Further Characterization of the Helicase Activity of eIF4A
2001; Elsevier BV; Volume: 276; Issue: 16 Linguagem: Inglês
10.1074/jbc.m007560200
ISSN1083-351X
AutoresGeorge W. Rogers, Walt F. Lima, William C. Merrick,
Tópico(s)Viral Infections and Immunology Research
ResumoEukaryotic 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. 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. The initiation of protein synthesis in eukaryotic systems is an intricate process involving at least 12 protein factors that work in concert to bring the mRNA, the initiating methionyl-tRNA, and the 40 S and 60 S ribosomal subunits together to form an 80 S complex capable of peptide chain elongation (see Refs. 1Merrick W.C. Hershey J.W.B. Matthews M.B. Sonenberg N. Hershey J.W.B. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar, 2Kozak M. Gene. 1999; 234: 187-208Crossref PubMed Scopus (1121) Google Scholar, 3Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (636) Google Scholar for reviews on translation initiation). In this process, eukaryotic initiation factor (eIF) 1The abbreviations used are:Met-tRNAiinitiator methionyl-tRNAeIFeukaryotic initiation factorCWRU MBCLCase Western Reserve University Molecular Biology Core LaboratoryPOphosphatePSphosphorothioateMOEmethoxyethylBSAbovine serum albuminMES2-(N-morpholino)ethanesulfonic acidssRNAsingle-stranded RNAdsRNAdouble-stranded RNAssDNAsingle-stranded DNAdsDNAdouble-stranded DNAbpbase pair(s)ntnucleotide(s) 4A works in conjunction with eIF4B, eIF4F, and eIF4H to promote the binding of mRNA to the 40 S ribosomal subunit (1Merrick W.C. Hershey J.W.B. Matthews M.B. Sonenberg N. Hershey J.W.B. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar, 3Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (636) Google Scholar, 4Richter N.J. Rogers Jr., G.W. Hensold J.O. Merrick W.C. J. Biol. Chem. 1999; 274: 35415-35424Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). eIF4A is proposed to function in this important regulatory step by unwinding secondary structure in the 5′-untranslated region of the mRNA, thus facilitating the binding of the 40 S ribosomal subunit to the mRNA and allowing for subsequent scanning to the initiator AUG codon.eIF4A is the archetype of the DEAD box family of proteins (5Linder P. Lasko P.F. Ashburner M. Leroy P. Nielsen P.J. Nishi K. Schnier J. Slonimski P.P. Nature. 1989; 337: 121-122Crossref PubMed Scopus (624) Google Scholar). DEAD box (and related DEXH box) proteins contain eight highly conserved amino acid sequence motifs and have been implicated in virtually every cellular process involving RNA unwinding and/or rearrangement. These include transcription, ribosomal biogenesis, pre-mRNA splicing, RNA export, translation, and RNA degradation (6de la Cruz J. Kressler D. Linder P. Trends Biochem. 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Nature. 2000; 403: 447-451Crossref PubMed Scopus (195) Google Scholar) have elegantly demonstrated that the NPH-II protein of vaccinia virus is able to function as a processive and directional RNA helicase.The eight conserved motifs of eIF4A span 400 amino acid residues and represent the core sequence of DEAD/DEXH proteins in the superfamily II group of helicases (5Linder P. Lasko P.F. Ashburner M. Leroy P. Nielsen P.J. Nishi K. Schnier J. Slonimski P.P. Nature. 1989; 337: 121-122Crossref PubMed Scopus (624) Google Scholar, 6de la Cruz J. Kressler D. Linder P. Trends Biochem. Sci. 1999; 24: 192-198Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 24Schmid S.R. Linder P. Mol. Microbiol. 