Effects of Oligonucleotide Length and Atomic Composition on Stimulation of the Atpase Activity of Translation Initiation Factor Eif4a

Home , Oligonucleotide

Effects of oligonucleotide length and atomic composition on stimulation of the ATPase activity of translation initiation factor eIF4A

MATTHEW L. PECK and DANIEL HERSCHLAG Department of Biochemistry, Stanford University, Stanford, California 94305-5307, USA

ABSTRACT Eukaryotic translation initiation factor 4A (eIF4A) has been proposed to use the energy of ATP hydrolysis to remove RNA structure in the 59 untranslated region (UTR) of mRNAs, helping the 43S ribosomal complex bind to an mRNA and scan to find the 59-most AUG initiator codon. We have examined the effect of changing the atomic composition and length of single-stranded oligonucleotides on binding to eIF4A and on stimulation of its ATPase activity once bound. Substitution of 29-OH groups with 29-H or 29-OCH3 groups reduces ATPase stimulation at least 100-fold, to background levels, without significantly affecting oligonucleotide affinity. These effects suggest that 29-OH groups par- ticipate in an eIF4A conformational change that occurs subsequent to oligonucleotide binding and is required for ATPase stimulation. Replacing nonbridging oxygen atoms in phosphodiester linkages with sulfur atoms to make phosphorothioate linkages has no significant effect on stimulation, while substantially increasing affinity. Extending the length of an RNA oligonucleotide from 4 to ;15 nt gradually increases oligonucleotide affinity and ATPase stimulation. Consistent with this observation, the increase in affinity and stimulation provided by phosphorothioate linkages and 29-OH groups is proportional to the number of these groups present within larger oligonucleotides. Further, changing the position of blocks of phosphorothioate linkages or 29-OH groups within a larger oligonucleotide does not affect affinity and has only a small effect on stimulation. These observations suggest that numerous interactions between the oligonucleotide and eIF4A contribute individually to binding and ATPase stimulation. Never- theless, significant stimulation is observed with as few as four RNA residues. These properties may allow eIF4A to operate within regions of 59 UTRs containing only short stretches of exposed single-stranded RNA. As stimulation increases when longer stretches of single-stranded RNA are available, it is possible that the accessibility of single- stranded RNA in a 59 UTR influences translation efficiency. Keywords: DEAD box; DExD/H box; helicase; phosphorothioate; Prp16

