Pathway of ATP utilization and duplex rRNA unwinding by the DEAD-box helicase, DbpA (original) (raw)
Related papers
The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA
2008
DEAD-box proteins are ATPase enzymes that destabilize and unwind duplex RNA. Quantitative knowledge of the ATPase cycle parameters is critical for developing models of helicase activity. However, limited information regarding the rate and equilibrium constants defining the ATPase cycle of RNA helicases is available, including the distribution and flux of populated biochemical intermediates, the catalytic step(s) that limits the enzymatic reaction cycle, and how ATP utilization and RNA interactions are linked. We present a quantitative kinetic and equilibrium characterization of the rRNA-activated ATPase cycle mechanism of DbpA, a DEAD-box rRNA helicase implicated in ribosome biogenesis. rRNA activates the ATPase activity of DbpA by promoting a conformational change after ATP binding that is associated with hydrolysis. Chemical cleavage of bound ATP is reversible and occurs via a gamma phosphate attack mechanism. ADP-P i and RNA binding display strong thermodynamic coupling, which causes DbpA-ADP-P i to bind rRNA with > 10-fold higher affinity than with bound ATP, ADP or in the absence of nucleotide. The rRNA-activated steady-state ATPase cycle of DbpA is limited both by ATP hydrolysis and P i release, which occur with comparable rates. Consequently, the predominantly populated biochemical states during steady-state cycling are the ATP-and ADP-P i -bound intermediates. Thermodynamic linkage analysis of the ATPase cycle transitions favors models in which rRNA duplex destabilization is linked to strong rRNA and nucleotide binding. The presented analysis of the DbpA ATPase cycle reaction mechanism provides a rigorous kinetic and thermodynamic foundation for developing testable hypotheses regarding the functions and molecular mechanisms of DEAD-box helicases.
Nucleic Acids Research, 2023
Ribosomes are ribozymes, hence correct folding of the rRNAs during ribosome biogenesis is crucial to ensure catalytic activity. RNA helicases, which can modulate RNA-RNA and RNA/protein interactions, are proposed to participate in rRNA tridimensional folding. Here, we analyze the biochemical properties of Dbp6, a DEAD-box RNA helicase required for the conversion of the initial 90S pre-ribosomal particle into the first pre-60S particle. We demonstrate that in vitro, Dbp6 shows ATPase as well as annealing and clamping activities negatively regulated by ATP. Mutations in Dbp6 core motifs involved in ATP binding and ATP hydrolysis are lethal and impair Dbp6 ATPase activity but increase its RNA binding and RNA annealing activities. These data suggest that correct regulation of these activities is important for Dbp6 function in vivo. Using in vivo cross-linking (CRAC) experiments, we show that Dbp6 interacts with 25S rRNA sequences located in the 5 domain I and in the peptidyl transferase center (PTC), and also crosslinks to snoRNAs hybridizing to the immature PTC. We propose that the ATPase and RNA clamping/annealing activities of Dbp6 modulate interactions of snoRNAs with the immature PTC and/or contribute directly to the folding of this region.
Journal of Molecular Biology, 2010
Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATPdependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k cat ), but not the affinity for ATP (K m ). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of γ-PO 4 of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the β,γ-phosphoanhydride bond of ATP.
Allosteric Activation of the ATPase Activity of the Escherichia coli RhlB RNA Helicase
Journal of Biological Chemistry, 2007
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, enthalpyfavored interaction between the helicase and RNase E-(696-762) with an equilibrium binding constant (K a) of at least 1 ؋ 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.