1992; 6: 283-291Crossref PubMed Scopus (448) Google Scholar). Extensive mutational analyses of the conserved regions of eIF4A and other DEAD/DEXH proteins have demonstrated that they are important for ATP binding, ATP hydrolysis, RNA binding, RNA unwinding, and coupling of these different activities (17Gross C.H. Shuman S. J. Virol. 1995; 69: 4727-4736Crossref PubMed Google Scholar, 25Pause A. Sonenberg N. EMBO J. 1992; 11: 2643-2654Crossref PubMed Scopus (529) Google Scholar, 26Pause A. Methot N. Sonenberg N. Mol. Cell. Biol. 1993; 13: 6789-6798Crossref PubMed Scopus (258) Google Scholar, 27Gross C.H. Shuman S. J. Virol. 1996; 70: 1706-1713Crossref PubMed Google Scholar, 28Kim D.W. Kim J. Gwack Y. Han J.H. Choe J. J. Virol. 1997; 71: 9400-9409Crossref PubMed Google Scholar, 29Wardell A.D. Errington W. Ciaramella G. Merson J. McGarvey M.J. J. Gen. Virol. 1999; 80: 701-709Crossref PubMed Scopus (87) Google Scholar, 30Lin C. Kim J.L. J. Virol. 1999; 73: 8798-8807Crossref PubMed Google Scholar). Two groups have presented partial crystal structures of yeast eIF4A (31Benz J. Trachsel H. Baumann U. Struct. Fold. Des. 1999; 15: 671-679Abstract Full Text Full Text PDF Scopus (110) Google Scholar, 32Johnson E.R. McKay D.B. RNA. 1999; 5: 1526-1534Crossref PubMed Scopus (73) Google Scholar). Both show that the three-dimensional fold, strand topology, and the orientation of conserved motifs in the N-terminal portion of eIF4A are strikingly similar to domain I of the HCV NS3, PcrA, and Rep helicases (31Benz J. Trachsel H. Baumann U. Struct. Fold. Des. 1999; 15: 671-679Abstract Full Text Full Text PDF Scopus (110) Google Scholar, 32Johnson E.R. McKay D.B. RNA. 1999; 5: 1526-1534Crossref PubMed Scopus (73) Google Scholar). Recently, the crystal structure of the C-terminal portion eIF4A has been solved and again shows that tertiary structure and positions of the conserved motifs in eIF4A have the same topology as the equivalent domains of the Rep, PcrA, and HCV NS3 helicases (33Caruthers J.M. Johnson E.R. McKay D.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13080-13085Crossref PubMed Scopus (243) Google Scholar). Such similarities strongly suggest a common structural and mechanistic theme among these helicases (34Bird L.E. Subramanya H.S. Wigley D.B. Curr. Opin. Struct. Biol. 1998; 8: 14-18Crossref PubMed Scopus (147) Google Scholar).eIF4A has been the subject of intense biochemical study. A detailed kinetic and thermodynamic analysis of the RNA-activated ATPase activity of eIF4A has been performed, which showed that the binding of RNA and ATP to eIF4A is coupled (35Lorsch J.R. Herschlag D. Biochemistry. 1998; 37: 2180-2193Crossref PubMed Scopus (171) Google Scholar). These data, along with limited proteolysis experiments, suggest that eIF4A undergoes a series of ligand-dependent conformational changes as it binds its substrates (RNA and ATP), hydrolyzes ATP, and releases products (36Lorsch J.R. Herschlag D. Biochemistry. 1998; 37: 2194-2206Crossref PubMed Scopus (134) Google Scholar). The effect of changing oligonucleotide length, backbone composition, and identity of the 2′-substituent of the ribose moiety has been investigated and shown to alter the nucleic acid binding and ATPase activities of eIF4A (37Peck M.L. Herschlag D. RNA. 1999; 5: 1210-1221Crossref PubMed Scopus (21) Google Scholar).Previously, we have demonstrated that eIF4A functions alone as an ATP-dependent RNA helicase (8Rogers Jr., G.W. Richter N.J. Merrick W.C. J. Biol. Chem. 1999; 274: 