INTRODUCTION the 59 UTR+ Eukaryotic initiation factor 4A (eIF4A) has been proposed to promote translation initiation by using To initiate translation, the 40S ribosomal subunit typi- the energy from ATP hydrolysis to facilitate the loading cally must find the 59-most AUG initiator codon in the 59 of the ribosomal subunit onto mRNA and the removal untranslated region (UTR) of an mRNA+ The ribosomal of structure within the 59 UTR (for reviews see Hershey, subunit is thought to accomplish this by first binding to 1991; Merrick & Hershey, 1996)+ an mRNA near the 59 cap and then scanning 59 to 39 Determining the biochemical capabilities of eIF4A is along the 59 UTR in search of the first AUG codon+ a necessary step toward understanding its biological These processes may be slowed by limited access to function and how it performs this function+ eIF4A is the archetypal member of the DEAD box protein family Reprint requests to: Daniel Herschlag, Department of Biochemis- (Linder et al+, 1989; Schmid & Linder, 1992)+ These try, B400 Beckman Center, Stanford University, Stanford, California 94305-5307, USA; e-mail: herschla@cmgm+stanford+edu+ proteins and the closely related DExH box proteins are Abbreviations: DTT: dithiothreitol; E: enzyme; EDTA: (ethylene- involved in essentially all cellular RNA processes in- dinitrilo)tetraacetic acid; eIF4A: eukaryotic initiation factor 4A; HEPES: cluding pre-mRNA splicing, RNA degradation, ribo- N-(2-hydroxyethyl)piperazine-N -(2-ethanesulfonic acid); MES: 2-(N- 9 some biogenesis, RNA transport, and RNA localization morpholino)ethanesulfonic acid; MS17: 17-mer oligonucleotide with sequence 59-GGGAACGUCGUCGUCGC-39; oligo: oligonucleotide; (Wassarman & Steitz, 1991; Schmid & Linder, 1992; : ; : ; Pi inorganic phosphate (P-O) phosphodiester backbone linkage Lüking et al+, 1998)+ eIF4A also shares sequence sim- (P-S): phosphorothioate backbone linkage; Tris: tris(hydroxymethyl)- ilarity with DNA helicases (Koonin, 1991; Gorbalenya & aminomethane; Un: oligouridine of n nucleotides length; UTR: untranslated region+ Koonin, 1993)+ These observations support an RNA 1210 Oligonucleotide stimulation of eIF4A ATPase activity 1211 unwinding function for eIF4A and other DEAD box pro- gonucleotides+ The results provide a biochemical un- teins, although alternative functions, such as main- derstanding of the distinct roles that features of nucleic taining the fidelity of biological processes by kinetic acid structure play in oligonucleotide binding and proofreading, rearranging RNA–protein interactions, and ATPase stimulation+ Aspects of these biochemical find- translocating complexes along RNA, have been pro- ings that may be important for eIF4A’s biological func- posed (Burgess & Guthrie, 1993; Staley & Guthrie, 1998; tion are discussed+ Lorsch & Herschlag, 1998a)+ eIF4A has been the focus of significant biochemical RESULTS characterization+ In vitro, eIF4A bidirectionally unwinds + duplexes containing single-stranded RNA overhangs Changing 29-OH groups to 29-H This unwinding activity requires ATP, and is enhanced and 29-OCH3 groups by the presence of another initiation factor, eIF4B, or by inclusion of eIF4A within the eIF4F complex The effects of changing the identity of the 29-substituent (eIF4A•eIF4E•eIF4G) (Ray et al+, 1985; Lawson et al+, on eIF4A’s ATPase activity were first examined by com- 1989; Rozen et al+, 1990; Rogers et al+, 1999)+ Muta- paring the stimulation provided by a mixed sequence genesis of motifs conserved among DEAD box pro- 17-mer oligonucleotide (MS17) containing 29-OH groups teins has helped to define the role of these motifs to the stimulation provided by analogous oligonucleo- + in this unwinding reaction, as well as in RNA and tides containing 29-H or 29-OCH3 groups The fraction ATP binding, ATP hydrolysis, and translation initiation of inorganic phosphate product formed at various times (Schmid & Linder, 1991; Pause & Sonenberg, 1992; during a reaction was measured using a radioactivity- Pause et al+, 1993, 1994)+ The effect of RNA structure, based assay described in Materials and Methods with other translation factors and ATP on RNA binding by ATP•Mg subsaturating with respect to eIF4A (Fig+ 1A)+ eIF4A has been assayed using cross-linking and fluo- The 29-OH-containing oligonucleotide, but not the 29-H- , rescence techniques (Grifo et al+, 1982; Ray et al+, 1985; or 29-OCH3-containing oligonucleotides stimulated ATP Goss et al+, 1987)+ A kinetic and thermodynamic frame- hydrolysis above the background rate observed in the work for RNA-dependent ATPase activity has been es- absence of added oligonucleotide+ ATPase activity was tablished (Lorsch & Herschlag, 1998a)+ This ATPase measured at several concentrations of each oligo- activity appears to drive a cycle of enzyme conforma- nucleotide (Fig+ 1B)+ The dependence of the ATPase tional changes that could promote RNA unwinding or activity on the concentration of 29-OH-containing oligo- perform other functions (Lorsch & Herschlag, 1998b)+ nucleotide exhibited saturation behavior+ The maximal An essential aspect of the biochemical characteriza- rate constant for ATP hydrolysis with enzyme saturated / ATP tion of eIF4A is understanding how nucleic acids stim- with oligonucleotide [(kcat Km) ] is 300-fold faster than ulate ATP hydrolysis+ Previously, it was demonstrated the rate constant measured in the absence of added +1 , that RNA, but not DNA, stimulates ATP hydrolysis (Grifo nucleic acid In contrast the 29-H- and 29-OCH3- et al+, 1984) and that this stimulation is not dependent containing oligonucleotides did not stimulate the ATPase on the sequence of the RNA (Abramson et al+, 1987)+ activity, even at concentrations as high as 100 mM+ Single-stranded RNA stimulates ATP hydrolysis much These results confirm the report that 29-H groups do better than duplexes, hairpins, and other structured not activate the ATPase activity (Grifo et al+, 1984)+ RNAs (e+g+, tRNA) (Abramson et al+, 1987; Lorsch & To determine whether the absence of stimulation from Herschlag, 1998a)+ Also, ATPase stimulation decreases these oligonucleotides arose from a lack of binding or when the length of oligo(A) is less than 12 nt (Abram- from an inability to stimulate once bound, we tested the son et al+, 1987)+ However, it was not determined ability of the 29-H- and 29-OCH3-containing oligonucle- whether changing RNA to DNA or modifying RNA length otides to inhibit the RNA-stimulated ATPase activity of affected ATP hydrolysis by reducing oligonucleotide eIF4A (Fig+ 2)+ Both oligonucleotides inhibited the binding or by reducing the stimulation provided by the ATPase activity with similar concentration dependen- bound oligonucleotide+ Distinguishing these effects is cies+ The curves through the data represent fits for critical for understanding the nature of individual reac- inhibition by a single oligonucleotide (Scheme 1)+ Al- tion steps and, ultimately, for obtaining a molecular de- though there is some deviation of the data in Figure 2, scription of the mechanism of action for this important these are the largest deviations observed in this study+ enzyme+ We have measured the effect of varying the length, 1 The saturation behavior of ATPase stimulation by oligonucleo- the 29-substituent of the ribose sugar, and the phos- tides was fit to a Michaelis-Menten model (Equation 2)+ The nomen- clature reflects that ATP•Mg was subsaturating in these experiments+ phate backbone of single-stranded oligonucleotides on Thus, the rate constant for reaction with subsaturating oligonucleotide oligo their ability to bind to and stimulate the ATPase activity (Equation 3) is called k3 rather than (kcat/Km) + Likewise, the rate + , constant for the reaction with enzyme saturated with oligonucleotide of eIF4A Further we have measured the effects of ATP ATP, app (Equation 4) is called (kcat/Km) rather than kcat+ (kcat/Km) is changing the number and position of groups that are the observed second-order rate constant for the reaction with sub- important for binding and stimulation within larger oli- saturating ATP and any specified oligonucleotide concentration+ 1212 M.L. Peck and D. Herschlag

FIGURE 2. MS17 oligonucleotides with 29-H and 29-OCH3 substitu- ATP, app ents inhibit ATPase stimulation+ ATPase activity, (kcat/Km) , with subsaturating ATP•Mg (1 mM), 0+25 mM eIF4A, and subsaturating U20 RNA (3 mM) in the presence of varying concentrations of 29-H- containing (▫ ) and 29-OCH3-containing (छ)MS17 oligonucleotides+ Normalized values were obtained by dividing the observed rate constants by the rate constant measured in the absence of inhibitory oligonucleotide+ Curves represent fits to a simple inhibition model oligo (Equation 5), yielding Ki ϭ 6+1 and 8+9 mM for the 29-H and 29- OCH3 oligonucleotides, respectively+

evidence for a single inhibitory site+ Finally, the rescue of inhibition by higher concentrations of stimulatory oligonucleotide and other control experiments (below and Materials and Methods) indicate that the inhibition is due to competitive binding of the inhibitory oligonucleotides+ oligo Inhibition constants (Ki )of6+1mM and 8+9 mM for the 29-H- and 29-OCH3-containing oligonucleotides, respectively (Fig+ 2), are within threefold of K oligo FIGURE 1. ATP hydrolysis is stimulated by MS17 oligonucleotide with m 29-OH substituents, but not by analogous oligonucleotides with 29-H (4+4 mM) for the 29-OH-containing oligonucleotide + : or 29-OCH3 substituents A Fraction ATP hydrolyzed versus time in (Fig+ 1B)+ K oligo values are expected to equal equilib- reactions containing subsaturating ATP•Mg (1 mM) and 0+25 mM i rium dissociation constants for these oligonucleotides, eIF4A with no added nucleic acid (᭹), 30 mM29-OH-containing MS17 (⅙), 25 mM29-H-containing MS17 (▫ ), or 25 mM29-OCH3-containing because the concentration of activating RNA was sub- छ + MS17 ( ) The slopes of linear fits give observed rate constants (kobs) saturating in the inhibition experiments+ Previous ki- of 2+8 ϫ 10Ϫ4, 8+6 ϫ 10Ϫ3, 2+5 ϫ 10Ϫ4 and 2+6 ϫ 10Ϫ4 minϪ1, re- ATP, app netic and thermodynamic results obtained under similar spectively+ B: Dependence of ATPase stimulation [(kcat/Km) ] , oligo on the concentration of 29-OH-containing MS17 (⅙) 29-H-containing reaction conditions strongly suggest that Km also MS17 (▫ ), and 29-OCH3-containing MS17 (छ) oligonucleotides+ The curve for the 29-OH data represents a fit to a Michaelis-Menten oligo model (Equation 2), yielding Km ϭ 4+4 mM and a maximal rate ATP 4 Ϫ1 Ϫ1 constant of (kcat/Km) ϭ 3+9 ϫ 10 M min + The 29-H and 29- OCH3-containing oligonucleotides did not provide significant stimulation+

