Cysteine Activation Is an Inherent in Vitro Property of Prolyl-tRNA Synthetases
2002; Elsevier BV; Volume: 277; Issue: 38 Linguagem: Inglês
10.1074/jbc.m206928200
ISSN1083-351X
AutoresIvan Ahel, Constantinos Stathopoulos, Alexandre Ambrogelly, Anselm Sauerwald, Helen S. Toogood, Thomas Hartsch, Dieter Söll,
Tópico(s)RNA Research and Splicing
ResumoAminoacyl-tRNA synthetases are well known for their remarkable precision in substrate selection during aminoacyl-tRNA formation. Some synthetases enhance the accuracy of this process by editing mechanisms that lead to hydrolysis of incorrectly activated and/or charged amino acids. Prolyl-tRNA synthetases (ProRSs) can be divided into two structurally divergent groups, archaeal-type and bacterial-type enzymes. A striking difference between these groups is the presence of an insertion domain (∼180 amino acids) in the bacterial-type ProRS. Because the archaeal-type ProRS enzymes have been shown to recognize cysteine, we tested selected ProRSs from all three domains of life to determine whether cysteine activation is a general property of ProRS. Here we show that cysteine is activated by recombinant ProRS enzymes from the archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus, from the eukaryote Saccharomyces cerevisiae, and from the bacteria Aquifex aeolicus, Borrelia burgdorferi, Clostridium sticklandii, Cytophaga hutchinsonii, Deinococcus radiodurans, Escherichia coli, Magnetospirillum magnetotacticum, Novosphingobium aromaticivorans, Rhodopseudomonas palustris, and Thermus thermophilus.This non-cognate amino acid was efficiently acylated in vitro onto tRNAPro, and the misacylated Cys-tRNAPro was not edited by ProRS. Therefore, ProRS exhibits a natural level of mischarging that is to date unequalled among the aminoacyl-tRNA synthetases. Aminoacyl-tRNA synthetases are well known for their remarkable precision in substrate selection during aminoacyl-tRNA formation. Some synthetases enhance the accuracy of this process by editing mechanisms that lead to hydrolysis of incorrectly activated and/or charged amino acids. Prolyl-tRNA synthetases (ProRSs) can be divided into two structurally divergent groups, archaeal-type and bacterial-type enzymes. A striking difference between these groups is the presence of an insertion domain (∼180 amino acids) in the bacterial-type ProRS. Because the archaeal-type ProRS enzymes have been shown to recognize cysteine, we tested selected ProRSs from all three domains of life to determine whether cysteine activation is a general property of ProRS. Here we show that cysteine is activated by recombinant ProRS enzymes from the archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus, from the eukaryote Saccharomyces cerevisiae, and from the bacteria Aquifex aeolicus, Borrelia burgdorferi, Clostridium sticklandii, Cytophaga hutchinsonii, Deinococcus radiodurans, Escherichia coli, Magnetospirillum magnetotacticum, Novosphingobium aromaticivorans, Rhodopseudomonas palustris, and Thermus thermophilus.This non-cognate amino acid was efficiently acylated in vitro onto tRNAPro, and the misacylated Cys-tRNAPro was not edited by ProRS. Therefore, ProRS exhibits a natural level of mischarging that is to date unequalled among the aminoacyl-tRNA synthetases. Aminoacyl-tRNA synthetases (AARSs) 1The abbreviations used are: AARS, aminoacyl-tRNA synthetase; ProRS, prolyl-tRNA synthetase; MOPS, 4-morpholinepropanesulfonic acid; ATP-PPi, ATP-pyrophosphate. ensure accuracy in the translation of the genetic code by precisely selecting and attaching their cognate amino acids to the corresponding tRNA species (1Ibba M. Söll D. Annu. Rev. Biochem. 2000; 69: 617-650Crossref PubMed Scopus (1115) Google Scholar). This is accomplished in a two-step process of amino acid activation and aminoacyl-tRNA formation. First, the enzyme-bound aminoacyl-adenylate is formed in the presence of ATP. The activated amino acid is subsequently transferred to the 3′ terminus of the cognate tRNA (1Ibba M. Söll D. Annu. Rev. Biochem. 2000; 69: 617-650Crossref PubMed Scopus (1115) Google Scholar). Although accurate amino acid recognition by AARSs is an efficient process, some misactivation or mischarging of non-cognate amino acids has been observed. The incorrect products are usually hydrolyzed during pre-transfer and/or post-transfer editing activities (2Jakubowski H. Goldman E. Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar). A well described editing model is the "double-sieve" mechanism (3Fersht A.R. Dingwall C. Biochemistry. 1979; 18: 2627-2631Crossref PubMed Scopus (121) Google Scholar, 4Fersht A.R. Science. 1998; 280: 541Crossref PubMed Scopus (41) Google Scholar). It suggests that some aminoacyl-tRNA synthetases choose their cognate amino acids primarily by size and then by specific chemical features. The active site for aminoacylation acts as the "coarse" sieve, activating at a significant rate only those amino acids that are the same size as or smaller than the desired one. The second sieve, the "fine sieve," is the editing site where the products of those amino acids that are smaller than the correct one are hydrolyzed. Editing activities have been demonstrated for many synthetases (5Jakubowski H. Fersht A.R. Nucleic Acids Res. 1981; 9: 3105-3117Crossref PubMed Scopus (136) Google Scholar, 6Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-582Crossref PubMed Scopus (316) Google Scholar, 7Dock-Bregeon A. Sankaranarayanan R. Romby P. Caillet J. Springer M. Rees B. Francklyn C.S. Ehresmann C. Moras D. Cell. 2000; 103: 877-884Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), most recently also for prolyl-tRNA synthetase (ProRS) (8Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8920Crossref PubMed Scopus (121) Google Scholar). ProRS is responsible for the formation of prolyl-tRNA. Progress in genome analysis has made available a large number of ProRS sequences. Their alignments and phylogenetic examination revealed the existence of two quite diverged groups (Fig. 1), archaeal-type and bacterial-type ProRS enzymes (9Woese C.R. Olsen G. Ibba M. Söll D. Microbiol. Mol. Biol. Rev. 2000; 64: 202-236Crossref PubMed Scopus (536) Google Scholar, 10Ribas de Pouplana L. Brown J.R. Schimmel P. J. Mol. Evol. 2001; 53: 261-268Crossref PubMed Scopus (20) Google Scholar, 11Yaremchuk A. Cusack S. Tukalo M. EMBO J. 2000; 19: 4745-4758Crossref PubMed Scopus (78) Google Scholar, 12Burke B. Lipman R.S. Shiba K. Musier-Forsyth K. Hou Y.M. J. Biol. Chem. 2001; 276: 20286-20291Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Bacterial-type ProRS, found in bacteria and mitochondria, is usually larger than the archaeal-type enzyme because of an insertion domain of ∼180 amino acids (Table I). However, ProRSs from Rhodopseudomonas palustris,Caulobacter crescentus, Rickettsia prowazekii, and a few other representatives of α-proteobacteria lack this insertion domain, yet they are bacterial-type enzymes based on phylogenetic analyses. In contrast, the archaeal-type ProRS lacks the insertion sequence but contains a conserved C-terminal extension of ∼100 amino acids including a totally conserved terminal tyrosine (Fig. 1). In addition to its occurrence in archaea and eukarya, the archaeal-type ProRS is also found in the bacterial domain in theThermus-Deinococcus group, theCytophaga-Flexibacter-Bacteroides group,Borrelia, some Clostridium species, and some α-proteobacteria.Table IProperties and kinetic parameters for proline and cysteine activation of various ProRS enzymesaCysteine activation was measured in the presence of 1 mg/ml of the appropriate unfractionated tRNA (see "Experimental Procedures"). Individual kinetic parameters are based on three independent determinations with standard deviations from 3–10%.Source of proSDomainbAbbreviations A, B, E refer to archaea, bacteria, and eukayote, respectively.ProRScA indicates archaeal-type ProRS; Bindicates bacterial-type ProRS.typeLengthdProRS length (number of amino acids). (amino acid)AeAbbreviation P is for amino acid proline and C is for cysteine., fThe C. sticklandii genome is not sequenced. As the closely related C. difficile, species possesses an archaeal-type and a bacterial-type proS gene, it is possible that C. sticklandii may also contain a secondproS gene.K Mmmk cats−1k cat/K MP/Cmms−1k cat/K MM. jannaschiiAA455P0.2863225107C0.090.1902.11M. thermautotrophicusAA481P0.263.41333C0.050.0200.40E. coliBB572P0.291448369C0.170.0220.13R. palustrisBB438P0.281450333C0.170.0250.15A. aeolicusBB570P0.0615250139C0.050.0901.80D. radioduransBA499P0.16322001428C0.010.0140.14C. sticklandii