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

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Effects of Oligonucleotide Length and Atomic Composition on Stimulation of the Atpase Activity of Translation Initiation Factor Eif4a RNA (1999), 5:1210–1221+ Cambridge University Press+ Printed in the USA+ Copyright © 1999 RNA Society+ 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 back- ground 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: un- translated 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
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