational Analysis ◽

Cold Shock Protein ◽

Null Mutant ◽

Rna Helicases ◽

Dead Box ◽

Rna Duplexes ◽

The Dead

ABSTRACT The Escherichia coli cold shock protein CsdA is a member of the DEAD box family of ATP-dependent RNA helicases, which share a core of nine conserved motifs. The DEAD (Asp-Glu-Ala-Asp) motif for which this family is named has been demonstrated to be essential for ATP hydrolysis. We show here that CsdA exhibits in vitro ATPase and helicase activities in the presence of short RNA duplexes with either 3′ or 5′ extensions at 15°C. In contrast to wild-type CsdA, a DQAD variant of CsdA (Glu-157→Gln) had no detectible helicase or ATPase activity at 15°C in vitro. A plasmid encoding the DQAD variant was also unable to suppress the impaired growth of the csdA null mutant at 15°C. Plasmid-encoded CsdAΔ444, which lacks most of the carboxy-terminal extension, enhanced the growth of a csdA null mutant at 25°C but not at 15°C; this truncated protein also has limited in vitro activity at 15°C. These results support the physiological function of CsdA as a DEAD box ATP-dependent RNA helicase at low temperature.

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