Characterization of DbpA, an Escherichia coli DEAD box protein with ATP independent RNA unwinding activity.

DbpA is a putative Escherichia coli ATP dependent RNA helicase belonging to the family of DEAD box proteins. It hydrolyzes ATP in the presence of 23S ribosomal RNA and 93 bases in the peptidyl transferase center of 23S rRNA are sufficient to trigger 100% of the ATPase activity of DbpA. In the present study we characterized the ATPase and RNA unwinding activities of DbpA in more detail. We report that-in contrast to eIF-4A, the prototype of the DEAD box protein family-the ATPase and the helicase activities of DbpA are not coupled. Moreover, the RNA unwinding activity of DbpA is not specific for 23S rRNA, since DbpA is also able to unwind 16S rRNA hybrids. Furthermore, we determined that the ATPase activity of DbpA is triggered to a significant extent not only by the 93 bases of the 23S rRNA previously reported but also by other regions of the 23S rRNA molecule. Since all these regions of 23S rRNA are either part of the 'functional core' of the 50S ribosomal subunit or involved in the 50S assembly, DbpA may play an important role in the ribosomal assembly process.     

Cloning and biochemical characterization of Bacillus subtilis YxiN, a DEAD protein specifically activated by 23S rRNA: delineation of a novel sub-family of bacterial DEAD proteins.

DEAD, DEAH and DExH proteins are involved in almost every facet of RNA biochemistry. Members of these protein families exhibit an RNA-dependent ATPase activity and some possess an ATP-dependent RNA helicase activity. Although genetic studies have identified specific functions for certain DEx(D)/(H)proteins from which an RNA substrate can be reasonably inferred, only DbpA from Escherichia coli has been shown to exhibit significant RNA specificity in vitro. Here we describe the characterization of YxiN from Bacillus subtilis, the second DEx(D)/(H)protein to show significant RNA specificity as an isolated, homogenous protein. The ATPase activity of YxiN, like that of DbpA, is stimulated by a 154 nt fragment of 23S rRNA. YxiN has a 2 nM apparent binding constant for this fragment, yet its ATPase activity shows 1800-fold RNA specificity. Along with the conserved motifs shared among all DEAD proteins, YxiN and DbpA have a conserved C-terminal extension. This extension is highly conserved in several additional DEAD proteins. We propose that the C-terminus identifies a protein sub-family whose members bind 23S rRNA and that proteins of this family are likely to function in rRNA maturation/ribosome biogenesis or an unappreciated aspect of translation.     

DbpA: a DEAD box protein specifically activated by 23s rRNA.

The Escherichia coli protein DbpA is a member of the 'DEAD box' family of putative RNA-dependent ATPases and RNA helicases, so called because they share the highly conserved motif Asp-Glu-Ala-Asp, together with several other conserved elements. We have investigated DbpA expression under conditions where an endogenous promoter is used. In this context, translation initiation does not occur at the previously identified AUG, but at an upstream, in-frame GUG. Mutation of the GUG initiation codon to AUG virtually abolishes DbpA expression, suggesting an unusual translation initiation mechanism. Using an inducible overexpression plasmid, we have purified milligram quantities of DbpA to homogeneity and shown that the purified protein hydrolyses ATP in an RNA-dependent manner. This ATPase activity is interesting in that, unlike that of other DEAD box proteins investigated to date, it absolutely requires a specific bacterial RNA, which we have identified as 23S rRNA. This observation is particularly significant since DbpA will bind other RNAs and DNA, but will only hydrolyse ATP in the presence of 23S rRNA.     

Escherichia coli DbpA is an RNA helicase that requires hairpin 92 of 23S rRNA

Escherichia coli DbpA is a member of the DEAD/H family of proteins which has been shown to have robust ATPase activity only in the presence of a specific region of 23S rRNA. A series of bimolecular RNA substrates were designed based on this activating region of rRNA and used to demonstrate that DbpA is also a non-processive, sequence-specific RNA helicase. The high affinity of DbpA for the RNA substrates allowed both single and multiple turnover helicase assays to be performed. Helicase activity of DbpA is dependent on the presence of ATP or dATP, the sequence of the loop of hairpin 92 of 23S rRNA and the position of the substrate helix with respect to hairpin 92. This work indicates that certain RNA helicases require particular RNA structures in order for optimal unwinding activity to be observed.     

