Energy-dependent degradation: Linkage between ClpX-catalyzed nucleotide hydrolysis and protein-substrate processing

ClpX requires ATP to unfold protein substrates and translocate them into the proteolytic chamber of ClpP for degradation. The steady-state parameters for hydrolysis of ATP and ATPgammaS by ClpX were measured with different protein partners and the kinetics of degradation of ssrA-tagged substrates were determined with both nucleotides. ClpX hydrolyzed ATPgammaS to ADP and thiophosphate at a rate (6/min) significantly slower than ATP hydrolysis (140/min), but the hydrolysis of both nucleotides was increased by ssrA-tagged substrates and decreased by ClpP. K(M) and k(cat) for hydrolysis of ATP and ATPgammaS were linearly correlated over a 200-fold range, suggesting that protein partners largely affect k(cat) rather than nucleotide binding, indicating that most bound ATP leaves the enzyme by hydrolysis rather than dissociation, and placing an upper limit of approximately 15 micro M on K(D) for both nucleotides. Competition studies with ClpX and fluorescently labeled ADP gave inhibition constants for ATPgammaS ( approximately 2 micro M) and ADP ( approximately 3 micro M) under the reaction conditions used for steady-state kinetics. In the absence of Mg(2+), where hydrolysis does not occur, the inhibition constant for ATP ( approximately 55 micro M) was weaker but very similar to the value for ATPgammaS ( approximately 45 micro M). Compared with ATP, ATPgammaS supported slow but roughly comparable rates of ClpXP degradation for two Arc-ssrA substrates and denatured GFP-ssrA, but not of native GFP-ssrA. These results show that the processing of protein substrates by ClpX is closely coupled to the maximum rate of nucleotide hydrolysis.     

Duplex unwinding and ATPase activities of the DEAD-box helicase eIF4A are coupled by eIF4G and eIF4B

Eukaryotic initiation factor (eIF) 4A is a DEAD-box helicase that stimulates translation initiation by unwinding mRNA secondary structure. The accessory proteins eIF4G, eIF4B, and eIF4H enhance the duplex unwinding activity of eIF4A, but the extent to which they modulate eIF4A activity is poorly understood. Here, we use real-time fluorescence assays to determine the kinetic parameters of duplex unwinding and ATP hydrolysis by these initiation factors. To ensure efficient duplex unwinding, eIF4B and eIF4G cooperatively activate the duplex unwinding activity of eIF4A. Our data reveal that eIF4H is much less efficient at stimulating eIF4A unwinding activity than eIF4B, implying that eIF4H is not able to completely substitute for eIF4B in duplex unwinding. By monitoring unwinding and ATPase assays under identical conditions, we demonstrate that eIF4B couples the ATP hydrolysis cycle of eIF4A with strand separation, thereby minimizing nonproductive unwinding events. Using duplex substrates with altered GC contents but similar predicted thermal stabilities, we further show that the rate of formation of productive unwinding complexes is strongly influenced by the local stability per base pair, in addition to the stability of the entire duplex. This finding explains how a change in the GC content of a hairpin is able to influence translation initiation while maintaining the overall predicted thermal stability.     

Wheat germ translation initiation factor eIF4B affects eIF4A and eIFiso4F helicase activity by increasing the ATP binding affinity of eIF4A

It has been proposed that, during translational initiation, structures in the 5' untranslated region of mRNA are unwound. eIF4A, a member of the DEAD box family of proteins (those that contain a DEAD amino acid sequence), separately or in conjunction with other eukaryotic initiation factors, utilizes the energy from ATP hydrolysis to unwind these structures. As a step in defining the mechanism of helicase activity in the wheat germ protein synthesis system, we have utilized direct fluorescence measurements, ATPase assays, and helicase assays. The RNA duplex unwinding activity of wheat germ eIF4A is similar to other mammalian systems; however, eIF4F or eIFiso4F is required, probably because of the low binding affinity of wheat germ eIF4A for mRNA. Direct ATP binding measurements showed that eIF4A had a higher binding affinity for ADP than ATP, resulting in a limited hydrolysis and procession along the RNA in the helicase assay. The addition of eIF4B resulted in a change in binding affinity for ATP, increasing it almost 10-fold while the ADP binding affinity was approximately the same. The data presented in this paper suggest that eIF4F or eIFiso4F acts to position the eIF4A and stabilize the interaction with mRNA. ATP produces a conformational change which allows a limited unwinding of the RNA duplex. The binding of eIF4B either prior to or after hydrolysis allows for increased affinity for ATP and for the cycle of conformational changes to proceed, resulting in further unwinding and processive movement along the mRNA.     