Recognition and Cooperation Between the ATP-dependent RNA Helicase RhlB and Ribonuclease RNase E
Journal of Molecular Biology, 2007
The Escherichia coli protein RhlB is an ATP-dependent motor that unfolds structured RNA for destruction by partner ribonucleases. In E. coli, and probably many other related γ-proteobacteria, RhlB associates with the essential endoribonuclease RNase E as part of the multi-enzyme RNA degradosome assembly. The interaction with RNase E boosts RhlB's ATPase activity by an order of magnitude. Here, we examine the origins and implications of this effect. The location of the interaction sites on both RNase E and RhlB are refined and analysed using limited protease digestion, domain cross-linking and homology modelling. These data indicate that RhlB's carboxy-terminal RecA-like domain engages a segment of RNase E that is no greater than 64 residues. The interaction between RhlB and RNase E has two important consequences: first, the interaction itself stimulates the unwinding and ATPase activities of RhlB; second, RhlB gains proximity to two RNA-binding sites on RNase E, with which it cooperates to unwind RNA. Our homology model identifies a pattern of residues in RhlB that may be key for recognition of RNase E and which may communicate the activating effects. Our data also suggest that the association with RNase E may partially repress the RNA-binding activity of RhlB. This repression may in fact permit the interplay of the helicase and adjacent RNA binding segments as part of a process that steers substrates to either processing or destruction, depending on context, within the RNA degradosome assembly. Abbreviations used: AR2, arginine-rich region 2 of RNase E (EC 3.1.26), corresponding to residues 798 to 819; DMS, dimethylsuberimidate; ODA, optimal docking area; PNPase, polynucleotide phosphorylase (EC 2.7.7.8); RBD, RNA-binding domain of RNase E, corresponding to residues 604 to 688; REP, repetitive extragenic palindrome; RhlB, RNA helicase B (EC 3.6.1); RhlB-CTD, carboxy terminal domain of RhlB helicase, corresponding to residues 267 to 421; RhlB-NTD, amino terminal domain of RhlB helicase, corresponding to residues 1 to 266; RhlB-ΔCT, derivative of RhlB lacking the arginine-rich carboxy-terminal residues 398 to 421; RNase E (628-843), a segment of ribonuclease RNase E corresponding to residues 628 to 843; RNase E (498-1061), segment of RNase E containing residues 1-26 and 498 to 1061;
The DEAD-box protein family of RNA helicases
Gene, 2006
RNA helicases of the DEAD-box protein family have been shown to participate in every aspect of RNA metabolism. They are present in most organisms where they work as RNA helicases or RNPases. The properties of these enzymes in vivo remains poorly described, however some were extensively characterized in vitro, and the solved crystal structures of a few are now available. Taken together, this information gives insight into the regulation of ATP and RNA binding as well as in the ATPase and helicase activities. This review will focus on the description of the molecular characteristics of members of the DEAD-box protein family and on the enzymatic activities they possess.
Potential Regulatory Interactions of Escherichia coli RraA Protein with DEAD-box Helicases
Journal of Biological Chemistry, 2013
Background: DEAD-box helicases in bacteria play a key role in cellular RNA metabolism. Results: The trimeric protein RraA binds to Escherichia coli DEAD-box proteins. Conclusion: The mechanism of interaction between RraA and SrmB is shown by x-ray crystallography. Significance: Structural basis of potential regulation of a bacterial DEAD-box helicase. Members of the DEAD-box family of RNA helicases contribute to virtually every aspect of RNA metabolism, in organisms from all domains of life. Many of these helicases are constituents of multicomponent assemblies, and their interactions with partner proteins within the complexes underpin their activities and biological function. In Escherichia coli the DEAD-box helicase RhlB is a component of the multienzyme RNA degradosome assembly, and its interaction with the core ribonuclease RNase E boosts the ATP-dependent activity of the helicase. Earlier studies have identified the regulator of ribonuclease activity A (RraA) as a potential interaction partner of both RNase E and RhlB. We present structural and biochemical evidence showing how RraA can bind to, and modulate the activity of RhlB and another E. coli DEAD-box enzyme, SrmB. Crystallographic structures are presented of RraA in complex with a portion of the natively unstructured C-terminal tail of RhlB at 2.8-Å resolution, and in complex with the C-terminal RecA-like domain of SrmB at 2.9 Å. The models suggest two distinct mechanisms by which RraA might modulate the activity of these and potentially other helicases. RraA 3 (Regulator of ribonuclease activity A) is a ring-shaped homotrimeric protein with the ability in vitro to influence the activity of the essential Escherichia coli ribonuclease, RNase E (1). As an inhibitor of RNase E, RraA has widespread effects on transcript levels in E. coli (2), although its