The Escherichia coli DEAD protein DbpA recognizes a small RNA hairpin in 23S rRNA

The Escherichia coli DEAD protein DbpA is an RNA-specific ATPase that is activated by a 153-nt fragment within domain V of 23S rRNA. A series of RNA subfragments and sequence changes were used to identify the recognition elements of this RNA-protein interaction. Reducing the size of the fully active 153-nt RNA yields compromised substrates in which both RNA and ATP binding are weakened considerably without affecting the maximal rate of ATP hydrolysis. All RNAs that stimulate ATPase activity contain hairpin 92 of 23S rRNA, which is known to interact with the 3' end of tRNAs in the ribosomal A-site. RNAs with base mutations within this hairpin fail to activate ATP hydrolysis, suggesting that it is a critical recognition element for DbpA. Although the isolated hairpin fails to activate DbpA, RNAs with an extension of approximately 15 nt on either the 5' or 3' side of hairpin 92 elicit full ATPase activity. These results suggest that the binding of DbpA to RNA requires sequence-specific interactions with hairpin 92 as well as nonspecific interactions with the RNA extension. A model relating the RNA binding and ATPase activities of DbpA is presented.     

The putative RNase P motif in the DEAD box helicase Hera is dispensable for efficient interaction with RNA and helicase activity

DEAD box helicases use the energy of ATP hydrolysis to remodel RNA structures or RNA/protein complexes. They share a common helicase core with conserved signature motifs, and additional domains may confer substrate specificity. Identification of a specific substrate is crucial towards understanding the physiological role of a helicase. RNA binding and ATPase stimulation are necessary, but not sufficient criteria for a bona fide helicase substrate. Here, we report single molecule FRET experiments that identify fragments of the 23S rRNA comprising hairpin 92 and RNase P RNA as substrates for the Thermus thermophilus DEAD box helicase Hera. Both substrates induce a switch to the closed conformation of the helicase core and stimulate the intrinsic ATPase activity of Hera. Binding of these RNAs is mediated by the Hera C-terminal domain, but does not require a previously proposed putative RNase P motif within this domain. ATP-dependent unwinding of a short helix adjacent to hairpin 92 in the ribosomal RNA suggests a specific role for Hera in ribosome assembly, analogously to the Escherichia coli and Bacillus subtilis helicases DbpA and YxiN. In addition, the specificity of Hera for RNase P RNA may be required for RNase P RNA folding or RNase P assembly.     

DbpA is a region‐specific RNA helicase

DbpA is a DEAD‐box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA‐like catalytic core responsible for double‐helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic core's grasp. In other words, it is not known if DbpA is a site‐specific enzyme or region‐specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region‐specific or a site‐specific enzyme. Our data suggest that DbpA is a region‐specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C‐terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double‐helices in its vicinity. The only requirement for a double‐helix to serve as a DbpA substrate is for the double‐helix to be positioned within the catalytic core's grasp.     

Interaction of Escherichia coli DbpA with 23S rRNA in different functional states of the enzyme

DEx^D/_H proteins catalyze structural rearrangements in RNA by coupling ATP hydrolysis to the destabilization of RNA helices or RNP complexes. The Escherichia coli DEx^D/_H protein DbpA specifically recognizes a region within the catalytic core of 23S rRNA. To better characterize the interaction of DbpA with this region and to identify changes in the complex between different nucleotide-bound states of the enzyme, RNase T1, RNase T2, kethoxal and DMS footprinting of DbpA on a 172 nt fragment of 23S rRNA were performed. A number of protections identified in helices 90 and 92 were consistent with biochemical experiments measuring the RNA binding and ATPase activity of DbpA with truncated RNAs. When DbpA was bound with AMPPNP, but not ADP, several additional footprints were detected in helix 93 and the single-stranded region 5′ of helix 90, suggesting binding of the helicase domains of DbpA at these sites. These results propose that DbpA can act at multiple sites and hint at the targets of its biological activity on rRNA.     