eIF4B and eIF4G Jointly Stimulate eIF4A ATPase and Unwinding Activities by Modulation of the eIF4A Conformational Cycle

Eukaryotic translation initiation factor 4A (eIF4A) is a DEAD-box protein that participates in translation initiation. As an ATP-dependent RNA helicase, it is thought to resolve secondary structure elements from the 5′-untranslated region of mRNAs to enable ribosome scanning. The RNA-stimulated ATPase and ATP-dependent helicase activities of eIF4A are enhanced by auxiliary proteins, but the underlying mechanisms are still largely unknown. Here, we have dissected the effect of eIF4B and eIF4G on eIF4A RNA-dependent ATPase- and RNA helicase activities and on eIF4A conformation. We show for the first time that yeast eIF4B, like its mammalian counterpart, can stimulate RNA unwinding by eIF4A, although it does not affect the eIF4A conformation. The eIF4G middle domain enhances this stimulatory effect and promotes the formation of a closed eIF4A conformation in the presence of ATP and RNA. The closed state of eIF4A has been inferred but has not been observed experimentally before. eIF4B and eIF4G jointly stimulate ATP hydrolysis and RNA unwinding by eIF4A and favor the formation of the closed eIF4A conformer. Our results reveal distinct functions of eIF4B and eIF4G in synergistically stimulating the eIF4A helicase activity in the mRNA scanning process.     

Further biochemical and kinetic characterization of human eukaryotic initiation factor 4H

A cDNA encoding human eukaryotic initiation factor (eIF) 4H was subcloned into a bacterial expression plasmid for purification of recombinant protein. Recombinant human eIF4H (heIF4H) was purified to greater than 95% homogeneity and shown to have similar physical characteristics to eIF4H purified from rabbit reticulocyte lysate as described previously. Functional studies have revealed that recombinant heIF4H functions identically to rabbit eIF4H in stimulating protein synthesis, and the ATP hydrolysis and helicase activities of eIF4A. More detailed enzymatic studies revealed that eIF4H increases the affinity of eIF4A for RNA by 2-fold, but has no effect on the binding of ATP by eIF4A. eIF4H stimulates the helicase activity of eIF4A at least 4-fold, and it is postulated that this stimulation occurs through increasing the processivity of eIF4A. Northern blot analysis shows that eIF4H is expressed ubiquitously in human tissues, and displays different levels of expression in given tissues relative to eIF4B. Secondary structure analysis of heIF4H by circular dichroism suggest that eIF4H has a mostly beta-sheet structure, which appears similar to other RNA recognition motif-containing proteins. Finally, it is suggested that eIF4H functions in translation initiation through protein-protein interactions that possibly stabilize conformational changes that occur in eIF4A during RNA binding, ATP hydrolysis, and RNA duplex unwinding.     

ATP binding,not hydrolysis,at the first nucleotide-binding domain of multidrug resistance-associated protein MRP1 enhances ADP.Vi trapping at the second domain

Multidrug resistance-associated protein (MRP1) transports solutes in an ATP-dependent manner by utilizing its two nonequivalent nucleotide binding domains (NBDs) to bind and hydrolyze ATP. We found that ATP binding to the first NBD of MRP1 increases binding and trapping of ADP at the second domain (Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2002) J. Biol. Chem. 277, 5110-5119). These results were interpreted as indicating that the binding of ATP at NBD1 causes a conformational change in the molecule and increases the affinity for ATP at NBD2. However, we did not distinguish between the possibilities that the enhancement of ADP trapping might be caused by either ATP binding alone or hydrolysis. We now report the following. 1) ATP has a much lesser effect at 0 degrees C than at 37 degrees C. 2) After hexokinase treatment, the nonhydrolyzable ATP analogue, adenyl 5'-(yl iminodiphosphate), does not enhance ADP trapping. 3) Another nonhydrolyzable ATP analogue, adenosine 5'-(beta,gamma-methylene)triphosphate, whether hexokinase-treated or not, causes a slight enhancement. 4) In contrast, the hexokinase-treated poorly hydrolyzable ATP analogue, adenosine 5'-O-(thiotriphosphate) (ATPgammaS), enhances ADP trapping to a similar extent as ATP under conditions in which ATPgammaS should not be hydrolyzed. We conclude that: 1) ATP hydrolysis is not required to enhance ADP trapping by MRP1 protein; 2) with nucleotides having appropriate structure such as ATP or ATPgammaS, binding alone can enhance ADP trapping by MRP1; 3) the stimulatory effect on ADP trapping is greatly diminished when the MRP1 protein is in a "frozen state" (0 degrees C); and 4) the steric structure of the nucleotide gamma-phosphate is crucial in determining whether binding of the nucleotide to NBD1 of MRP1 protein can induce the conformational change that influences nucleotide trapping at NBD2.     