Figure 3B is more representative of the data obtained in analogous experiments with several other inhibitory oligonucleotides and shows data that closely follow the dependence expected for inhibition by a single inhibitory oligonucleotide+ Further, ATPase activity was inhibited with 1:1 stoichiometry when eIF4A was titrated with a tight-binding inhibitory oligonucleotide (data not SCHEME 1. Model for competitive binding of activating (oligoact) and shown, see Materials and Methods), providing strong inhibitory (oligoinhib) oligonucleotides+ Oligonucleotide stimulation of eIF4A ATPase activity 1213

thioate backbone [mixed stereoisomers, abbreviated (P-S)] was measured at several oligonucleotide concentrations (Fig+ 3A)+ Substitution of the (P-O) backbone with a (P-S) backbone had only a two- to threefold ATP effect on the maximal rate constant (kcat/Km) , oligo whereas Km decreased at least 50-fold+ Consistent with this result, U20 DNA with a (P-S) backbone [abbreviated U20:29-H,(P-S)] was a much stronger inhibitor of ATPase stimulation than the analogous (P-O)-containing oligonucleotide (Fig+ 3B)+ Because a minimum of 0+25 mM eIF4A was required to avoid significant loss of enzyme to tube walls (see Materials and Methods), only limits for the binding of the (P-S)-containing oligonucleotides could be obtained from these data+ This is because inhibition reflects the titration of enzyme with oligonucleotide at concentrations of oligonucleotide lower than the enzyme concentration+ Additional experiments were therefore performed to determine the actual binding constants for these oligonucleotides+ An inhibition constant can be determined from the effect of an inhibitory oligonucleotide, in this case , oligo U20:29-H,(P-S) on Km for an activating oligonucleotide (Scheme 1)+ In the absence of inhibitory oligonucleotide, the activating oligonucleotide binds eIF4A to

form the stimulated eIF4A•oligoact complex with an oligo affinity described by Km + The presence of inhibitory oligonucleotide sequesters eIF4A into the inactive

eIF4A•oligoinhib complex, thereby increasing the ob- oligo,app served value of the Michaelis constant (Km )as described by Equation 1a, which was derived from oligo oligo,app Scheme 1+ Ki can then be calculated from Km , oligo FIGURE 3. Oligonucleotides with phosphorothioate backbones bind the actual Km , and the concentration of inhibitory + : , / ATP, app, tightly to eIF4A A ATPase activity (kcat Km) with subsaturat- oligonucleotide according to Equation 1b, a rearranged ing ATP•Mg (1 mM), 0+25 mM eIF4A and varying concentrations of form of Equation 1a+ Using K oligo values obtained from U15 RNA with a phosphodiester (Ⅲ ) or a phosphorothioate backbone m oligo (▫ )+ Curves represent fit to Michaelis-Menten model (Equation 2) the data in Figure 4A, this analysis provides a Ki to yield K oligo ϭ 13 M for the phosphodiester oligonucleotide oligo m m of 24 nM for U : , + This K value is 500-fold oligo + + 20 29-H (P-S) i and Km , 0 25 mM for the phosphorothioate oligonucleotide oligo B: ATPase activity with subsaturating ATP•Mg (1 mM), 0+25 mM eIF4A smaller than the value of Ki (12 mM) for the analo- and subsaturating U20 RNA (3 mM) is inhibited by addition of U20 DNA gous (P-O)-containing oligonucleotide (Fig+ 3B)+ As ex- with a phosphodiester (Ⅲ ) or a phosphorothioate backbone (▫ )+ Nor- pected for a competitive inhibitor, the presence of malized values were obtained by dividing the observed rate constants by the rate constant measured in the absence of inhibitory U20:29-H,(P-S) did not affect the rate constant with satu- oligonucleotide+ Curves represent fits to simple inhibition model (Equa- rating poly(U) RNA+ oligo tion 5), yielding Ki ϭ 12 mM for the phosphodiester oligonucleotide and K oligo , 0+25 mM for the phosphorothioate oligonucleotide+ i [oligo ] oligo,app ϭ oligo ͩ ϩ inhib ͪ Km Km 1 oligo (1a) Ki equals the oligonucleotide’s dissociation constant oligo [oligoinhib] (Lorsch & Herschlag, 1998a)+ Thus, substituting the K ϭ , (1b) i K oligo app , m Ϫ 29-OH groups with 29-H or 29-OCH3 groups has at oligo 1 most, a small effect on binding, but once bound, these Km oligonucleotides do not provide measurable ATPase oligo,app + Km stimulation K oligo ϭ (1c) m [oligo ] 1 ϩ inhib K oligo Substituting phosphodiester linkages i with phosphorothioate linkages An analogous strategy was used to determine the oligo The ATPase activity provided by U15 RNA with either a value of Km for U15 RNA with a (P-S) backbone + phosphodiester [abbreviated (P-O)] or a phosphoro- [abbreviated U15:29-OH,(P-S)] Stimulation provided by 1214 M.L. Peck and D. Herschlag

, + , oligo U20:29-H,(P-S) (24 nM determined from Fig 4A) Km for + U15:29-OH,(P-S) was calculated to be 0 12 mM according to oligo Equation 1c+ Km for this (P-S)-containing oligonucle- oligo otide is ;100-fold smaller than Km for the analogous (P-O)-containing oligonucleotide (13 mM, Fig+ 3A)+

Another DEAD box protein, PRP16, also binds tightly to phosphorothioate oligonucleotides Given the unexpectedly strong interaction between eIF4A and phosphorothioate-substituted oligonucleotides, it was of interest to determine if other DEAD box proteins also bind (P-S)-containing oligonucleotides tightly+ The DEAD box protein Prp16 is an RNA- dependent ATPase involved in pre-mRNA splicing (Schwer & Guthrie, 1991; Wang & Guthrie, 1998)+