12236-12244Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Results indicated that the initial rate of eIF4A-dependent unwinding decreased as the stability of the duplex is increased and that eIF4A helicase activity is nonprocessive (8Rogers Jr., G.W. Richter N.J. Merrick W.C. J. Biol. Chem. 1999; 274: 12236-12244Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). A simple model of unwinding was proposed suggesting that eIF4A affects strand separation by unwinding a small number of base pairs, consequently making the duplex behave as a shorter, less stable duplex in a thermodynamic sense. If there is sufficient destabilization of the duplex by eIF4A, complete stand separation occurs (less stable duplex substrates). Conversely, if the duplex is not sufficiently destabilized by eIF4A, the partially unwound strands reanneal (i.e. snap back), and complete strand separation does not occur (more stable duplex substrates) (8Rogers Jr., G.W. Richter N.J. Merrick W.C. J. Biol. Chem. 1999; 274: 12236-12244Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). In addition, the helicase activity of eIF4A may be stimulated by either eIF4B or eIF4H or if eIF4A exists as part of the eIF4F complex (4Richter N.J. Rogers Jr., G.W. Hensold J.O. Merrick W.C. J. Biol. Chem. 1999; 274: 35415-35424Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 8Rogers Jr., G.W. Richter N.J. Merrick W.C. J. Biol. Chem. 1999; 274: 12236-12244Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar,38Rozen F. Edery I. Meerovitch K. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1990; 10: 1134-1144Crossref PubMed Scopus (497) Google Scholar). However, it has not been determined if there is a minimal size of the single-stranded region adjacent to the duplex region of the substrate needed to promote helicase activity or if eIF4A alone may function bidirectionally. Furthermore, the effect of changing the 2′-substituents of the ribose sugars or the phosphate backbone of the duplex on the helicase activity of eIF4A is unknown. In a continuing effort to understand the mechanism by which eIF4A and other DEAD/DEXH proteins unwind nucleic acid duplexes, an investigation of the helicase activity of eIF4A with respect to duplex substrate specificity was performed.EXPERIMENTAL PROCEDURESMaterialsReagents were purchased from the following suppliers: rabbit reticulocyte lysate from Green Hectares, Oregon, WI; ATP and BSA from Sigma; [γ-32P]ATP and [α-32P]CTP from PerkinElmer Life Sciences; DNA oligonucleotides PTT-23, PTB-44, and D-14, D-15, D-16, D-17, D-20, and D-20C from the Molecular Biology Core Laboratory, Case Western Reserve University; DNA oligonucleotides PTB-41SD, PTB-41S-, PTB-41S+, PTB-44L+, and d-44 from IDT, Inc., Coralville, IA; RNA oligonucleotides R-10, R-11, R-12, R-13, R-14, and R-15 from Cybersyn, Lenni, PA; RNA oligonucleotides R-17–5′, R-19–5′, R-21–5′, R-23–5′, R-28–5′, R-38–5′, R-17–3′, R-19–3′, R-21–3′, R-23–3′, R-28–3′, R-38–3′ R-17C, R-13C, R-13SD, R-13S-, R-13S+, and R-16L+ from Dharmacon, Inc, Boulder, CO; MegashortscriptTM In Vitro transcription kit from Ambion; and T4 polynucleotide kinase from New England Biolabs. DNA-PS, 2′-MOE, and 2′-MOE/PS oligonucleotides were supplied by Isis Pharmaceuticals, Carlsbad, CA.MethodsPurification of eIF4A from Rabbit Reticulocyte LysatePurification of eIF4A follows the standard procedure used to purify protein translation initiation factors that have been previously published by this laboratory (39Merrick W.C. Methods Enzymol. 