Translational control by a small RNA: dendritic BC1 RNA targets the eukaryotic initiation factor 4A helicase mechanism

Translational repressors, increasing evidence suggests, participate in the regulation of protein synthesis at the synapse, thus providing a basis for the long-term plastic modulation of synaptic strength. Dendritic BC1 RNA is a non-protein-coding RNA that represses translation at the level of initiation. However, the molecular mechanism of BC1 repression has remained unknown. Here we identify the catalytic activity of eukaryotic initiation factor 4A (eIF4A), an ATP-dependent RNA helicase, as a target of BC1-mediated translational control. BC1 RNA specifically blocks the RNA duplex unwinding activity of eIF4A but, at the same time, stimulates its ATPase activity. BC200 RNA, the primate-specific BC1 counterpart, targets eIF4A activity in identical fashion, as a result decoupling ATP hydrolysis from RNA duplex unwinding. In vivo, BC1 RNA represses translation of a reporter mRNA with 5' secondary structure. The eIF4A mechanism places BC RNAs in a central position to modulate protein synthesis in neurons.     

Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: magnesium provides a bridge for ATP to fuel unwinding

This study investigates the role of magnesium ions in coupling ATP hydrolysis to the nucleic acid unwinding catalyzed by the NS3 protein encoded by the hepatitis C virus (HCV). Analyses of steady-state ATP hydrolysis rates at various RNA and magnesium concentrations were used to determine values for the 15 dissociation constants describing the formation of a productive enzyme-metal-ATP-RNA complex and the four rate constants describing hydrolysis of ATP by the possible enzyme-ATP complexes. These values coupled with direct binding studies, specificity studies and analyses of site-directed mutants reveal only one ATP binding site on HCV helicase centered on the catalytic base Glu291. An adjacent residue, Asp290, binds a magnesium ion that forms a bridge to ATP, reorienting the nucleotide in the active site. RNA stimulates hydrolysis while decreasing the affinity of the enzyme for ATP, magnesium, and MgATP. The binding scheme described here explains the unusual regulation of the enzyme by ATP that has been reported previously. Binding of either free magnesium or free ATP to HCV helicase competes with MgATP, the true fuel for helicase movements, and leads to slower hydrolysis and nucleic acid unwinding.     

Interaction between the NH2-terminal domain of eIF4A and the central domain of eIF4G modulates RNA-stimulated ATPase activity

The eukaryotic translation factor 4A (eIF4A) is a member of DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples ATP hydrolysis to RNA binding and duplex separation. eIF4A participates in the initiation of translation by unwinding secondary structure in the 5'-untranslated region of mRNAs and facilitating scanning by the 40 S ribosomal subunit for the initiation codon. eIF4A alone has only weak ATPase and helicase activities, but these are stimulated by eIF4G, eIF4B, and eIF4H. eIF4G has two eIF4A-binding sites, one in the central domain (cp(C3)) and one in the COOH-terminal domain (cp(C2)). In the current work, we demonstrate that these two eIF4G domains have different effects on the RNA-stimulated ATPase activity of eIF4A. cp(C3) stimulates ATP-hydrolytic efficiency by about 40-fold through two mechanisms: lowering K(m)(RNA) by 10-fold and raising k(cat) by 4-fold. cp(C3) also stimulates RNA cross-linking to eIF4A in an ATP-independent manner. Studies with eIF4G and eIF4A variants suggest a model by which cp(C3) alters the conformation of the catalytic site to favor RNA binding. cp(C2) does not stimulate ATPase activity and furthermore increases both K(m)(ATP) (at saturating RNA concentrations) and K(m)(RNA) (at subsaturating ATP concentrations). Both cp(C3) and cp(C2) directly interact with the NH(2)-terminal domain of eIF4A, which possesses conserved ATP- and oligonucleotide-binding motifs, but not with the COOH-terminal domain.     

Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F.