U20:29-H,(P-S) inhibited the ability of poly(U) RNA to stimulate Prp16’s ATPase activity .3,000-fold more than the analogous (P-O)-containing oligonucleotide (data not shown)+ The rate constant measured for Prp16 saturated with poly(U) RNA was not affected by the presence of the inhibitory oligonucleotide, consistent with competitive inhibition+ Thus, Prp16, like eIF4A, appears to bind (P-S)-containing oligonucleotides considerably more tightly than (P-O)-containing oligonucleotides+

Dependence of oligonucleotide affinity on the number and position of phosphorothioate linkages within an oligonucleotide Inhibition constants were measured for oligo(U) DNA of varying lengths with (P-O) or (P-S) backbones FIGURE 4. Binding constants for oligonucleotides with phosphoro- (Table 1)+ For both (P-O)- and (P-S)-containing oligo- thioate backbones determined by competitive inhibition+ A: ATPase nucleotides, affinity increases as length is increased+ ATP, app activity, (kcat/Km) , with subsaturating ATP (1 mM), 0+25 mM eIF4A and various concentrations of poly(U) RNA measured in the absence The ratio of the inhibition constants for (P-O) and (P-S) (⅙) and presence (Ⅲ )of0+75 mMU20:29-H,(P-S)+ Curves represent fits oligonucleotides of the same length isolates the effect oligo ϭ + to a Michaelis-Menten model (Equation 2) and yield Km 1 5 mM of substituting (P-O) linkages with (P-S) linkages from oligo,app and Km ϭ 70 mM, respectively+ These values were used in oligo the overall effect of changing oligonucleotide length Equation 1b to calculate the value of Ki ϭ 24 nM for U20:29-H,(P-S)+ ATP, app , + B: ATPase activity, (kcat/Km) , with subsaturating ATP•Mg (ratio Table 1) Substituting the nine (P-O) linkages in (1 mM), 0+25 mM eIF4A and various concentrations of U15:29-OH,(P-S) measured in the absence (ࡗ) and presence of 1 mM(▫)or2+5mM (⅙)ofU20:29-H,(P-S) inhibitory oligonucleotide+ Curves represent fits to oligo,app the Michaelis-Menten model (Equation 2) and yield Km ϭ 5+7 and 11 mM, respectively+ These values were used in Equation 1c to TABLE 1+ Inhibition of eIF4A ATPase by oligo(U) DNA of differing oligo +a calculate values of Km ϭ 0+14 and 0+10 mM for U15:29-OH,(P-S)+ lengths and backbone composition

oligo oligo Ki (P-O) Ki (P-S) dUn oligonucleotide oligonucleotide ratio ϭ (P-O)/ (P-S) n (mM) (mM) (Ki Ki )

5 .250 .30 — U15:29-OH,(P-S) was measured in the presence of an in- + + , , 10 160 2 0(38) 60 hibitory oligonucleotide U20:29-H,(P-S) which was in- 15 45 (0+080) 500 cluded in the reaction to compete with U15:29-OH,(P-S) for 20 12 (16) (0+024) 580 oligo,app binding to eIF4A and thus increase Km (Scheme a oligo Reactions with subsaturating ATP•Mg (1 mM)+ Ki values in 1)+ Figure 4B shows the effect of this inhibitor on the oligo parentheses were obtained by comparing the Km values for U15:29-OH,(P-S) concentration dependence for ATP stim- poly(U) RNA that were measured in the presence and absence of oligo,app inhibitory oligonucleotide (as in Fig+ 4A; see Results)+ All other K oligo ulation, giving observed Km values of 5+7 and i + + values were obtained by measuring the concentration dependence 11 mM with 1 and 2 5 mMU20:29-H,(P-S) present With of the inhibition of RNA-dependent ATPase stimulation (as in Fig+ 2; oligo,app oligo these Km values and the value of Ki for see Materials and Methods)+ Oligonucleotide stimulation of eIF4A ATPase activity 1215

U10 DNA with (P-S) linkages increased binding ;60- ATPase stimulation within the enzyme•oligonucleotide fold+ This binding advantage increased to 500 upon complex, the Michaelis-Menten parameters for ATPase substitution of the 14 linkages in U15, suggesting that stimulation were determined for oligo(U) RNA of vary- multiple (P-S) linkages make contributions that in- ing length (Table 2)+ crease binding+ The effect of increasing length on k3 [representing We also measured the inhibition constants for a se- reaction starting with free eIF4A, ATP, and oligonucle- ATP ries of U20 DNA oligonucleotides containing a varying otide (Equation 3)], (kcat/Km) [representing reaction number of (P-S) linkages in an otherwise (P-O) back- starting with enzyme•oligonucleotide complex and free oligo bone (Fig+ 5)+ As the number of (P-S) linkages in- ATP (Equation 4)], and 1/Km appears to fall into two creases, there is an increase in affinity, with an effect of regimes (Fig+ 6, data from Table 2)+ As length is in- four- to eightfold per added block of four or five (P-S) creased from 4 to 15 nt, the constants increase steadily, linkages+ In addition, the inhibition constants for differ- but there is little or no additional effect as the length is ent oligonucleotides containing equal numbers of (P-S) extended beyond 15 nt+ For example, k3 increases 420- linkages are the same within error, suggesting that (P-S) fold as length is extended from 7 to 15 nt, but increases linkages at different locations within the oligonucleo- only sixfold as the length is extended from 15 to tides provide similar increases in binding affinity+ To- ;200 nt [poly(U)]+ In addition, there are effects of length oligo ATP gether, these observations suggest that (P-S) linkages on both Km and (kcat/Km) , and these effects are of distributed throughout most or all of a 20-mer oligonu- similar magnitudes+ For example, increasing length from ATP oligo cleotide contribute to the increased binding+ U7 to U15 increases (kcat/Km) and 1/Km 30- and 15-fold, respectively+ Consistent with previous results (Abramson et al+, 1987; Goss et al+, 1987), these ob- Changing oligonucleotide length servations suggest that oligonucleotide binding and Previously, it was found that the ATPase stimulation ATPase stimulation occur within a site that accommo- + , provided by oligonucleotides is length-dependent dates 10–15 nt Nevertheless substantial stimulation + (Abramson et al+, 1987)+ To distinguish the effect of is observed with shorter oligonucleotides length on oligonucleotide binding from its effect on Dependence of stimulation on the number and position of 29-OH groups within oligonucleotides