1979; 60: 101-108Crossref PubMed Scopus (67) Google Scholar, 40Grifo J.A. Tahara S.M. Leis J.P. Morgan M.A. Shatkin A.J. Merrick W.C. J. Biol. Chem. 1982; 257: 5246-5252Abstract Full Text PDF PubMed Google Scholar).DNA and RNA OligonucleotidesDNA oligonucleotides synthesized by the Molecular Biology Core Laboratory, Case Western Reserve University, were oligonucleotide purification cartridge-purified and stored in distilled H2O. DNA oligonucleotides synthesized by IDT, Inc., were polyacrylamide gel electrophoresis-purified, lyophilized, and resuspended in distilled H2O. RNA oligonucleotides synthesized by Cybersyn were polyacrylamide gel electrophoresis-purified, lyophilized, and resuspended in distilled H2O. RNA oligonucleotides synthesized by Dharmacon were deprotected per the manufacturer's instructions, lyophilized, and resuspended in distilled H2O. Quantitation of each oligonucleotide was performed by UV spectroscopy, and a value of 33 μg per 1A260 was used in determining concentration. Integrity and proper size of each oligonucleotide was assessed by32P-end-labeling each oligonucleotide (described below) and analysis on a denaturing (7 m urea) 20% polyacrylamide gel with known size standards.Transcription of RNAR-44, -41SD, -41S-, -41S+, and -44L+ (see Table I) were synthesized by in vitro transcription using T7 RNA polymerase. The templates for transcription were composed of PTT-23 annealed to PTB-44, -41SD, -41S-, -41S+, or -44L+ (see TableI). Transcription reactions were performed using Ambion's MegashortscriptTM transcription kit per the manufacturer's instructions and included 1 μl of [α-32P]CTP (specific activity 3000 Ci/mmol, 10 mCi/ml) as a tracer label. Products of the reactions were purified on a denaturing (7 m urea) 14% polyacrylamide (19:1 acrylamide:bisacrylamide) gel. The proper bands were located by autoradiography, excised from the gel, and the RNA eluted from the gel slices in 400 μl of 0.5 m sodium acetate, pH 5.2, 1 mm EDTA, and 3% phenol:chloroform (v/v) for 3–4 h at 4 °C. The gel slices were removed from the solution by centrifugation through quick-sep columns, and the recovered RNAs were phenol:chloroform-extracted once and ether-extracted twice, followed by ethanol (1 ml) precipitation for 12–24 h at −20 °C. The precipitated RNAs were recovered by centrifugation at 14,000 rpm at 4 °C for 60 min. The supernatant was removed, and the RNA pellets washed in ice-cold 70% ethanol, dried, and resuspended in distilled H2O. Quantitation of each purified transcript was performed by UV spectroscopy as described above.Table ICharacteristics of DNA and RNA oligonucleotidesOligonucleotideLengthSequence 5′ → 3′DNA templates for transcriptionPTT-2323gaatttaatacgactcactatagPTB-4466gcgtgctttacggtgctagttttgttttgttttgtttttctccctatagtgagtcgtattaaattcPTB-41SD63ccgttgaagcatcgttgttttgttttgttttgttttctccctatagtgagtcgtattaaattcPTB-41S−63ccgattacgatacgttgttttgttttgttttgttttctccctatagtgagtcgtattaaattcPTB-41S+63cctgttgccatgcgttgttttgttttgttttgttttctccctatagtgagtcgtattaaattcPTB-44L+66ccgaattatacgttacgttgttttgttttgttttgttttctccctatagtgagtcgtattaaattcLong strands of duplex substrates1-aR designates RNA, D designates DNA, PS designates a phosphorothiate backbone, 2′-MOE indicates the substitution of 2′-MOE for 