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.     

YxiN is a modular protein combining a DEx(D/H) core and a specific RNA-binding domain

DEx(D/H) proteins, typically described as RNA helicases, participate in rearrangement of RNA-RNA and possibly RNA-protein complexes in the cell. Aside from the conserved DEx(D/H) core, members of this protein family often contain N- and C-terminal extensions that are responsible for additional functions. The Bacillus subtilis DEx(D/H)-box protein YxiN and its Escherichia coli ortholog DbpA contain an approximately 80 amino acid C-terminal extension that has been proposed to specifically interact with a region of 23 S ribosomal RNA including hairpin 92. In this study, the DEx(D/H)-box core and the C-terminal domain of YxiN were expressed and characterized as separate proteins. The isolated DEx(D/H)-box core, YxCat, had weak, nonspecific RNA binding activity and showed RNA-stimulated ATPase activity with a Km(ATP) that resembled several non-specific DEx(D/H) proteins. The isolated C-terminal domain, YxRBD, bound RNA with the high affinity and specificity seen with full-length YxiN. Thus, YxiN is a modular protein combining the activities of the YxCat and YxRBD domains. Footprinting of YxiN and YxRBD on a 172-nucleotide fragment of 23 S rRNA was used to identify the sites of interaction of the C-terminal and helicase domains with the RNA.     

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.     

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.     

The Bacillus subtilis RNA Helicase YxiN is Distended in Solution

The Bacillus subtilis YxiN protein is a modular three-domain RNA helicase of the DEx(D/H)-box protein family. The first two domains form the highly conserved helicase core, and the third domain confers RNA target binding specificity. Small angle x-ray scattering on YxiN and two-domain fragments thereof shows that the protein has a distended structure in solution, in contrast to helicases involved in replication processes. These data are consistent with a chaperone activity in which the carboxy-terminal domain of YxiN tethers the protein to the vicinity of its targets and the helicase core is free to transiently interact with RNA duplexes, possibly to melt out misfolded elements of secondary structure.     

The Thermus thermophilus DEAD box helicase Hera contains a modified RNA recognition motif domain loosely connected to the helicase core

DEAD box family helicases consist of a helicase core that is formed by two flexibly linked RecA-like domains. The helicase activity can be regulated by N- or C-terminal extensions flanking the core. Thermus thermophilus heat resistant RNA-dependent ATPase (Hera) is the first DEAD box helicase that forms a dimer using a unique dimerization domain. In addition to the dimerization domain, Hera contains a C-terminal RNA binding domain (RBD) that shares sequence homology only to uncharacterized proteins of the Deinococcus/Thermus group. The crystal structure of Hera_RBD reveals the fold of an altered RNA recognition motif (RRM) with limited structural homology to the RBD of the DEAD box helicase YxiN from Bacillus subtilis. Comparison with RRM/RNA complexes shows that a RNA binding mode different than that suggested for YxiN, but similar to U1A, can be inferred for Hera. The orientation of the RBD relative to the helicase core was defined in a second crystal structure of a Hera fragment including the C-terminal RecA domain, the dimerization domain, and the RBD. The structures allow construction of a model for the entire Hera helicase dimer. A likely binding surface for large RNA substrates that spans both RecA-like domains and the RBD is identified.     

Structure of the RNA Binding Domain of a DEAD-Box Helicase Bound to Its Ribosomal RNA Target Reveals a Novel Mode of Recognition by an RNA Recognition Motif

DEAD-box RNA helicases of the bacterial DbpA subfamily are localized to their biological substrate when a carboxy-terminal RNA recognition motif domain binds tightly and specifically to a segment of 23S ribosomal RNA (rRNA) that includes hairpin 92 of the peptidyl transferase center. A complex between a fragment of 23S rRNA and the RNA binding domain (RBD) of the Bacillus subtilis DbpA protein YxiN was crystallized and its structure was determined to 2.9 Å resolution, revealing an RNA recognition mode that differs from those observed with other RNA recognition motifs. The RBD is bound between two RNA strands at a three-way junction. Multiple phosphates of the RNA backbone interact with an electropositive band generated by lysines of the RBD. Nucleotides of the single-stranded loop of hairpin 92 interact with the RBD, including the guanosine base of G2553, which forms three hydrogen bonds with the peptide backbone. A G2553U mutation reduces the RNA binding affinity by 2 orders of magnitude, confirming that G2553 is a sequence specificity determinant in RNA binding. Binding of the RBD to 23S rRNA in the late stages of ribosome subunit maturation would position the ATP-binding duplex destabilization fragment of the protein for interaction with rRNA in the peptidyl transferase cleft of the subunit, allowing it to “melt out” unstable secondary structures and allow proper folding.     

The domain of the Bacillus subtilis DEAD-box helicase YxiN that is responsible for specific binding of 23S rRNA has an RNA recognition motif fold

The YxiN protein of Bacillus subtilis is a member of the DbpA subfamily of prokaryotic DEAD-box RNA helicases. Like DbpA, it binds with high affinity and specificity to segments of 23S ribosomal RNA as short as 32 nucleotides (nt) that include hairpin 92. Several experiments have shown that the 76-residue carboxy-terminal domain of YxiN is responsible for the high-affinity RNA binding. The domain has been crystallized and its structure has been solved to 1.7 Angstroms resolution. The structure reveals an RNA recognition motif (RRM) fold that is found in many eukaryotic RNA binding proteins; the RRM fold was not apparent from the amino acid sequence. The domain has two solvent exposed aromatic residues at sites that correspond to the aromatic residues of the ribonucleoprotein (RNP) motifs RNP1 and RNP2 that are essential for RNA binding in many RRMs. However, mutagenesis of these residues (Tyr404 and Tyr447) to alanine has little effect on RNA affinity, suggesting that the YxiN domain binds target RNAs in a manner that differs from the binding mode commonly found in many eukaryotic RRMs.     

