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

2015 ◽  
Vol 396 (8) ◽  
pp. 849-865 ◽  
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.

scholarly journals The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

2009 ◽  
Vol 390 (12) ◽  
Author(s):  
Manuel Hilbert ◽  
Anne R. Karow ◽  
Dagmar Klostermeier
Keyword(s):  
Rna Binding ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Current Data ◽  
Protein Activity ◽  
Closed Conformation ◽  
Dead Box ◽  
Rna Duplexes ◽  
The Dead ◽  
Rna Unwinding

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.

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

Biochemistry ◽  
2009 ◽  
Vol 48 (45) ◽  
pp. 10679-10681 ◽  
Author(s):  
Regula Aregger ◽  
Dagmar Klostermeier
Keyword(s):  
Atp Hydrolysis ◽  
Closed Conformation ◽  
Dead Box ◽  
The Dead

scholarly journals PRH75, a new nucleus-localized member of the DEAD-box protein family from higher plants.

1997 ◽  
Vol 17 (4) ◽  
pp. 2257-2265 ◽  
Author(s):  
Z J Lorković ◽  
R G Herrmann ◽  
R Oelmüller
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Protein Family ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Rna Helicases ◽  
Nuclear Targeting ◽  
Dead Box ◽  
Cell Extracts ◽  
The Dead

The putative RNA helicases of the DEAD-box protein family are involved in pre-mRNA splicing, rRNA maturation, ribosome assembly, and translation. Members of this protein family have been identified in organisms from Escherichia coli to humans, but except for the translation initiation factor 4A, there have been no reports on the characterization of other DEAD-box proteins from plants. Here we report on a novel member of the DEAD-box protein family, the plant RNA helicase 75 (PRH75). PRH75 is localized in the nucleus and contains two domains for RNA binding. One is located at the C terminus and is similar to RGG RNA-binding domains of nucleus-localized RNA-binding proteins. The other one is located between amino acids 308 and 622, a region containing the conserved motif VI characteristic of DEAD-box proteins and known as the RNA-binding site of eIF-4A. The N-terminal 81 amino acids are sufficient for nuclear targeting of the protein. Northern and Western blot analyses show that PRH75 is mainly expressed in young and rapidly developing tissues. The purified recombinant PRH75 has a weak ATPase activity which is barely stimulated by RNA ligands. The fractionation of spinach whole-cell extracts by glycerol gradient centrifugation and gel filtration on a Superdex 200 column shows that the protein exists in a complex of about 500 kDa. Possible biological functions of PRH75 as well as structure-function relationships in the context of its modular primary structure are discussed.

The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis

1993 ◽  
Vol 13 (11) ◽  
pp. 6789-6798 ◽  
Author(s):  
A Pause ◽  
N Méthot ◽  
N Sonenberg
Keyword(s):  
Atpase Activity ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Rna Helicase ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Cross Linking ◽  
Atp Binding ◽  
Eukaryotic Translation ◽  
The Dead

eIF-4A is a eukaryotic translation initiation factor that is required for mRNA binding to ribosomes. It exhibits single-stranded RNA-dependent ATPase activity, and in combination with a second initiation factor, eIF-4B, it exhibits duplex RNA helicase activity. eIF-4A is the prototype of a large family of proteins termed the DEAD box protein family, whose members share nine highly conserved amino acid regions. The functions of several of these conserved regions in eIF-4A have previously been assigned to ATP binding, ATPase, and helicase activities. To define the RNA-binding region of eIF-4A, a UV-induced cross-linking assay was used to analyze binding of mutant eIF-4A proteins to RNA. Mutants carrying mutations in the ATP-binding region (AXXXXGKT), ATPase region (DEAD), helicase region (SAT), and the most carboxy-terminal conserved region of the DEAD family, HRIGRXXR, were tested for RNA cross-linking. We show that mutations, either conservative or not, in any one of the three arginines in the HRIGRXXR sequence drastically reduced eIF-4A cross-linking to RNA. In addition, all the mutations in the HRIGRXXR region abrogate RNA helicase activity. Some but not all of these mutations affect ATP binding and ATPase activity. This is consistent with the hypothesis that the HRIGRXXR region is involved in the ATP hydrolysis reaction and would explain the coupling of ATPase and RNA-binding/helicase activities. Our results show that the HRIGRXXR region, which is QRXGRXXR or QXXGRXXR in the RNA and DNA helicases of the helicase superfamily II, is involved in ATP hydrolysis-dependent RNA interaction during unwinding. We also show that mutations in other regions of eIF-4A that abolish ATPase activity sharply decrease eIF-4A cross-linking to RNA. A model is proposed in which eIF-4A first binds ATP, resulting in a change in eIF-4A conformation which allows RNA binding that is dependent on the HRIGRXXR region. Binding of RNA induces ATP hydrolysis, leading to a more stable interaction with RNA. This process is then linked to unwinding of duplex RNA in the presence of eIF-4B.