2′-OH.R-4444gggagaaaaacaaaacaaaacaaaacuagcaccguaaagcacgcR-38–5′38aaaacaaaacaaaacaaaacaaaauagcaccguaaagcR-28–5′28aaaacaaaacaaaauagcaccguaaagcR-23–5′23aaaacaaaauagcaccguaaagcR-21–5′21aacaaaauagcaccguaaagcR-19–5′19caaaauagcaccguaaagcR-17–5′17aaauagcaccguaaagcR-38–3′38gcuuuacggugcuuaaaacaaaacaaaacaaaacaaaaR-28–3′28gcuuuacggugcuuaaaacaaaacaaaaR-23–3′23gcuuuacggugcuuaaaacaaaaR-21–3′21gcuuuacggugcuuaaaacaaR-19–3′19gcuuuacggugcuuaaaacR-17–3′17gcuuuacggugcuuaaaR-13C13agcaccguaaagcR-41SD41gggagaaaacaaaacaaaacaaaacaacgaugcuucaacggR-41RE41ggcaacuucguagcaacaaaacaaaacaaaacaaaagagggR-41S−41gggagaaaacaaaacaaaacaaaacaacguaucguaaucggR-41S+41gggagaaaacaaaacaaaacaaaacaacgcauggcaacaggR-44L+44gggagaaaacaaaacaaaacaaaacaacguaacguauaauucggD-4444gggagaaaaacaaaacaaaacaaaactagcaccgtaaagcacgcD-2020gtgcgtgctttacggtgctaShort strands of duplex substrates1-aR designates RNA, D designates DNA, PS designates a phosphorothiate backbone, 2′-MOE indicates the substitution of 2′-MOE for 2′-OH.R-1010gcuuuacgguR-1111gcuuuacggugR-1212gcuuuacggugcR-1313gcuuuacggugcuR-1414gcuuuacggugcuaR-13SD13ccguugaagcaucR-13S−13ccgauuacgauacR-13S+13ccuguugccaugcR-16L+16ccgaauuauacguuacR-17C17uuuaagcaccguaaagcD-1414gctttacggtgctaD-1515tgctttacggtgctaD-1616gtgctttacggtgctaD-1717cgtgctttacggtgctaD-20C20tagcaccgtaaagcacgcacD-PS-161-bIndicates that all of the nucleotides in the oligonucleotide contain the indicated modification.16gtgctttacggtgcta2′-MOE-111-bIndicates that all of the nucleotides in the oligonucleotide contain the indicated modification.11gctttacggtg2′-MOE-131-bIndicates that all of the nucleotides in the oligonucleotide contain the indicated modification.13gctttacggtgct2′-MOE/PS-141-bIndicates that all of the nucleotides in the oligonucleotide contain the indicated modification.14gctttacggtgcta2′-MOE/PS-161-bIndicates that all of the nucleotides in the oligonucleotide contain the indicated modification.16gtgctttacggtgcta1-a R designates RNA, D designates DNA, PS designates a phosphorothiate backbone, 2′-MOE indicates the substitution of 2′-MOE for 2′-OH.1-b Indicates that all of the nucleotides in the oligonucleotide contain the indicated modification. Open table in a new tab 32P-End-labeling of OligonucleotidesForty picomoles of either R-10, -11, -12, -13, -14, -15, -13SD, -13S-, -13S+, -16L+, and -13C or D-14, -15, -16, and -17 oligonucleotides (see TableI) and 10 pmol of [γ-32P]ATP (specific activity 3000 Ci/mmol, 10 mCi/ml) were combined with 10 units of T4 polynucleotide kinase (10 μl final volume), and reactions were performed as described (41Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 11.31-11.32Google Scholar). Percent incorporation of the label into these oligonucleotides is 90–95%, and specific activities of the oligonucleotides are on the order of 1 × 106cpm/pmol. Forty picomoles of DNA-PS, 2′-MOE, or 2′-MOE/PS oligonucleotides (see Table I) and 100 pmol of [γ-32P]ATP (specific activity 6000 Ci/mmol, 150 mCi/ml) were combined with 10 units of T4 polynucleotide kinase (10 μl final volume) and reactions were performed as described above. Percent incorporation of the label into these oligonucleotides is 1–5%, and specific activities of the oligonucleotides are on the order of 5×105 cpm/pmol.Helicase SubstratesDuplexes used in the helicase reactions are made by combining long oligonucleotides with appropriate complementary 32P-labeled short oligonucleotides (see TableI for individual oligonucleotide lengths and sequences and figures for duplexes used in specific experiments) in a 1.25:1 ratio, respectively. This excess of long strand is to ensure that essentially all of the labeled short oligonucleotide is in the duplex species. The complementary strands are combined in 1× hybridization buffer (1× Tris-EDTA (TE) plus 100 mm KCl), and the concentration of duplex was 0.5 pmol/μl. Samples were heated to 95 °C for 5 min and then slow-cooled to 4 °C over 90 min (0.1 °C/5 s) using a programmable thermocycling instrument. Under these conditions, ∼90–95% of the labeled bottom (short) strand hybridizes to the top (long) strand. The duplex was stored at −20 °C and diluted in 1× hybridization buffer for use in the helicase assay.dsRNA and dsDNA Competitor DuplexesThe R-17-3′/R-17C and D-20/D-20C duplexes used in the competition assay (Fig. 4) were prepared as above except the R-17-3′ and R-17C or d-20 andd-20C oligonucleotides were combined in a 1:1 ratio at a concentration of 105 or 89 pmol/μl, respectively. Control experiments showed that no significant strand separation occurred at the lowest final concentration of duplex used in the competition experiment (10 nm) over the time course of the reaction (data not shown).Helicase AssayThe helicase assay was performed as described in detail previously (8Rogers Jr., G.W. Richter N.J. Merrick W.C. J. Biol. Chem. 1999; 274: 12236-12244Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Unwinding of duplex substrates was monitored by following displacement of the radiolabeled short strand from the duplex. In general, 20-μl reactions contained 20 mm HEPES-KOH, pH 7.5 (final pH 7.2), 70 mm KCl, 2 mm dithiothreitol, 1 mg/ml BSA, and 1 mmmagnesium acetate (Buffer A). Buffer B′ contained 20 mmMES-KOH, pH 6.0, 15 mm KCl, 15 mm potassium acetate, 2 mm dithiothreitol, 1 mg/ml BSA, and 1 mm magnesium acetate. The concentration of ATP was 1 mm; duplex concentration was 1.8–1.9 nm(36–38 fmol), and eIF4A concentration was 0.8 μm (16 pmol), unless stated otherwise. All reactions were mixed in siliconized tubes to minimize material sticking to tube walls and kept on ice throughout the assembly process. Reactions were incubated at 35 °C (unless otherwise stated) for the duration of the time course. In most circumstances, eIF4A was the last component added to the reaction, just before incubation at 35 °C; however, equivalent results were obtained when duplex or competitor was added as the last component. Reactions were terminated by adding 5 μl of a solution containing 50% glycerol, 2% SDS, 20 mm EDTA, and 0.05% bromphenol blue and xylene cyanol dyes. The products of unwinding reactions were analyzed by separation of displaced labeled short strand from duplex species by electrophoresis on 15 or 18% native polyacrylamide gels (19:1 acrylamide:bisacrylamide) at 4 °C for 2–3 h at 200 V in 1× TBE (TBE, Tris borate/EDTA) buffer. Gels were pre-electrophoresed at 4 °C for 30 min. After electrophoresis, gels were scanned directly using an Ambis radioanalytical scanner. Gels were then exposed to Kodak X-Omat AR film at −80 °C for using intensifying screens.Quantitation of the Helicase AssayQuantitation of the helicase assay was as described previously (8Rogers Jr., G.W. Richter N.J. Merrick W.C. J. Biol. Chem. 1999; 274: 12236-12244Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). In brief, quantitation of counts/min in duplex and displaced/single-stranded RNA bands was performed using the Ambis software. The degree of unwinding for each reaction was determined by measuring the percent of counts/min in duplex and displaced/single-stranded RNA bands using the following formulas: % duplex = cpm duplex/(cpm duplex + cpm displaced strand) and % displaced strand = cpm displaced strand/(cpm duplex + cpm displaced strand). This method of quantitation was used to account for slightly different yields in total counts/min due to variations in pipetting and gel loading