Eukaryotic initiation factor (eIF) 4A is a DEAD box RNA helicase that works in conjunction with eIF4B, eIF4H, or as a subunit of eIF4F to unwind secondary structure in the 5'-untranslated region of mRNA, which facilitates binding of the mRNA to the 40 S ribosomal subunit. This study demonstrates how the helicase activity of eIF4A is modulated by eIF4B, eIF4H, or as a subunit of eIF4F. Results indicate that a linear relationship exists between the initial rate or amplitude of unwinding and duplex stability for all factor combinations tested. eIF4F, like eIF4A, behaves as a non-processive helicase. Either eIF4B or eIF4H stimulated the initial rate and amplitude of eIF4A-dependent duplex unwinding, and the magnitude of stimulation is dependent on duplex stability. Furthermore, eIF4A (or eIF4F) becomes a slightly processive helicase in the presence of eIF4B or eIF4H. All combinations of factors tested indicate that the rate of duplex unwinding is equivalent in the 5' --> 3' and 3' --> 5' directions. However, the optimal rate of unwinding was dependent on the length of the single-stranded region of the substrate when different combinations of factors were used. The combinations of eIF4A, eIF4A + eIF4B, eIF4A + eIF4H, and eIF4F showed differences in their ability to unwind chemically modified duplexes. A simple model of how eIF4B or eIF4H affects the duplex unwinding mechanism of eIF4A is proposed.     

RNA aptamers to initiation factor 4A helicase hinder cap-dependent translation by blocking ATP hydrolysis

The mammalian translation initiation factor 4A (eIF4A) is a prototype member of the DEAD-box RNA helicase family that couples ATPase activity to RNA binding and unwinding. In the crystal form, eIF4A has a distended "dumbbell" structure consisting of two domains, which probably undergo a conformational change, on binding ATP, to form a compact, functional structure via the juxtaposition of the two domains. Moreover, additional conformational changes between two domains may be involved in the ATPase and helicase activity of eIF4A. The molecular basis of these conformational changes, however, is not understood. Here, we generated RNA aptamers with high affinity for eIF4A by in vitro RNA selection-amplification. On binding, the RNAs inhibit ATP hydrolysis. One class of RNAs contains members that exhibit dissociation constant of 27 nM for eIF4A and severely inhibit cap-dependent in vitro translation. The binding affinity was increased on Arg substitution in the conserved motif Ia of eIF4A, which probably improves a predicted arginine network to bind RNA substrates. Selected RNAs, however, failed to bind either domain of eIF4A that had been split at the linker site. These findings suggest that the selected RNAs interact cooperatively with both domains of eIF4A, either in the dumbbell or the compact form, and entrap it into a dead-end conformation, probably by blocking the conformational change of eIF4A. The selected RNAs, therefore, represent a new class of specific inhibitors that are suitable for the analysis of eukaryotic initiation, and which pose a potential therapeutic against malignancies that are caused by aberrant translational control.     

Magnesium influences the discrimination and release of ADP by human RAD51

hRAD51 lacks cooperative DNA-dependent ATPase activity and appears to function with 5-10-fold less Mg2+ compared to RecA. We have further explored the effect of Mg2+ on adenosine nucleotide binding, ATPase, and DNA strand exchange activities. hRAD51 was saturated with the poorly hydrolyzable analog of ATP, ATPgammaS, at approximately 0.08 mM Mg2+. In contrast, > 0.5 mM Mg2+ was required to saturate hRAD51 with ADP. We found ADP to be a significantly less effective competitive inhibitor of the hRAD51 ATPase at low Mg2+ concentrations (0.08 mM). Mg2+ did not appear to affect the ability of ATPgammaS to competitively inhibit the hRAD51 ATPase. Low Mg2+ (0.08-0.12 mM) enhanced the steady-state ATPase of hRAD51 while higher Mg2+ concentration (> 0.3 mM) was inhibitory. At low Mg2+, hRAD51 appeared capable of nearly complete hydrolysis of available ATP, suggesting a lack of ADP product inhibition. There was a strong correlation between the amount of Mg2+ required for stable ADP binding and the inhibition of hRad51 strand exchange activity. Simultaneous inclusion of exogenous ATP and chelation of Mg2+ with EDTA significantly enhanced ADP-->ATP exchange by hRAD51. These studies are consistent with the hypothesis that Mg2+ influences the discrimination and release of ADP, which may sequentially impose an important regulatory step in the hRAD51 ATPase cycle.     