Michaelis-Menten parameters for MS17 oligonucleotides with mixtures of 29-OH and 29-OCH3 groups were determined (Table 3)+ Oligonucleotides with only one or two 29-OH groups did not stimulate the ATPase above background+ However, the ATPase stimulation pro-

vided by m6r3m8, which contains only three 29-OH-

TABLE 2+ Kinetic parameters for stimulation of eIF4A ATPase activity by oligo(U) RNA of varying lengths+a

ATP oligo oligo b rUn k3 (kcat/Km) Km (or Ki ) n ϭ (MϪ2minϪ1) (MϪ1minϪ1) (mM)

42ϫ106 .1+5 ϫ 103 (.700) 64ϫ106 .1+3 ϫ 103 (.300) 72+6ϫ107 5+2 ϫ 103 200 81+6ϫ108 1+2 ϫ 104 74 10 5+8 ϫ 108 4+5 ϫ 104 78 15 1+1 ϫ 1010 1+5 ϫ 105 13 20 1+6 ϫ 1010 1+7 ϫ 105 14 FIGURE 5. The number but not the position of phosphorothioate ;200 5+6 ϫ 1010 c 0+8 ϫ 105 1+5c linkages in U20 DNA oligonucleotides affects oligonucleotide affinity+ Inhibition constants for oligonucleotides containing a mixture of phos- a Reactions with subsaturating ATP•Mg (1 mM)+ Constants are de- phorothioate linkages (depicted by the black background) and phos- fined in Equations 2–4 and were measured as described in Materials phodiester linkages (depicted by the white background) were and Methods+ b oligo oligo determined by competitive inhibition, as described in Results (see Ki values are in parentheses, all others are Km values+ rel c Fig+ 4A for representative data)+ The values for (1/Ki) were calcu- Concentration of poly(U) RNA and thermodynamic constants in- oligo oligo lated by dividing observed 1/Ki values by the 1/Ki value of the volving poly(U) RNA are given in 20-mer units (i+e+, the nucleotide phosphodiester oligonucleotide+ Note that there are 19 linkages in a concentration divided by 20) to facilitate comparisons with the oligo- 20-mer+ nucleotides+ 1216 M.L. Peck and D. Herschlag

TABLE 3+ Stimulation of eIF4A ATPase activity by MS17 oligonucleotides composed of combinations of 29-OH a and 29-OCH3 residues+

ATP oligo oligo c k3 (kcat/Km) Km (or Ki ) 29-compositionb (1010 MϪ2minϪ1) (104 MϪ1minϪ1) (mM)

d m17 —NS (8+9) d m8r1m8 —NS(,10) d m7r2m8 —NS(,10) m6r3m8 0+10 0+23+5 m3r11m 3 1+33+83+5 r17 0+89 3+94+4

aConstants are defined in Equations 2–4 and were measured as described in Materials and Methods+ Oligonucleotide sequence is 59-GGGAACGUCGUCGUCGC-39+ b Identity of 29-substituents are noted 59 to 39, with r ϭ 29-OH and m ϭ 29-OCH3+ For example, m6r3m8 is composed of (59 to 39) six 29-OCH3 nucleotides, followed by three 29-OH nucleotides, and then followed by eight additional 29-OCH3 nucleotides+ c oligo oligo Ki values are in parentheses, all others are Km values+ d NS ϭ not stimulated+ These oligonucleotides did not measurably stimulate ATP hydrolysis above the rate observed in the absence of added oligonucleotide+

ATP (kcat/Km) ]+ Thus, a small number of 29-OH groups can significantly stimulate the ATPase activity of eIF4A+ ATP The values of (kcat/Km) for m3r11m 3 and U10 are similar, suggesting that the stimulation provided by

29-OH groups is not inhibited by the flanking 29-OCH3 nucleotides+ Further, k3, representing RNA binding and stimulation, is 20-fold greater for m3r11m 3 than for U10, indicating that the 29-OCH3-containing residues promote productive binding (Tables 2 and 3)+ Thus, even

though 29-OCH3 residues do not alone provide stimulation in the bound complex, the flanking 29-OCH3- containing residues facilitate binding of the neighboring 29-OH groups in an active mode+ To test if the position of 29-OH groups within an oligo- ATP nucleotide affects ATPase stimulation, (kcat/Km) was measured for a series of U15 oligonucleotides (Fig+ 7)+ Each oligonucleotide contains six 29-OH nucleotides at different positions within an otherwise 29-H oligonucleotide+ All four oligonucleotides gave significant ATPase stimulation+ Nevertheless, positioning the 29-OH groups near the middle of the oligonucleotide, rather than at the edge of the oligonucleotide, gave three- to fivefold greater stimulation, although there was no positional effect on binding affinity+ These observations, combined with the above length dependence data, suggest

ATP oligo that contacts within the stimulatory site are distributed FIGURE 6. Dependence of k3 (A), (kcat/Km) (B), and 1/Km (or oligo over ;15 residues, with 29-OH groups possibly provid- 1/Ki )(C) on oligo(U) RNA length+ Data are from Table 2+ Curves through data are shown as guides and do not represent a fit to a ing somewhat greater stimulation when positioned cen- specific model+ Arrows in B and C indicate that these values are + ATP trally in the nucleic acid site limits+ The dashed line in B represents the background (kcat/Km) value measured in the absence of added nucleic acid+ DISCUSSION

Biochemical features of oligonucleotide containing nucleotides flanked by 29-OCH -containing 3 binding and ATPase stimulation nucleotides, was within 20-fold of the stimulation provided by r17, the corresponding oligonucleotide with all The analysis herein of the ATPase activity of eIF4A in 17 residues containing 29-OH groups [Table 3, k3 and the presence of a series of single-stranded oligonucle- Oligonucleotide stimulation of eIF4A ATPase activity 1217

taining some 29-OH groups suggests that there is flex- ibility within the region of eIF4A that interacts with the 29-substituents, such that unfavorable interactions are avoided+ This observation has implications for the biological function of eIF4A, as discussed below+