steps. The total yield in counts/min among different reactions did not vary by more than ±10% of the average counts/min value of all the reactions in the given experiment. Furthermore, all reagents and protein preparations used in the assay were judged to be free of RNase and phosphatase contamination. A single reaction without (−) eIF4A was incubated at 4 °C during the course of each experiment, and the amount of duplex in this control reaction (typically 36–38 fmol) was normalized to 100%, and the amount of (background) single-stranded RNA (typically 2–4 fmol) was normalized to 0%. All other reactions were scaled to these values. For the competition experiments (Fig. 4), the percentage of inhibition is given by 100 − ((% unwinding of R-41S-/R-13S- in the presence of x μm inhibitor)/(% unwinding of R-41S-/R-13S- in the presence of 0.0 μminhibitor) × 100).Data TreatmentTreatment of data has been described previously (8Rogers Jr., G.W. Richter N.J. Merrick W.C. J. Biol. Chem. 1999; 274: 12236-12244Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). All data reported are the average of 3–5 separate experiments. Standard errors were less than ±10% in all experiments. Plots and graphs were constructed using Prism software by GraphPad or SlideWrite 4.0. Duplex stability (ΔG) values for RNA/RNA duplex substrates were calculated for 35 °C using the nearest neighbor method of analysis as described by Turner and Sugimoto (42Turner D.H. Sugimoto N. Annu. Rev. Biophys. Biophys. Chem. 1988; 17: 167-192Crossref PubMed Scopus (579) Google Scholar). Duplex stability (ΔG) values for RNA/DNA and DNA/RNA duplex substrates were calculated for 35 °C using the nearest neighbor method of analysis as described by Sugimoto et al. (43Sugimoto N. Nakano S. Katoh M. Matsumura A. Nakamuta H. Ohmichi T. Yoneyama M. Sasaki M. Biochemistry. 1995; 34: 11211-11216Crossref PubMed Scopus (559) Google Scholar). Duplex stability (ΔG) values for DNA/DNA duplex substrates were calculated for 35 °C using the nearest neighbor method of analysis as described by SantaLucia et al. (44SantaLucia J. Allawi H.T. Seneviratne P.A. Biochemistry. 1996; 35: 3555-3562Crossref PubMed Scopus (697) Google Scholar). Changing the nonbridging oxygen atom in the oligonucleotide phosphodiester (PO) backbone to sulfur (phosphorothioate (PS) backbone) decreases the stability of the duplex by 0.3 kcal/mol per modification (45Crooke S. Landes R.G. Therapeutic Applications of Oligonucleotides. Austin, TX. 1995; : 53-62Google Scholar). Changing the 2′-group of the ribose sugar to a methoxyethyl (MOE) group increases the stability of the duplex by 0.2 kcal/mol per modification (46Altmann K.H. Fabbro D. Dean N.M. Geiger T. Monia B.P. Muller M. Nicklin P. Biochem. Soc. Trans. 1996; 24: 630-637Crossref PubMed Scopus (101) Google Scholar, 47Altmann K.H. Nicholas N.D. Fabbro D. Freier S.M. Geiger T. Haner R. Husker D. Martin P. Monia B.P. Muller M. Natt F. Nicklin P. Phillips J. Pieles U. Sasmor H. Moser H.E. Chimia. 1996; 50: 168-176Google Scholar, 48Freier S.M. Altmann K.H. Nucleic Acids Res. 1997; 25: 4429-4443Crossref PubMed Scopus (616) Google Scholar).Initial rates of unwinding duplex substrates by eIF4A were determined by measuring the amount of unwinding in the linear (0–2 min) portion of the reaction. Linear fits were applied, and the initial rate (in fmol/min) was taken from the slope of this line (data not shown). The amplitude (maximum % duplex unwound) of duplex unwinding was deter
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