Hera from Thermus thermophilus: the first thermostable DEAD-box helicase with an RNase P protein motif

DEAD-box proteins have been implicated in a wide array of cellular processes ranging from initiation of protein synthesis and ribosome biogenesis to mRNA splicing. Here, we report the isolation, biochemical characterization and crystallization of the first thermophilic DEAD box protein, Hera (heat-resistant RNA-dependent ATPase) from Thermus thermophilus HB8. The molecular mass of the deduced Hera protein sequence (510 amino acid residues) is 55.95 kDa. Hera possesses all of the conserved motifs found among the, DEAD-box RNA helicases. In addition, it also has a motif characteristic of the protein component of ribonuclease P at its C-terminal region (residues 372-386). Hera appears to be non-specific with respect to the RNA species that triggers ATPase activity. Nevertheless, at high temperature, ATPase activity is at a maximum when bacterial 16 S rRNA or 23 S rRNA are used as the substrates. Moreover, a deletion of the RNase P protein motif significantly reduces the ability of Hera to hydrolyze ATP in the presence of RNase P RNA. Hera has a specific ATPase activity of 480 units/microg and therefore, displays the highest ATPase specific activity reported for a protein of the RNA helicase family. We determined that Hera shows helix-destabilizing activity, and that the RNA-unwinding or helix-destabilizing activity of Hera is coupled to ATP hydrolysis. Since Hera is a stable thermophilic protein and we have obtained crystals of it diffracting beyond 2.6 A, the possibilities for structure determination of a full-length RNA-helicase are open.     

Cooperative binding of ATP and RNA substrates to the DEAD/H protein DbpA

Unlike most DEAD/H proteins, the purified Escherichia coli protein DbpA demonstrates high specificity for its 23S rRNA substrate in vitro. Here we describe several assays designed to characterize the interaction of DbpA with its RNA and ATP substrates. Electrophoretic mobility shift assays reveal a sub-nanomolar binding affinity for a 153 nucleotide RNA substrate (R153) derived from the 23S rRNA. High affinity RNA binding requires both hairpin 92 and helix 90, as substrates lacking these structures bind DbpA with lower affinity. AMPPNP inhibition assays and ATP/ADP binding assays provide binding constants for ATP and ADP to DbpA with and without RNA substrates. These data have been used to describe a minimal thermodynamic scheme for the binding of the RNA and ATP substrates to DbpA, which reveals cooperative binding between larger RNAs and ATP with cooperative energies of approximately 1.3 kcal mol(-1). This cooperativity is lost upon removal of helix 89 from R153, suggesting this helix is either the preferred target for DbpA's helicase activity or is a necessary structural element for organization of the target site within R153.     

The carboxy-terminal domain of the DExDH protein YxiN is sufficient to confer specificity for 23S rRNA

DE x DH proteins are believed to modulate the structures of RNAs and ribonucleoprotein complexes by disrupting RNA helices and RNA-protein interactions. All DE x DH proteins contain a two-domain catalytic core that enables their RNA-dependent ATPase and RNA helicase activities. The catalytic core may be flanked by ancillary domains that are proposed to confer substrate specificity and facilitate the unique functions of individual proteins. The Escherichia coli DE x DH protein DbpA and its Bacillus subtilis ortholog YxiN have similar 75aa carboxy-terminal domains, and both proteins are specifically targeted to 23S rRNA. Here we demonstrate that the carboxy-terminal domain of YxiN is sufficient to confer RNA specificity by characterizing a chimera in which this domain is appended to the core domains of E.coli SrmB, a DE x DH protein with no apparent substrate specificity. Both the RNA-dependent ATPase and RNA helicase activities of the chimera are specifically activated by 23S rRNA and abolished by sequence changes within hairpin 92, a critical recognition element for Y x iN. These data support a model in which the carboxy-terminal domain binds hairpin 92 to target the protein to 23S rRNA.     

