Rearrangement of structured RNA via branch migration structures catalysed by the highly related DEAD-box proteins p68 and p72

RNA helicases, like their DNA-specific counterparts, can function as processive enzymes, unwinding RNA with a defined step size in a unidirectional fashion. Recombinant nuclear DEAD-box protein p68 and its close relative p72 are reported here to function in a similar fashion, though the processivity of both RNA helicases appears to be limited to only a few consecutive catalytic steps. The two proteins resemble each other also with regard to other biochemical properties. We have found that both proteins exhibit an RNA annealing in addition to their helicase activity. By using both these activities the enzymes are able in vitro to catalyse rearrangements of RNA secondary structures that otherwise are too stable to be resolved by their low processive helicase activities. RNA rearrangement proceeds via protein induced formation and subsequent resolution of RNA branch migration structures, whereby the latter step is dependent on ATP hydrolysis. The analysed DEAD-box proteins are reminiscent of certain DNA helicases, for example those found in bacteriophages T4 and T7, that catalyse homologous DNA strand exchange in cooperation with the annealing activity of specific single strand binding proteins.     

Helicase structure and mechanism

Structural information on helicase proteins has expanded recently beyond the DNA helicases Rep and PcrA, and the hepatitis C virus RNA helicase to include UvrB, members of the DEA(D/H)-box RNA helicase family, examples of DnaB-related helicases and RuvB. The expanding database of structures has clarified the structural 'theme and variations' that relate the different helicase families. Furthermore, information is emerging on the functions of the conserved helicase motifs and their participation in the mechanisms by which these proteins catalyze the remodeling of DNA and RNA in ATP-dependent activities.     

Binding and unwinding: SF3 viral helicases

The SF3 helicases, distinct from the more prevalent SF1 and SF2 helicases, were originally identified in the genomes of small DNA and RNA viruses. The first crystal structures of SF3 helicases have been determined, revealing a closer structural relationship to AAA+ proteins than to RecA, consistent with their participation in replication initiation. In conjunction with origin-binding domains, SF3 helicases are responsible for distorting DNA before replication forks can be assembled. At these forks, the SF3 helicases act as replicative helicases. The simian virus 40 SF3 helicase forms a hexameric ring, anticipated to be characteristic of the entire superfamily.     

A novel superfamily of nucleoside triphosphate-binding motif containing proteins which are probably involved in duplex unwinding in DNA and RNA replication and recombination

A statistically significant similarity was demonstrated between the amino acid sequences of 4 Escherichia coli helicases and helicase subunits, a family of non-structural proteins of eukaryotic positive-strand RNA viruses and 2 herpesvirus proteins all of which contain an NTP-binding sequence motif. Based on sequence analysis and secondary structure predictions, a generalized structural model for the ATP-binding core is proposed. It is suggested that all these proteins constitute a superfamily of helicases (or helicase subunits) involved in NTP-dependent duplex unwinding during DNA and RNA replication and recombination.     

Yeast and human mitochondrial helicases

Mitochondria are semiautonomous organelles which contain their own genome. Both maintenance and expression of mitochondrial DNA require activity of RNA and DNA helicases. In Saccharomyces cerevisiae the nuclear genome encodes four DExH/D superfamily members (MSS116, SUV3, MRH4, IRC3) that act as helicases and/or RNA chaperones. Their activity is necessary for mitochondrial RNA splicing, degradation, translation and genome maintenance. In humans the ortholog of SUV3 (hSUV3, SUPV3L1) so far is the best described mitochondrial RNA helicase. The enzyme, together with the matrix-localized pool of PNPase (PNPT1), forms an RNA-degrading complex called the mitochondrial degradosome, which localizes to distinct structures (D-foci). Global regulation of mitochondrially encoded genes can be achieved by changing mitochondrial DNA copy number. This way the proteins involved in its replication, like the Twinkle helicase (c10orf2), can indirectly regulate gene expression. Here, we describe yeast and human mitochondrial helicases that are directly involved in mitochondrial RNA metabolism, and present other helicases that participate in mitochondrial DNA replication and maintenance. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization

The identification of G-quadruplex (G4) binding proteins and insights into their mechanism of action are important for understanding the regulatory functions of G4 structures. Here, we performed an unbiased affinity-purification assay coupled with mass spectrometry and identified 30 putative G4 binding proteins from the fission yeast Schizosaccharomyces pombe. Gene ontology analysis of the molecular functions enriched in this pull-down assay included mRNA binding, RNA helicase activity, and translation regulator activity. We focused this study on three of the identified proteins that possessed putative arginine-glycine-glycine (RGG) domains, namely the Stm1 homolog Oga1 and the DEAD box RNA helicases Dbp2 and Ded1. We found that Oga1, Dbp2, and Ded1 bound to both DNA and RNA G4s in vitro. Both Dbp2 and Ded1 bound to G4 structures through the RGG domain located in the C-terminal region of the helicases, and point mutations in this domain weakened the G4 binding properties of the helicases. Dbp2 and Ded1 destabilized less thermostable G4 RNA and DNA structures, and this ability was independent of ATP but dependent on the RGG domain. Our study provides the first evidence that the RGG motifs in DEAD box helicases are necessary for both G4 binding and G4 destabilization.     

Crystal structure of the BstDEAD N-terminal domain: a novel DEAD protein from Bacillus stearothermophilus

Most cellular processes requiring RNA structure rearrangement necessitate the action of Asp-Glu-Ala-Asp (DEAD) proteins. Members of the family, named originally for the conserved DEAD amino acid sequence, are thought to disrupt RNA structure and facilitate its rearrangement by unwinding short stretches of duplex RNA. BstDEAD is a novel 436 amino acid representative of the DEAD protein family from Bacillus stearothermophilus that contains all eight conserved motifs found in DEAD proteins and is homologous with other members of the family. Here, we describe the 1.85 A resolution structure of the N-terminal domain (residues 1-211) of BstDEAD (BstDEAD-NT). Similar to the corresponding domains of related helicases, BstDEAD-NT adopts a parallel alpha/beta structure with RecA-like topology. In general, the conserved motifs superimpose on closely related DEAD proteins and on more distantly related helicases such as RecA. This affirms the current belief that the core helicase domains, responsible for mechanistic activity, are structurally similar in DEAD proteins. In contrast, however, the so-called Walker A P-loop, which binds the beta- and gamma-phosphates of ATP, adopts a rarely seen "closed" conformation that would sterically block ATP binding. The closed conformation may be indicative of a general regulatory feature among DEAD proteins (and RNA helicases) that differs from that used by DNA helicases. BstDEAD also contains a unique extension of approximately 60 residues at the C terminus that is highly basic, suggesting that it might bind nucleic acids and, in so doing, confer specificity to the helicase activity of the core region.     

A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands

Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5′ to 3′ DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures.     

The replicative helicases of bacteria, archaea, and eukarya can unwind RNA-DNA hybrid substrates

Replicative helicases are hexameric enzymes that unwind DNA during chromosomal replication. They use energy from nucleoside triphosphate hydrolysis to translocate along one strand of the duplex DNA and displace the complementary strand. Here, the ability of a replicative helicase from each of the three domains, bacteria, archaea, and eukarya, to unwind RNA-containing substrate was determined. It is shown that all three helicases can unwind DNA-RNA hybrids while translocating along the single-stranded DNA. No unwinding could be observed when the helicases were provided with a single-stranded RNA overhang. Using DNA, RNA, and DNA-RNA chimeric oligonucleotides it was found that whereas the enzymes can bind both DNA and RNA, they could translocate only along DNA and only DNA stimulates the ATPase activity of the enzymes. Recent observations suggest that helicases may interact with enzymes participating in RNA metabolism and that RNA-DNA hybrids may be present on the chromosomes. Thus, the results presented here may suggest a new role for the replicative helicases during chromosomal replication or in other cellular processes.     

The DEAD box proteins DDX5 (p68) and DDX17 (p72): Multi-tasking transcriptional regulators

Members of the DEAD box family of RNA helicases, which are characterised by the presence of twelve conserved motifs (including the signature D-E-A-D motif) within a structurally conserved ‘helicase’ core, are involved in all aspects of RNA metabolism. Apart from unwinding RNA duplexes, which established these proteins as RNA helicases, DEAD box proteins have been shown to also catalyse RNA annealing and to displace proteins from RNA. DEAD box proteins generally act as components of large multi-protein complexes and it is thought that interactions, via their divergent N- and C-terminal extensions, with other factors in the complexes may be responsible for the many different functions attributed to these proteins.     