fBA481P0.0560120031C0.010.39039T. thermophilusBA477P0.1535233111C0.020.0422.10B. burgdorferiBA488P0.175.834283C0.180.0220.12C. hutchinsoniiBA491P0.0819238541C0.190.0830.44N. aromaticivoransBA515P0.1426186564C0.200.0650.33M. magnetotacticumBA500P0.1435250595C0.260.1100.42S. cerevisiaeEA688P0.115.651510C0.030.0030.10a Cysteine activation was measured in the presence of 1 mg/ml of the appropriate unfractionated tRNA (see "Experimental Procedures"). Individual kinetic parameters are based on three independent determinations with standard deviations from 3–10%.b Abbreviations A, B, E refer to archaea, bacteria, and eukayote, respectively.c A indicates archaeal-type ProRS; Bindicates bacterial-type ProRS.d ProRS length (number of amino acids).e Abbreviation P is for amino acid proline and C is for cysteine.f The C. sticklandii genome is not sequenced. As the closely related C. difficile, species possesses an archaeal-type and a bacterial-type proS gene, it is possible that C. sticklandii may also contain a secondproS gene. Open table in a new tab Although the ProRS enzymes from Escherichia coli andThermus aquaticus have been purified and partially characterized many years ago, all 20 canonical amino acids had not been tested for their ability to be substrates (13Lee M.L. Muench K.H. J. Biol. Chem. 1969; 244: 223-230Abstract Full Text PDF PubMed Google Scholar, 14Rivera J.A. Wang Q.S. Wong J.T. Can. J. Biochem. Cell Biol. 1984; 62: 507-515Crossref PubMed Scopus (4) Google Scholar). Thus, it was surprising that archaeal ProRS enzymes were shown to be capable of activating cysteine in addition to the cognate proline (15Stathopoulos C., Li, T. Longman R. Vothknecht U.C. Becker H.D. Ibba M. Söll D. Science. 2000; 287: 479-482Crossref PubMed Scopus (64) Google Scholar, 16Lipman R.S.A. Sowers K.R. Hou Y.M. Biochemistry. 2000; 39: 7792-7798Crossref PubMed Scopus (44) Google Scholar). This appeared to be a desirable property as the methanogenic archaeaMethanocaldococcus jannaschii and Methanothermobacter thermautotrophicus lack a canonical cysteinyl-tRNA synthetase in their genomes (17Bult C.J. White O. Olsen G.J. Zhou L. Fleischmann R.D. Sutton G.G. Blake J.A. FitzGerald L.M. Clayton R.A. Gocayne J.D. Kerlavage A.R. Dougherty B.A. Tomb J.F. Adams M.D. Reich C.I. Overbeek R. Kirkness E.F. Weinstock K.G. Merrick J.M. Glodek A. Scott J.L. Geoghagen N.S.M. Venter J.C. Science. 1996; 273: 1058-1073Crossref PubMed Scopus (2291) Google Scholar, 18Smith D.R. Doucette-Stamm L.A. Deloughery C. Lee H. Dubois J. Aldredge T. Bashirzadeh R. Blakely D. Cook R. Gilbert K. Harrison D. Hoang L. Keagle P. Lumm W. Pothier B. Qiu D. Spadafora R. Vicaire R. Wang Y. Wierzbowski J. Gibson R. Jiwani N. Caruso A. Bush D. Reeve J.N. J. Bacteriol. 1997; 179: 7135-7155Crossref PubMed Scopus (1040) Google Scholar), and Cys-tRNACys formation was reported to be carried out by a dual specificity ProRS (15Stathopoulos C., Li, T. Longman R. Vothknecht U.C. Becker H.D. Ibba M. Söll D. Science. 2000; 287: 479-482Crossref PubMed Scopus (64) Google Scholar, 16Lipman R.S.A. Sowers K.R. Hou Y.M. Biochemistry. 2000; 39: 7792-7798Crossref PubMed Scopus (44) Google Scholar). Furthermore, the binding sites for proline and cysteine greatly overlap in M. jannaschii ProRS (19Stathopoulos C. Jacquin-Becker C. Becker H.D., Li, T. Ambrogelly A. Longman R. Söll D. Biochemistry. 2001; 40: 46-52Crossref PubMed Scopus (22) Google Scholar). Recently, however, cysteine activation was also detected for human (20Beuning P.J. Musier-Forsyth K. J. Biol. Chem. 2001; 276: 30779-30785Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), Giardia lamblia(21Bunjun S. Stathopoulos C. Graham D. Min B. Kitabatake M. Wang A.L. Wang C.C. Vivares C.P. Weiss L.M. Söll D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12997-13002Crossref PubMed Scopus (39) Google Scholar), Thermus thermophilus (22Feng L. Stathopoulos C. Ahel I. Mitra A. Tumbula-Hansen D. Hartsch T. Söll D. Extremophiles. 