physiological role in ribonuclease regulation is debated. Surprisingly, in vitro RraA does not inhibit the catalytic activity of RNase E directly, but appears to act indirectly by occluding RNA binding regions in the C-terminal domain of the ribonuclease (1). These RNA binding domains are adjacent to a site that recruits the DEADbox helicase RhlB, in a multienzyme assembly known as the RNA degradosome. As a component of the RNA degradosome, RhlB contributes to mRNA decay and RNA processing (3-5). We have previously shown that RraA can directly interact with RhlB, and it is possible that RraA plays two distinct roles in modulating the degradosome, by inhibiting both its ribonuclease and helicase activities (1). In the Górna et al. (1) study, it was shown that RraA is able to interact in vitro with two other DEAD-box proteins from Escherichia coli, namely RhlE and SrmB. SrmB is one of the five DEAD-box proteins in E. coli and is known to contribute to ribosome biogenesis along with CsdA (DeaD), RhlE, and DbpA (6). SrmB targets 23S rRNA in vivo and forms a ribonucleoprotein complex with ribosomal proteins L4 and L24 (7). SrmB is believed to act as a chaperone by preventing 23S rRNA structures from misfolding during ribosome assembly, preventing their spurious interaction with 5S rRNA (6, 8). It also helps to prevent interactions between rRNA decay intermediates and nascent 50S ribosome subunits (9). Although RhlB is the canonical helicase component of the RNA degradosome, other DEAD-box helicases may be recruited into the assembly depending on growth conditions. For instance, under conditions of cold stress, CsdA may be recruited to the degradosome (10), and SrmB becomes associated during stationary growth phase (11). Moreover, the functional interplay between helicases and RNase E may be important in vivo, as genetic screens show that mutations of CsdA suppress the phenotype of RNase E defects (12). Here we further investigate the interaction between RraA and the DEAD-box helicases of E. coli. We present biochemical and structural data to characterize the interaction between RraA and SrmB, and provide structural data on the interaction of RhlB with RraA and compare the two complexes. Our data show that RraA can modulate SrmB activity and provide structural insight into the mechanism. Finally, we elaborate on possible functions of the interaction between RraA and DEAD-box helicases. EXPERIMENTAL PROCEDURES Protein Purification E. coli BL21(DE3) transformed with protein expression vectors were grown at 37°C in 2x YT medium (Formedium) sup-* This work was supported in part by the Wellcome Trust.
Rna-a Publication of The Rna Society, 2010
The Escherichia coli endoribonuclease RNase E is an essential enzyme having key roles in mRNA turnover and the processing of several structured RNA precursors, and it provides the scaffold to assemble the multienzyme RNA degradosome. The activity of RNase E is inhibited by the protein RraA, which can interact with the ribonuclease's degradosome-scaffolding domain. Here, we report that RraA can bind to the RNA helicase component of the degradosome (RhlB) and the two RNA-binding sites in the degradosome-scaffolding domain of RNase E. In the presence of ATP, the helicase can facilitate the exchange of RraA for RNA stably bound to the degradosome. Our data suggest that RraA can affect multiple components of the RNA degradosome in a dynamic, energy-dependent equilibrium. The multidentate interactions of RraA impede the RNA-binding and ribonuclease activities of the degradosome and may result in complex modulation and rerouting of degradosome activity.
Journal of Biological Chemistry, 2002
The Escherichia coli RNA degradosome is a multicomponent ribonucleolytic complex consisting of three major proteins that assemble on a scaffold provided by the C-terminal region of the endonuclease, RNase E. Using an E. coli two-hybrid system, together with BIAcore apparatus, we investigated the ability of three proteins, polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase, a glycolytic protein, to interact physically and functionally independently of RNase E. Here we report that Rh1B can physically bind to PNPase, both in vitro and in vivo, and can also form homodimers with itself. However, binding of RhlB or PNPase to enolase was not detected under the same conditions. BIAcore analysis revealed real-time, direct binding for bimolecular interactions between Rh1B units and for the RhlB interaction with PNPase. Furthermore, in the absence of RNase E, purified RhlB can carry out ATP-dependent unwinding of double-stranded RNA and consequently modulate degradation of double-stranded RNA together with the exonuclease activity of PNPase. These results provide evidence for the first time that both functional and physical interactions of individual degradosome protein components can occur in the absence of RNase E and raise the prospect that the RNase E-independent complexes of RhlB RNA helicase and PNPase, detected in vivo, may constitute mini-machines that assist in the degradation of duplex RNA in structures physically distinct from multicomponent RNA degradosomes.