Contacts appear to be distributed throughout a nucleic acid site that accommodates 10–15 residues Extending the length of single-stranded oligo(U) RNA to 10–15 nt increases both oligonucleotide affinity for oligo eIF4A (1/Km ) and ATPase stimulation in the bound FIGURE 7. Effect of changing the location of 29-OH groups within a / ATP , / ATP+ , state [(kcat Km) ] but extension beyond 15 nt has U15 oligonucleotide on (kcat Km) Regions containing 29-OH (P-O) + nucleotides are depicted by the black background and regions con- little effect This suggests that the interactions between taining 29-H, (P-S) nucleotides are depicted by the white background+ the oligonucleotide and the enzyme that promote oli- The linkages between regions of 29-OH and 29-H residues are phos- ATP oligo gonucleotide binding and ATPase stimulation are con- phodiester bonds+ Values of (kcat/Km) and Km were obtained from fits of the dependencies of ATPase activity on oligonucleotide tained within and distributed throughout a site that concentration (Equation 2) in the presence of 1 mM ATP•Mg and accommodates 10–15 nt+ Results with oligonucleo- 0+25 mM eIF4A (data not shown)+ Values in parentheses represent oligo tides containing blocks of residues with 29-OH groups Ki values measured by the competitive inhibition method de- / scribed in Results and shown in Figure 4A+ and or phosphorothioate linkages (see below) further support the model that interactions involved in binding and stimulation are distributed throughout this site+ otides confirms and extends previous results (Grifo et al+, 1984; Abramson et al+, 1987)+ Phosphorothioates increase oligonucleotide affinity for eIF4A and other proteins Replacing the phosphodiester backbone with a phos- The involvement of 29-OH groups in ATPase phorothioate backbone increases oligonucleotide affin- stimulation, but not oligonucleotide binding, ity for eIF4A without significantly affecting ATPase indicate an eIF4A conformational change stimulation+ Increased binding correlates with the num- Substituting the 29-OH groups of an oligonucleotide with ber of phosphorothioate groups in an oligonucleotide, 29-H or 29-OCH3 groups has little or no effect on oligo- and does not appear to depend on the position of the nucleotide binding, but reduces ATPase stimulation be- phosphorothioates within an otherwise phosphodiester low measurable levels+ However, as few as three 29-OH backbone+ These observations suggest that, on aver- groups within a longer oligonucleotide are sufficient for age, an individual phosphorothioate linkage has a mod- measurable stimulation+ The simplest interpretation of est effect on binding of ;0+3 kcal/mol+2 The large overall these observations gives the following physical model+ increase in binding of 500-fold (3+7 kcal/mol) upon full In the oligonucleotide•eIF4A complex, the 29-sub- phosphorothioate substitution of U15 DNA arises from stituents of the oligonucleotide do not interact with eIF4A+ the combined effect of the multiple phosphorothioates + Thus, oligonucleotides with 29-OH, 29-H, and 29-OCH3 distributed throughout the ;15-nt active site groups bind equally well+ Following the formation of the Consistent with a role for the phosphate backbone in bound state, eIF4A undergoes an unfavorable confor- oligonucleotide binding, the crystal structures of the mational change to a conformation that is stabilized by DEAD box protein Hepatitis C Virus NS3 (Kim et al+, interactions with the 29-OH groups, and this conforma- 1998) and of the related protein Escherichia coli Rep tion gives stimulated ATP hydrolysis+ Thus, only oligo- helicase (Korolev et al+, 1997) reveal an extensive in- nucleotides with 29-OH groups, and not those with 29-H terface between the enzymes and the phosphate back- + , and 29-OCH3 groups, stimulate ATP hydrolysis+ It is not bone of complexed oligonucleotides In both structures known if the interactions between eIF4A and the 29-OH most of the hydrogen bonding between the enzyme groups are direct or indirect, although an interaction and oligonucleotide appears to involve the phosphate based solely on the sugar-pucker preference of the backbone+ ribose ring is unlikely, as 29-OCH3 substitutions favor the same sugar pucker (Uesugi et al+, 1979; Guschl- 2 Larger effects from particular stereoisomers at particular posi- bauer & Jankowski, 1980)+ Finally, the absence of in- tions may have been obscured because the oligonucleotides are mixtures of stereoisomers (2N isomers for an oligonucleotide with N hibitory effects on ATPase stimulation from 29-H and linkages) and because the oligonucleotides can bind to eIF4A in especially 29-OCH3 groups within oligonucleotides con- different registers+ 1218 M.L. Peck and D. Herschlag