Motif III in Superfamily 2 “Helicases” Helps Convert the Binding Energy of ATP into a High-Affinity RNA Binding Site in the Yeast DEAD-Box Protein Ded1

2010 ◽  
Vol 396 (4) ◽  
pp. 949-966 ◽  
Author(s):  
Josette Banroques ◽  
Monique Doère ◽  
Marc Dreyfus ◽  
Patrick Linder ◽  
N. Kyle Tanner
Keyword(s):  
Binding Energy ◽  
Binding Site ◽  
Rna Binding ◽  
High Affinity ◽  
Dead Box

scholarly journals DEAD-Box Helicases: Sensors, Regulators, and Effectors for Antiviral Defense

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 181 ◽  
Author(s):  
Frances Taschuk ◽  
Sara Cherry
Keyword(s):  
Rna Binding ◽  
Viral Infections ◽  
Rna Binding Proteins ◽  
Large Family ◽  
Cellular Functions ◽  
Rna Helicases ◽  
Independent Manner ◽  
Dead Box ◽  
The Dead ◽  
Immune Sensing

DEAD-box helicases are a large family of conserved RNA-binding proteins that belong to the broader group of cellular DExD/H helicases. Members of the DEAD-box helicase family have roles throughout cellular RNA metabolism from biogenesis to decay. Moreover, there is emerging evidence that cellular RNA helicases, including DEAD-box helicases, play roles in the recognition of foreign nucleic acids and the modulation of viral infection. As intracellular parasites, viruses must evade detection by innate immune sensing mechanisms and degradation by cellular machinery while also manipulating host cell processes to facilitate replication. The ability of DEAD-box helicases to recognize RNA in a sequence-independent manner, as well as the breadth of cellular functions carried out by members of this family, lead them to influence innate recognition and viral infections in multiple ways. Indeed, DEAD-box helicases have been shown to contribute to intracellular immune sensing, act as antiviral effectors, and even to be coopted by viruses to promote their replication. However, our understanding of the mechanisms underlying these interactions, as well as the cellular roles of DEAD-box helicases themselves, is limited in many cases. We will discuss the diverse roles that members of the DEAD-box helicase family play during viral infections.

scholarly journals Dynamics of the DEAD-box ATPase Prp5 RecA-like domains provide a conformational switch during spliceosome assembly

2019 ◽  
Vol 47 (20) ◽  
pp. 10842-10851 ◽  
Author(s):  
David H Beier ◽  
Tucker J Carrocci ◽  
Clarisse van der Feltz ◽  
U Sandy Tretbar ◽  
Joshua C Paulson ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Conformational Dynamics ◽  
Spliceosome Assembly ◽  
Single Molecule Fret ◽  
Dead Box ◽  
U2 Snrnp ◽  
The Dead

Abstract The DEAD-box family of proteins are ATP-dependent, RNA-binding proteins implicated in many aspects of RNA metabolism. Pre-mRNA splicing in eukaryotes requires three DEAD-box ATPases (Prp5, Prp28 and Sub2), the molecular mechanisms of which are poorly understood. Here, we use single molecule FRET (smFRET) to study the conformational dynamics of yeast Prp5. Prp5 is essential for stable association of the U2 snRNP with the intron branch site (BS) sequence during spliceosome assembly. Our data show that the Prp5 RecA-like domains undergo a large conformational rearrangement only in response to binding of both ATP and RNA. Mutations in Prp5 impact the fidelity of BS recognition and change the conformational dynamics of the RecA-like domains. We propose that BS recognition during spliceosome assembly involves a set of coordinated conformational switches among U2 snRNP components. Spontaneous toggling of Prp5 into a stable, open conformation may be important for its release from U2 and to prevent competition between Prp5 re-binding and subsequent steps in spliceosome assembly.

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

eLife ◽  
2016 ◽  
Vol 5 ◽  
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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