Further characterization of the helicase activity of eIF4A. Substrate specificity

Eukaryotic initiation factor (eIF) 4A is the archetypal member of the DEAD box family of RNA helicases and is proposed to unwind structures in the 5'-untranslated region of mRNA to facilitate binding of the 40 S ribosomal subunit. The helicase activity of eIF4A has been further characterized with respect to substrate specificity and directionality. Results confirm that the initial rate and amplitude of duplex unwinding by eIF4A is dependent on the overall stability, rather than the length or sequence, of the duplex substrate. eIF4A helicase activity is minimally dependent on the length of the single-stranded region adjacent to the double-stranded region of the substrate. Interestingly, eIF4A is able to unwind blunt-ended duplexes. eIF4A helicase activity is also affected by substitution of 2'-OH (RNA) groups with 2'-H (DNA) or 2'-methoxyethyl groups. These observations, taken together with results from competitive inhibition experiments, suggest that eIF4A may interact directly with double-stranded RNA, and recognition of helicase substrates occurs via chemical and/or structural features of the duplex. These results allow for refinement of a previously proposed model for the mechanism of action of eIF4A helicase activity.     

Molecular Dynamics Simulation of the Allosteric Regulation of eIF4A Protein from the Open to Closed State,Induced by ATP and RNA Substrates

Background

Eukaryotic initiation factor 4A (eIF4A) plays a key role in the process of protein translation initiation by facilitating the melting of the 5′ proximal secondary structure of eukaryotic mRNA for ribosomal subunit attachment. It was experimentally postulated that the closed conformation of the eIF4A protein bound by the ATP and RNA substrates is coupled to RNA duplex unwinding to promote protein translation initiation, rather than an open conformation in the absence of ATP and RNA substrates. However, the allosteric process of eIF4A from the open to closed state induced by the ATP and RNA substrates are not yet fully understood.

Methodology

In the present work, we constructed a series of diplex and ternary models of the eIF4A protein bound by the ATP and RNA substrates to carry out molecular dynamics simulations, free energy calculations and conformation analysis and explore the allosteric properties of eIF4A.

Results

The results showed that the eIF4A protein completes the conformational transition from the open to closed state via two allosteric processes of ATP binding followed by RNA and vice versa. Based on cooperative allosteric network analysis, the ATP binding to the eIF4A protein mainly caused the relative rotation of two domains, while the RNA binding caused the proximity of two domains via the migration of RNA bases in the presence of ATP. The cooperative binding of ATP and RNA for the eIF4A protein plays a key role in the allosteric transition.     

Studies of the phosphoenzyme intermediate of the yeast plasma membrane proton-translocating ATPase

The yeast plasma membrane proton-pumping ATPase forms a phosphorylated intermediate during the hydrolysis of ATP. The fraction of enzyme phosphorylated during steady-state ATP hydrolysis was studied as a function of substrate concentration (MgATP), Mg2+ concentration, and pH. The dependence of the fraction of enzyme phosphorylated on the concentration of MgATP is sigmoidal, and the isotherms can be fit with parameters and mechanisms similar to those used to describe ATP hydrolysis. The isotherm is significantly more sigmoidal at pH 5.5 than at pH 6.0, with the limiting percentage (100.mol of phosphate/mol of enzyme) of enzyme phosphorylated being 70% and 6%, respectively, at the two pH values. The maxima in the steady-state rate of ATP hydrolysis occur at higher concentrations of Mg2+ and higher pH than the maxima in the fraction of enzyme phosphorylated. This suggests that the rate-determining step for ATP hydrolysis is different from that for enzyme phosphorylation and the hydrolysis of phosphoenzyme is enhanced by Mg2+ and high pH. The rate of phosphoenzyme formation was investigated with the quenched-flow method, but only a lower bound of 140 s-1 could be obtained for the rate constant at MgATP concentrations greater than 2.5 mM. Since the turnover number for ATP hydrolysis under similar conditions is 14 s-1, the rate-determining step in ATP hydrolysis occurs after enzyme phosphorylation.     