We also found that Prp16, a DEAD box family protein single-stranded RNA 10–15 nt in length+ This would be involved in pre-mRNA splicing, has an increased affin- particularly problematic for highly structured 59 UTRs, ity for phosphorothioate-substituted oligonucleotides+ which are presumably most in need of the unwinding Strengthened binding upon phosphorothioate substitu- activity of eIF4A+ tion has been reported for polyanion binding proteins The results herein reveal features of eIF4A’s oligo- on the cell surface (Stein & Cheng, 1993) and other nucleotide site that may ensure its binding to and stim- nucleic acid binding proteins (for examples, see Mu- ulation by 59 UTRs+ First, it appears that binding and jumdar et al+, 1989; Gao et al+, 1992; Weidner et al+, stimulation are only gradually lessened as the length of 1995; Cheng et al+, 1997; Tramontano et al+, 1998)+ single-stranded RNA is reduced, such that eIF4A can Thus, phosphorothioates may increase binding by a be significantly stimulated by as few as four RNA res- general effect, such as easing the removal of associ- idues+ Second, it appears that contacts at different po- ated solvent and metal ions and/or increasing oligonu- sitions within eIF4A’s nucleic-acid site can stimulate cleotide rigidity (Mujumdar et al+, 1989; Gao et al+, 1992; ATP hydrolysis, suggesting that eIF4A may be able to Benimetskaya et al+, 1995; White et al+, 1996; Tramon- accommodate a number of the structural and steric tano et al+, 1998)+ It is also possible that differences in constraints of the RNAs it encounters+ charge distribution, size, and polarizability of phospho- Although it appears to be advantageous for eIF4A to rothioates fortuitously improve interactions within the be stimulated by short stretches of single-stranded RNA, binding sites (Frey & Sammons, 1985; Gao et al+, 1992; the stimulation provided increases with longer RNAs+ It Tramontano et al+, 1998)+ has previously been noted that the thermodynamic sta- The increased affinity provided by phosphorothio- bility of structures in the 59 UTR may influence the ates may provide a tool for studies of eIF4A and other efficiency of translation initiation for a particular mRNA nucleic acid-binding proteins+ In both functional and (Pelletier & Sonenberg, 1985; Kozak, 1986)+ The length structural studies, phosphorothioates could be used to of single-stranded RNA regions within a 59 UTR could increase the affinity between an enzyme and a nucleic be used to set or regulate the ATPase activity of eIF4A, acid and to localize the enzyme at a particular position thereby providing an additional mechanism for influ- within the nucleic acid+ Phosphorothioates could also encing the rate that a particular mRNA is translated+ provide inhibitors that aid the biochemical dissection of complex processes, such as translation initiation, in re- constituted systems and might provide a starting point MATERIALS AND METHODS for the development of tight-binding inhibitors for drug targets such as the Hepatitis C virus NS3 helicase (Mar- Enzymes shall & Caruthers, 1993; Kim et al+, 1998)+ The vector pET-4A for overexpression of eIF4AI (Mouse)in E. coli was the generous gift of N+ Methot and N+ Sonenberg+ Implications of the biochemical features of Overexpression and purification of eIF4A were performed RNA-dependent ATPase stimulation for following published methods (Pause & Sonenberg, 1992; eIF4A’s role in translation initiation Lorsch & Herschlag, 1998a), with the following modifications+ After the MonoQ, HiTrap Blue and HiTrap Heparin columns, eIF4A is thought to use its ATPase activity to promote fractions containing eIF4A were dialyzed against purification translation initiation by removing structure within the 59 buffer (20 mM Tris-Cl, pH 7+4, 0+1 mM EDTA, 2mMDTT,10% UTRs of mRNAs+ To stimulate its ATPase activity, eIF4A glycerol) plus 100 mM KCl and applied to a Source 15Q must encounter regions of single-stranded RNA, as anion-exchange column (Pharmacia)+ With a linear gradient eIF4A apparently cannot bind to or be stimulated by from 0+1 to 1 M KCl in purification buffer, eIF4A eluted from + + structured RNA (Abramson et al+, 1987; Lorsch & the column at ;0 3 M KCl eIF4A was dialyzed into purifica- , + +, tion buffer with 80 mM potassium acetate (enzyme storage Herschlag 1998a) Previous work (Abramson et al Ϫ + ; +, buffer) for storage at 80 8C Enzyme was .97% pure as 1987 Goss et al 1987) and our results suggest that judged by Coomassie staining of polyacrylamide gels+ eIF4A’s nucleic-acid site accommodates 10–15 nt of Purified, recombinant Saccharomyces cerevisiae Prp16 was + single-stranded RNA There are several nucleic acid the generous gift of Y+ Wang and C+ Guthrie+ enzymes, such as restriction endonucleases and poliovirus RNA polymerase, that have severely reduced activity in the presence of nucleic-acids substrates that Nucleic acids are only 1 or 2 nt shorter than their respective site sizes Poly(U) (Pharmacia) was purified by gel electrophoresis+ Oli- (Lesser et al+, 1990; Alves et al+, 1995; Beckman & , + , gonucleotides synthesized using solid-phase methods were Kirkegaard 1998) However a similar strictly defined obtained from the Stanford PAN Facility and Dharmacon Phar- site size for eIF4A would be problematic for its biolog- maceuticals (Boulder, Colorado)+ Phosphorothioate linkages, ical function+ This is because the prevalence of RNA with a single nonbridging oxygen replaced with a sulfur, were secondary structure and RNA binding proteins renders made without controlling for stereochemistry+ Thus, these oli- the 59 UTRs of mRNAs unlikely to possess stretches of gonucleotides are mixtures of 2N stereoisomers (N ϭ the Oligonucleotide stimulation of eIF4A ATPase activity 1219