DEAD-box helicases as integrators of RNA,nucleotide and protein binding

DEAD-box helicases perform diverse cellular functions in virtually all steps of RNA metabolism from Bacteria to Humans. Although DEAD-box helicases share a highly conserved core domain, the enzymes catalyze a wide range of biochemical reactions. In addition to the well established RNA unwinding and corresponding ATPase activities, DEAD-box helicases promote duplex formation and displace proteins from RNA. They can also function as assembly platforms for larger ribonucleoprotein complexes, and as metabolite sensors. This review aims to provide a perspective on the diverse biochemical features of DEAD-box helicases and connections to structural information. We discuss these data in the context of a model that views the enzymes as integrators of RNA, nucleotide, and protein binding. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

DEAD-box proteins unwind duplexes by local strand separation

DEAD-box proteins catalyze ATP-driven, local structural changes in RNA or RNA-protein complexes (RNP) during which only few RNA base pairs are separated. It is unclear how duplex unwinding by DEAD-box proteins differs from unwinding by canonical helicases, which can separate many base pairs by directional and processive translocation on the nucleic acid, starting from a helical end. Here, we show that two different DEAD-box proteins, Ded1p and Mss116p, can unwind RNA duplexes from internal as well as terminal helical regions and act on RNA segments as small as two nucleotides flanked by DNA. The data indicate that duplex unwinding by DEAD-box proteins is based on local destabilization of RNA helical regions. No directional movement of the enzymes through the duplex is involved. We propose a three-step mechanism in which DEAD-box proteins unwind duplexes as "local strand separators." This unwinding mode is well-suited for local structural changes in complex RNA or RNP assemblies.     

Functional assignment to maize group 1 LEA protein EMB564 within the cell nucleus using computational analysis

Maize protein EMB564 is a member of group 1 LEA (late embryogenesis abundant) proteins. Currently, the molecular functions of group 1 LEA proteins remain largely unclear. We here report on the functional assignment to EMB564 by computational analysis. EMB564 is predicted as nuclear localization by five different predictors including CELLO, Plant-mPLoc, WoLF PSORT, Predotar and TargetP. EMB564 is found to be remote homologous with DNA/RNA helicases and single-stranded DNA-binding proteins, and their sequences contains similar DNA/RNA binding sites. Furthermore, the three-dimensional (3D) model of EMB564 structurally resembles a variety of nuclear and DNA/RNA-binding proteins, especially those involving in the regulation of cell division, chromosomal replication and DNA unwinding or repairing. Our results reveal that EMB564 protein is most likely to function within the cell nucleus.     

The long unwinding road of RNA helicases

RNA helicases comprise a large family of enzymes that are thought to utilize the energy of NTP binding and hydrolysis to remodel RNA or RNA-protein complexes, resulting in RNA duplex strand separation, displacement of proteins from RNA molecules, or both. These functions of RNA helicases are required for all aspects of cellular RNA metabolism, from bacteria to humans. We provide a brief overview of the functions of RNA helicases and highlight some of the recent key advances that have contributed to our current understanding of their biological function and mechanism of action.     

RNA structural rearrangement via unwinding and annealing by the cyanobacterial RNA helicase, CrhR

Rearrangement of RNA secondary structure is crucial for numerous biological processes. RNA helicases participate in these rearrangements through the unwinding of duplex RNA. We report here that the redox-regulated cyanobacterial RNA helicase, CrhR, is a bona fide RNA helicase possessing both RNA-stimulated ATPase and bidirectional ATP-stimulated RNA helicase activity. The processivity of the unwinding reaction appears to be low, because RNA substrates containing duplex regions of 41 bp are not unwound. CrhR also catalyzes the annealing of complementary RNA into intermolecular duplexes. Uniquely and in contrast to other proteins that perform annealing, the CrhR-catalyzed reactions require ATP hydrolysis. Through a combination of the unwinding and annealing activities, CrhR also catalyzes RNA strand exchange resulting in the formation of RNA secondary structures that are too stable to be resolved by helicase activity. RNA strand exchange most probably occurs through the CrhR-dependent formation and resolution of an RNA branch migration structure. Demonstration that another cyanobacterial RNA helicase, CrhC, does not catalyze annealing indicates that this activity is not a general biochemical characteristic of RNA helicases. Biochemically, CrhR resembles RecA and related proteins that catalyze strand exchange and branch migration on DNA substrates, a characteristic that is reflected in the recently reported structural similarities between these proteins. The data indicate the potential for CrhR to catalyze dynamic RNA secondary structure rearrangements through a combination of RNA helicase and annealing activities.