A Structural Model for the DEAD Box Helicase YxiN in Solution: Localization of the RNA Binding Domain

DEAD box proteins consist of a common helicase core formed by two globular RecA domains that are separated by a cleft. The helicase core acts as a nucleotide-dependent switch that alternates between open and closed conformations during the catalytic cycle of duplex separation, thereby providing basic helicase activity. Flanking domains can direct the helicase core to a specific RNA substrate by mediating high-affinity or high-specificity RNA binding. In addition, they may position RNA for the helicase core or may directly contribute to unwinding. While structures of different helicase cores have been determined previously, little is known about the orientation of flanking domains relative to the helicase core.YxiN is a DEAD box protein that consists of a helicase core and a C-terminal RNA binding domain (RBD) that mediates specific binding to hairpin 92 in 23S rRNA. To provide a framework for understanding the functional cooperation of the YxiN helicase core and the RBD, we mapped the orientation of the RBD in single-molecule fluorescence resonance energy transfer experiments. We present a model for the global conformation of YxiN in which the RBD lies above a slightly concave patch that is formed by flexible loops on the surface of the C-terminal RecA domain. The orientation of the RBD is different from the orientations of flanking domains in the Thermus thermophilus DEAD box protein Hera and in Saccharomyces cerevisiae Mss116p, in line with the different functions of these DEAD box proteins and of their RBDs. Interestingly, the corresponding patch on the C-terminal RecA domain that is covered by the YxiN RBD is also part of the interface between the translation factors eIF4A and eIF4G. Possibly, this region constitutes an adaptable interface that generally allows for the interaction of the helicase core with additional domains or interacting factors.     

The DbpA catalytic core unwinds double-helix substrates by directly loading on them

DbpA is a DEAD-box RNA helicase implicated in the assembly of the large ribosomal subunit. Similar to all the members of the DEAD-box family, the DbpA protein has two N-terminal RecA-like domains, which perform the RNA unwinding. However, unlike other members of this family, the DbpA protein also possesses a structured C-terminal RNA-binding domain that mediates specific tethering of DbpA to hairpin 92 of the Escherichia coli 23S ribosomal RNA. Previous studies using model RNA molecules containing hairpin 92 show that the RNA molecules support the DbpA protein''s double-helix unwinding activity, provided that the double helix has a 3′ single-stranded region. The 3′ single-stranded region was suggested to be the start site of the DbpA protein''s catalytic unwinding activity. The data presented here demonstrate that the single-stranded region 3′ of the double-helix substrate is not required for the DbpA protein''s unwinding activity and the DbpA protein unwinds the double-helix substrates by directly loading on them.     

Mutation of the arginine finger in the active site of Escherichia coli DbpA abolishes ATPase and helicase activity and confers a dominant slow growth phenotype

Escherichia coli DEAD-box protein A (DbpA) is an ATP-dependent RNA helicase with specificity for 23S ribosomal RNA. Although DbpA has been extensively characterized biochemically, its biological function remains unknown. Previous work has shown that a DbpA deletion strain is viable with little or no effect on growth rate. In attempt to elucidate a phenotype for DbpA, point mutations were made at eleven conserved residues in the ATPase active site, which have exhibited dominant-negative phenotypes in other DExD/H proteins. Biochemical analysis of these DbpA mutants shows the expected decrease in RNA-dependent ATPase activity and helix unwinding activity. Only the least biochemically active mutation, R331A, produces small colony phenotype and a reduced growth rate. This dominant slow growth mutant will be valuable to determine the cellular function of DbpA.     

A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit

Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg²⁺ exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.     

Nuclear export of the DEAD box An3 protein by CRM1 is coupled to An3 helicase activity

We have recently identified the Xenopus laevis An3 protein as a bona fide substrate for the nuclear export receptor CRM1 (Exportin 1). An3 binds directly to CRM1 with high affinity via a leucine-rich nuclear export signal located in the extreme N terminus. An3 is a member of the DEAD box family of RNA helicases, which unwind RNA duplexes. RNA unwinding is coupled to hydrolysis of nucleoside triphosphates by the helicase, and the ATPase activity of several helicases is greatly stimulated by various polynucleotides. Here we report that dATP hydrolysis by An3 is stimulated approximately 6-fold by total RNA from X. laevis oocytes, whereas poly(U) RNA fails to enhance hydrolysis, suggesting the existence of a specific RNA activator for An3. Kinetic analysis reveals that a mutation within the conserved DEAD box motif reduces the rate of dATP hydrolysis by approximately 6-fold. In accordance with this, the DEAD box mutant is unable to unwind double-stranded RNA. Microinjection of the An3 DEAD box mutant into X. laevis oocytes nuclei reveals a significantly lower export rate as compared with wild-type An3 protein. This is not because the mutant has lower affinity toward CRM1, nor is it due to altered RNA binding capacity. This suggests that nuclear export of An3 protein by CRM1 is coupled to An3 helicase activity.     