number of phosphorothioate linkages in the oligonucleotide)+ obtained from kobs by first subtracting the rate constant mea- Oligonucleotides were purified by anion-exchange high- sured in the absence of oligonucleotide from kobs and then performance liquid chromatography (HPLC) using a Dionex dividing this difference by the enzyme concentration+ ATP, app NucleoPac column with aqueous ammonium acetate as the The dependence of (kcat/Km) on oligonucleotide con- eluant or by polyacrylamide gel electrophoresis+ Following centration followed Michaelis-Menten saturation behavior purification, oligonucleotides were ethanol precipitated in so- (Equation 2; Segel, 1993)+ Previous results obtained under dium acetate, resuspended in water, and stored at Ϫ20 8C+ similar reaction conditions suggest that the Michaelis con- oligo Controls with several oligonucleotides indicated that the stant (Km ) represents the equilibrium dissociation constant oligo method of purification had no effect on kinetic parameters for (Kd ) for oligonucleotide binding to enzyme (Lorsch & ATP activation and inhibition+ To assess purity, several oligonucle- Herschlag, 1998a)+ The third-order rate constant (kcat/Km) / 32 oligo otides were radiolabeled with [g- P] ATP using T4 polynucle- K m (hereafter k3) represents reaction of enzyme (E) with otide kinase and gel electrophoresed+ Oligonucleotides purified free ATP and free oligonucleotide (oligo), as described by ATP by HPLC or gel electrophoresis were .95% or .90% homo- Equation 3+ (kcat/Km) is a second-order rate constant rep- geneous in length, respectively+ resenting reaction of enzyme and ATP in the limiting case with enzyme saturated with oligonucleotide, but not ATP (Equa- tion 4)+ ATPase assays and kinetic analyses (k /K )ATP [oligonucleotide] / ATP, app ϭ cat m General ATPase assay conditions (kcat Km ) oligo (2) Km ϩ [oligonucleotide] [g-32P] ATP (ICN) and unlabeled ultrapure ATP (Pharmacia) in stoichiometric complex with MgCl2 (hereafter ATP•Mg) were k3 E ϩ oligo ϩ ATP & E ϩ oligo ϩ ADP ϩ P (3) prepared as previously described (Lorsch & Herschlag, 1998a)+ i ATPase assays were performed using trace [g-32P] ATP / ATP + (kcat Km) (,25 nM) with varying concentrations of unlabeled ATP•Mg E•oligo ϩ ATP & E•oligo ϩ ADP ϩ Pi (4) eIF4A ATPase reactions were carried out at 25 8Cin 25 mM MES-KOH, 5 mM Tris-Cl, 30 mM potassium acetate, ATP oligo Typically, k3, (kcat/Km) and Km were obtained by fit- 2+5 mM MgCl2, 1+5mMDTT,2+5% glycerol, and 25 mM EDTA at pH 6+0 (these concentrations include contributions from ting the dependence of ATPase stimulation on oligonucleo- + , the enzyme storage buffer)+ As reported previously, eIF4A is tide concentration to Equation 2 In such cases oligonucleotide lost on tube walls even when using siliconized polypropylene concentration was kept in excess of enzyme concentration oligo+ tubes (Lorsch & Herschlag, 1998a)+ Thus, a minimum of and was varied at least fivefold above and below Km 0+25 mM eIF4A was used in all reactions to avoid significant When saturation of enzyme with a particular oligonucleotide , losses to tube walls+ Reactions (20 mL total volume) were was not attained k3 was obtained from the linear depen- / ATP, app initiated by the addition of 5 mL of eIF4A diluted to desired dence of (kcat Km) on oligonucleotide concentration and oligo + concentrations with enzyme storage buffer+ Aliquots were re- only limiting values for Km could be obtained moved from the reactions at five to eight specific times and mixed with 1 vol of 25 mM EDTA, pH 8+0, to stop the reaction+ Radiolabeled inorganic phosphate (Pi) was separated from Oligonucleotide inhibition assays ATP using PEI-cellulose TLC plates (Baker) and the fraction Inhibition constants were obtained by measuring ATPase ac- P was quantified using a PhosphorImager as previously de- i tivity, (k /K )ATP, app, stimulated by subsaturating U RNA scribed (Lorsch & Herschlag, 1998a)+ Because of strong prod- cat m 20 (3 mM) in the presence of varying concentrations of inhibitory uct inhibition by ADP (Lorsch & Herschlag, 1998a), initial oligonucleotide+ The inhibition constant (K oligo ) was obtained reactions (,10% completion) were followed [under these con- i by fitting the dependence of (k /K )ATP, app on inhibitory oli- ditions K ADP is equal to 2 and 10 mM with subsaturating and cat m i gonucleotide concentration to an equation for simple com- saturating poly(U), respectively (data not shown)]+ Thus, the petitive inhibition (Equation 5)+ To simplify comparisons, (k / observed rate constant for ATP hydrolysis (k ) was ob- cat obs K )ATP, app values were normalized by dividing the observed tained by taking the slope of fraction P plotted against time+ m i rate constants by the rate constant measured in the absence Prp16 ATPase reactions were performed as above, but of inhibitory oligonucleotide+ The concentration of inhibitory were carried out in 40 mM HEPES-KOH, 100 mM potassium oligonucleotide was varied at least fivefold above and below acetate, 5mMKCl,0+5 mM MgCl , 1mMDTT,1% glycerol, 2 K oligo and was kept in excess of enzyme concentration, ex- and 20 mMEDTAatpH7+9+ i cept in cases that are noted as limits+ As the concentration of oligo activating U20 RNA was subsaturating, Ki is expected to oligo+ Kinetic analysis of ATPase stimulation equal Kd by oligonucleotides oligo ATP, app Ki The ATPase stimulation provided by various oligonucleotides normalized (kcat/Km ) ϭ (5) K oligo ϩ [oligo ] was measured in the presence of several concentrations of i inhib ATP oligonucleotide and subsaturating ATP•Mg [1 mM; Km is equal to 4 and 20 mM with saturating and subsaturating poly(U) Because a minimum of 0+25 mM eIF4A was required to RNA, respectively (data not shown)]+ The apparent second- avoid significant loss of enzyme to tube walls (see General ATP, app order rate constant for ATP hydrolysis, (kcat/Km) , was ATPase assay conditions), competitive inhibition assays were 1220 M.L. Peck and D. Herschlag

oligo oligo used to measure values of Ki and Km smaller than ACKNOWLEDGMENTS 0+25 mM+ To determine inhibition constants, the dependence of ATPase activity on poly(U) RNA concentration was deter- We are grateful to Yan Wang and Christine Guthrie for pro- , mined in the absence and presence of a constant concen- viding purified Prp16 protein Nahum Sonenberg for provid- , tration of the inhibitory oligonucleotide to obtain K oligo and ing the eIF4A overexpression plasmid and Gabriel Waksman m + K oligo,app, respectively+ These Michaelis constants were used for providing coordinates for the Rep helicase structure We m , to calculate K oligo according to Equation 1b, which was de- thank Jon Lorsch for invaluable advice and discussion and i , , rived from Scheme 1 in Results+ All nucleic acid concentra- Dave McKay Olke Uhlenbeck and members of the Herschlag oligo laboratory for comments on the manuscript and discussion+ tions were kept in excess of enzyme concentration, and Ki values obtained directly and by this indirect assay agreed This work was supported by a David and Lucile Packard + + within twofold+ Consistent with competitive inhibition, inhibi- Foundation Fellowship in Science and Engineering to D H and a National Science Foundation Predoctoral Fellowship to tory oligonucleotides had less than a twofold effect on (kcat/ ATP M+L+P+ Km) , the maximal ATPase activity with saturating poly(U)+ oligo An analogous strategy was used to obtain Km for a tight- + binding stimulatory oligonucleotide The dependence of the Received April 20, 1999; returned for revision May 12, ATPase activity on activating oligonucleotide concentration 1999; revised manuscript received June 2, 1999 was determined in the presence of a specific concentration of inhibitory oligonucleotide+ The inhibitory oligonucleotide oligo competes for binding, thereby increasing the observed Km oligo,app REFERENCES (Km ) to a value above the enzyme concentration (see + oligo,app, Scheme 1 and Results) This measured value of Km Abramson RD, Dever TE, Lawson TG, Ray BK, Thach RE, Merrick oligo the known Ki , and the concentration of the inhibitory oli- WC+ 1987+ The ATP-dependent interaction of eukaryotic initiation gonucleotide were used to determine K oligo according to factors with mRNA+ J Biol Chem 262:3826–3832+ m , , + + Equation 1c, derived from Scheme 1 (Results)+ Alves J Selent U Wolfes H 1995 Accuracy of the EcoRV restriction endonuclease: Binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition se- quences+ Biochemistry 34:11191–11197+ Beckman MTL, Kirkegaard K+ 1998+ Site size of cooperative single- Controls for competitive inhibition stranded RNA binding by poliovirus RNA-dependent RNA polymerase+ J Biol Chem 273:6724–6730+ The ATPase activity of eIF4A is very sensitive to changes in Benimetskaya L, Tonkinson JL, Koziolkiewicz M, Karwowksi B, Guga salt conditions (Lorsch & Herschlag, 1998a; 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