Quantitative analysis of binding of single-stranded DNA by Escherichia coli DnaB helicase and the DnaB x DnaC complex

DnaB helicase is responsible for unwinding duplex DNA during chromosomal DNA replication and is an essential component of the DNA replication apparatus in Escherichia coli. We have analyzed the mechanism of binding of single-stranded DNA (ssDNA) by the DnaB x DnaC complex and DnaB helicase. Binding of ssDNA to DnaB helicase was significantly modulated by nucleotide cofactors, and the modulation was distinctly different for its complex with DnaC. DnaB helicase bound ssDNA with a high affinity [Kd = (5.09 +/- 0.32) x 10(-8) M] only in the presence of ATPgammaS, a nonhydrolyzable analogue of ATP, but not other nucleotides. The binding was sensitive to ionic strength but not to changes in temperature in the range of 30-37 degrees C. On the other hand, ssDNA binding in the presence of ADP was weaker than that observed with ATPgammaS, and the binding was insensitive to ionic strength. DnaC protein hexamerizes to form a 1:1 complex with the DnaB hexamer and loads it onto the ssDNA by forming a DnaB6 x DnaC6 dodecameric complex. Our results demonstrate that the DnaB6 x DnaC6 complex bound ssDNA with a high affinity [Kd = (6.26 +/- 0.65) x 10(-8) M] in the presence of ATP, unlike the DnaB hexamer. In the presence of ATPgammaS or ADP, binding of ssDNA by the DnaB6 x DnaC6 complex was a lower-affinity process. In summary, our results suggest that in the presence of ATP in vivo, the DnaB6 x DnaC6 complex should be more efficient in binding DNA as well as in loading DnaB onto the ssDNA than DnaB helicase itself.     

ATP utilization by yeast replication factor C. II. Multiple stepwise ATP binding events are required to load proliferating cell nuclear antigen onto primed DNA.

Binding of adenosine (3-thiotriphosphate) (ATPgammaS), a nonhydrolyzable analog of ATP, to replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) was studied by filter binding. RFC alone bound 1.8 ATPgammaS molecules. However, when either PCNA or primer-template DNA were also present 2.6 or 2.7 ATPgammaS molecules, respectively, were bound. When both PCNA and DNA were present 3.6 ATPgammaS molecules were bound per RFC. Order of addition experiments using surface plasmon resonance indicate that RFC forms an ATP-mediated binary complex with PCNA prior to formation of a ternary DNA.PCNA.RFC complex. An ATP-mediated complex between RFC and DNA was not competent for binding PCNA, and the RFC.DNA complex dissociated with hydrolysis of ATP. Based on these experiments a model is proposed in which: (i) RFC binds two ATPs (RFC.ATP(2)); (ii) this complex binds PCNA (PCNA.RFC.ATP(2)), which goes through a conformational change to reveal a binding site for one additional ATP (PCNA.RFC.ATP(3)); (iii) this complex can bind DNA to yield DNA.PCNA.RFC.ATP(3); (iv) a conformational change in the latter complex reveals a fourth binding site for ATP; and (v) the DNA.PCNA.RFC.ATP(4) complex is finally competent for completion of PCNA loading and release of RFC upon hydrolysis of ATP.     

Differential specificities and simultaneous occupancy of human MutSalpha nucleotide binding sites

We have examined the permissible nucleotide occupancy states of human MutSalpha. The MSH2.MSH6 heterodimer binds 1 mol of ADP and 1 mol of adenosine 5'-O-(thiotriphosphate) (ATPgammaS), with a K(d) for each nucleotide of about 1 microm. Anisotropy measurements using BODIPY TR and BODIPY FL fluorescent derivatives of ADP and 5'-adenylyl-beta,gamma-imidodiphosphate (AMPPNP) also indicate an interaction stoichiometry of 1 mol of ADP and 1 mol of triphosphate analogue per MutSalpha heterodimer. Di- and triphosphate sites can be simultaneously occupied as judged by sequential filling of the two binding site classes with differentially radiolabeled ADP and ATPgammaS and by fluorescence resonance energy transfer between BODIPY TR- and BODIPY FL-labeled ADP and AMPPNP. ATP hydrolysis by MutSalpha is accompanied by a pre-steady-state burst of ADP formation, and analysis of MutSalpha-bound nucleotide during the first turnover has demonstrated the presence of both ADP and ATP. Simultaneous presence of ADP and a nonhydrolyzable ATP analogue modulates MutSalpha.heteroduplex interaction in a manner that is distinct from that observed in the presence of ADP or nonhydrolyzable triphosphate alone, and it is unlikely that this effect is due to the presence of a mixed population of binary complexes between MutSalpha and ADP or a triphosphate analogue. These findings imply that MutSalpha has two nucleotide binding sites with differential specificities for ADP and ATP and suggest that the ADP.MutSalpha.ATP ternary complex has an important role in mismatch repair.     

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.