Allosteric activation of the ATPase activity of the Escherichia coli RhlB RNA helicase

Helicase B (RhlB) is one of the five DEAD box RNA-dependent ATPases found in Escherichia coli. Unique among these enzymes, RhlB requires an interaction with the partner protein RNase E for appreciable ATPase and RNA unwinding activities. To explore the basis for this activating effect, we have generated a di-cistronic vector that overexpresses a complex comprising RhlB and its recognition site within RNase E, corresponding to residues 696-762. Complex formation has been characterized by isothermal titration calorimetry, revealing an avid, enthalpy-favored interaction between the helicase and RNase E-(696-762) with an equilibrium binding constant (Ka) of at least 1 x 10(8) m(-1). We studied ATPase activity of mutants with substitutions within the ATP binding pocket of RhlB and on the putative interaction surface that mediates recognition of RNase E. For comparisons, corresponding mutations were prepared in two other E. coli DEAD box ATPases, RhlE and SrmB. Strikingly, substitutions at a phenylalanine near the Q-motif found in DEAD box proteins boosts the ATPase activity of RhlB in the absence of RNA, but completely inhibits it in its presence. The data support the proposal that the protein-protein and RNA-binding surfaces both communicate allosterically with the ATPase catalytic center. We conjecture that this communication may govern the mechanical power and efficiency of the helicases, and is tuned in individual helicases in accordance with cellular function.     

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

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.     

The RNA helicase DbpA exhibits a markedly different conformation in the ADP-bound state when compared with the ATP- or RNA-bound states

The motor enzymes that belong to the family of RNA helicases catalyze the strand separation of duplex RNA via ATP hydrolysis. Among these enzymes, Escherichia coli DbpA is a unique RNA helicase because it possesses ATPase-specific activity toward the peptidyl transferase center in 23 S ribosomal RNA. For this reason, it has been the subject of numerous biochemical and structure-function studies. The ATP-stimulated unwinding activity of DbpA toward specific and nonspecific RNA duplexes has been demonstrated. However, the underlying molecular and structural basis, which facilitates its helicase activities, is presently not known. We combined time-dependent limited proteolysis digestion, fluorescence spectroscopy, and three-dimensional structural homology modeling techniques to study the structural conformations of DbpA with respect to its binding to stoichiometric ratios of RNA and cofactors. We show that the conformational state of DbpA is markedly different in the ADP-bound state than in any other state (ATP- or RNA-bound). These results, together with structural homology studies, suggest that a hinge region located in the core domain of DbpA mediates such conformational changes.     

Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A.

eIF-4A is a translation initiation factor that exhibits bidirectional RNA unwinding activity in vitro in the presence of another translation initiation factor, eIF-4B and ATP. This activity is thought to be responsible for the melting of secondary structure in the 5' untranslated region of eukaryotic mRNAs to facilitate ribosome binding. eIF-4A is a member of a fast growing family of proteins termed the DEAD family. These proteins are believed to be RNA helicases, based on the demonstrated in vitro RNA helicase activity of two members (eIF-4A and p68) and their homology in eight amino acid regions. Several related biochemical activities were attributed to eIF-4A: (i) ATP binding, (ii) RNA-dependent ATPase and (iii) RNA helicase. To determine the contribution of the highly conserved regions to these activities, we performed site-directed mutagenesis. First we show that recombinant eIF-4A, together with recombinant eIF-4B, exhibit RNA helicase activity in vitro. Mutations in the ATPase A motif (AXXXXGKT) affect ATP binding, whereas mutations in the predicted ATPase B motif (DEAD) affect ATP hydrolysis. We report here that the DEAD region couples the ATPase with the RNA helicase activity. Furthermore, two other regions, whose functions were unknown, have also been characterized. We report that the first residue in the HRIGRXXR region is involved in ATP hydrolysis and that the SAT region is essential for RNA unwinding. Our results suggest that the highly conserved regions in the DEAD box family are critical for RNA helicase activity.