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.     

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.     

Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains

RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.     

Function of the C-terminal domain of the DEAD-box protein Mss116p analyzed in vivo and in vitro

The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are general RNA chaperones that function in splicing mitochondrial group I and group II introns and in translational activation. Both proteins consist of a conserved ATP-dependent RNA helicase core region linked to N and C-terminal domains, the latter with a basic tail similar to many other DEAD-box proteins. In CYT-19, this basic tail was shown to contribute to non-specific RNA binding that helps tether the core helicase region to structured RNA substrates. Here, multiple sequence alignments and secondary structure predictions indicate that CYT-19 and Mss116p belong to distinct subgroups of DEAD-box proteins, whose C-terminal domains have a defining extended α-helical region preceding the basic tail. We find that mutations or C-terminal truncations in the predicted α-helical region of Mss116p strongly inhibit RNA-dependent ATPase activity, leading to loss of function in both translational activation and RNA splicing. These findings suggest that the α-helical region may stabilize and/or regulate the activity of the RNA helicase core. By contrast, a truncation that removes only the basic tail leaves high RNA-dependent ATPase activity and causes only a modest reduction in translation and RNA splicing efficiency in vivo and in vitro. Biochemical analysis shows that deletion of the basic tail leads to weaker non-specific binding of group I and group II intron RNAs, and surprisingly, also impairs RNA-unwinding at saturating protein concentrations and nucleotide-dependent tight binding of single-stranded RNAs by the RNA helicase core. Together, our results indicate that the two sub-regions of Mss116p's C-terminal domain act in different ways to support and modulate activities of the core helicase region, whose RNA-unwinding activity is critical for both the translation and RNA splicing functions.     

DEAD-box RNA helicase domains exhibit a continuum between complete functional independence and high thermodynamic coupling in nucleotide and RNA duplex recognition

DEAD-box helicases catalyze the non-processive unwinding of double-stranded RNA (dsRNA) at the expense of adenosine triphosphate (ATP) hydrolysis. Nucleotide and RNA binding and unwinding are mediated by the RecA domains of the helicase core, but their cooperation in these processes remains poorly understood. We therefore investigated dsRNA and nucleotide binding by the helicase cores and the isolated N- and C-terminal RecA domains (RecA_N, RecA_C) of the DEAD-box proteins Hera and YxiN by steady-state and time-resolved fluorescence methods. Both helicases bind nucleotides predominantly via RecA_N, in agreement with previous studies on Mss116, and with a universal, modular function of RecA_N in nucleotide recognition. In contrast, dsRNA recognition is different: Hera interacts with dsRNA in the absence of nucleotide, involving both RecA domains, whereas for YxiN neither RecA_N nor RecA_C binds dsRNA, and the complete core only interacts with dsRNA after nucleotide has been bound. DEAD-box proteins thus cover a continuum from complete functional independence of their domains, exemplified by Mss116, to various degrees of inter-domain cooperation in dsRNA binding. The different degrees of domain communication and of thermodynamic linkage between dsRNA and nucleotide binding have important implications on the mechanism of dsRNA unwinding, and may help direct RNA helicases to their respective cellular processes.     

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.     

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.     

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.     

Hydrodynamic characterization of the DEAD-box RNA helicase DbpA

The Escherichia coli DEAD-box protein A (DbpA) belongs to the highly conserved superfamily-II of nucleic acid helicases that play key roles in RNA metabolism. A central question regarding helicase activity is whether the process of coupling ATP hydrolysis to nucleic acid unwinding requires an oligomeric form of the enzyme. We have investigated the structural and functional properties of DbpA by multi-angle laser light-scattering, size-exclusion chromatography, analytical ultracentrifugation, chemical cross-linking and hydrodynamic modeling. DbpA is monomeric in solution up to a concentration of 25 microM and over the temperature range of 4 degrees C to 22 degrees C. Binding of neither nucleotide (ATP or ADP) nor peptidyl transferase center (PTC) RNA, the presumed physiological RNA substrate, favor oligomerization. The hydrodynamic parameters were used together with hydrodynamic bead modeling and structural homology in conjunction with ab initio structure prediction methods to define plausible shapes of DbpA. Collectively, the results favor models where DbpA functions as an active monomer that possesses two distinct RNA binding sites, one in the helicase core domain and the other in the carboxyl-terminal domain that recognizes 23S rRNA and interacts specifically with hairpin 92 of the PTC.     

Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro

RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNA_i^Met. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNA_i^Met turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNA_i^Met turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities.     

Escherichia coli DbpA is a 3' --> 5' RNA helicase

Previous work has demonstrated that Escherichia coli DbpA is a nonprocessive RNA helicase that can disrupt short RNA helices on either the 5' side or 3' side of hairpin 92 of 23S rRNA. Here the directionality of the helicase activity of DbpA was determined by using substrates containing a short reporter helix in the presence of a second adjacent helix of varying stability placed either 5' or 3' of the reporter helix. When the second helix was on the 5' side of the reporter helix, it had no effect on the dissociation rate of the reporter helix. However, when the second helix was on the 3' side of the reporter helix, its dissociation rate determined the dissociation rate of the reporter helix. This defines DbpA as a 3' --> 5' helicase. Like other helicases, DbpA requires a single-stranded RNA loading site on the 3' side of the duplex for disruption to be observed. Since the loading site could be on either strand of the helix that was disrupted, hairpin 92 does not influence the directionality of the helicase but only aids in targeting RNA substrates.