2002; 6: 167-174Crossref PubMed Scopus (3) Google Scholar), and even the bacterial-typeE. coli ProRS (20Beuning P.J. Musier-Forsyth K. J. Biol. Chem. 2001; 276: 30779-30785Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 23Jacquin-Becker C. Ahel I. Ambrogelly A. Ruan B. Söll D. Stathopoulos C. FEBS Lett. 2002; 514: 34-36Crossref PubMed Scopus (8) Google Scholar). This immediately raised the question whether cysteine activation is an inherent in vitro property of all ProRS enzymes. To provide an answer, we examined ProRS enzymes from a number of representative organisms for amino acid activation and tRNA charging with cysteine. Proline and cysteine were purchased from Sigma and were analyzed for purity by the Keck Foundation Research Biotechnology Resource Laboratory at Yale University. [35S]Cysteine (1075 Ci/mmol) and [32P]pyrophosphate (PPi) (15 Ci/mmol) were from PerkinElmer Life Sciences, and [3H]proline (104 Ci/mmol), [14C]proline (248 mCi/mmol), and [3H]alanine (52 Ci/mmol) were fromAmersham Biosciences. Nickel-nitrilotriacetic acid matrix was from Qiagen. GF/C glass microfiber filters were from Whatman. Nitrocellulose filters (0.45 μm) were from Schleicher & Schuell. Inorganic pyrophosphatase (0.2 units/μl) was from Roche Molecular Biochemicals. The TOPO-TA cloning kit was from Invitrogen. Epicurian coli© BL21-CodonPlusTM competent cells were purchased from Stratagene. Bulk mature E. coli tRNA was purchased from Sigma, and bulk mature yeast tRNA was from Roche Molecular Biochemicals. Unfractionated mature tRNA fromM. jannaschii was prepared as described previously (24Curnow A.W. Tumbula D.L. Pelaschier J.T. Min B. Söll D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12838-12843Crossref PubMed Scopus (131) Google Scholar). EF1A-purified E. coli tRNACysand T. thermophilus tRNACys were prepared as described (see Ref. 45Ambrogelly A. Ahel I. Polycarpo C. Bunjun-Srihari S. Krett B. Jacquin-Becker C. Ruan B. Köhrer C. Stathopoulos C. RajBhandary U.L. Söll D. J. Biol. Chem. 2002; 277: 34749-34754Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Partial separation of E. coli tRNAs was performed at 4 °C by chromatography on benzoylated DEAE-cellulose at pH 7.0 in the presence of Mg2+ as described previously (25Gillam I. Millward D.B. von Tigerstrom M. Wimmer E. Tener G.M. Biochemistry. 1967; 6: 3043-3056Crossref PubMed Scopus (495) Google Scholar). tRNA (40 mg) was adsorbed to a benzoylated DEAE-cellulose column (2.5 × 8 cm) in 50 mm MOPS-HCl, pH 7.0, containing 10 mm MgCl2. Elution was performed with a linear gradient of NaCl (0–1.0 m, 400 ml of total volume) at a flow rate of 1 ml/min maintained with 5-ml fractions collected. The fractions were assayed for presence of tRNAPro and tRNACys by charging with E. coli ProRS and cysteinyl-tRNA synthetase. A fraction enriched in tRNAPro(∼150 pmol of proline/A 260) and devoid of tRNACys was used in this study. E. coli (CGG) and M. jannaschii (UGG) tRNAPro genes were synthesized as described previously (15Stathopoulos C., Li, T. Longman R. Vothknecht U.C. Becker H.D. Ibba M. Söll D. Science. 2000; 287: 479-482Crossref PubMed Scopus (64) Google Scholar,19Stathopoulos C. Jacquin-Becker C. Becker H.D., Li, T. Ambrogelly A. Longman R. Söll D. Biochemistry. 2001; 40: 46-52Crossref PubMed Scopus (22) Google Scholar) and then transcribed from NsiI orBstNI-linearized plasmids using T7 polymerase. E. coli transcript was prepared as a ribozyme construct, because the first base is cytosine, which is highly unfavorable for T7 polymerase (26Fechter P. Rudinger J. Giegé R. Théobald-Dietrich A. FEBS Lett. 1998; 436: 99-103Crossref PubMed Scopus (107) Google Scholar). Digested template DNA was extracted with phenol and chloroform and precipitated with ethanol. The transcription reaction was incubated at 37 °C for 3 h and included 40 mm Tris-HCl, pH 8.1, 22 mm MgCl2, 2 mm spermidine, 10 mm dithiothreitol, 4 mm of each NTP (Sigma), 10 mm GMP (Sigma), 0.05% Triton X-100, 10 mg/ml yeast inorganic pyrophosphatase, 0.1 mg/ml digested template, and 50 μg/ml T7 polymerase. Reactions were stopped by phenol and chloroform extraction, precipitated with ethanol, and resuspended in gel loading buffer (8 m urea, 20% sucrose, 0.1% bromphenol blue, 0.1% xylene cyanol). For ribozyme constructs before the phenol extraction, reaction mixtures were diluted five times and incubated for 1 h at 60 °C to enhance autocatalytic cleavage. The transcripts were purified on a denaturing polyacrylamide gel (12% acrylamide:bisacrylamide (19:1), 8 m urea, 89 mm Tris borate, pH 8.3, 2 mm EDTA) and electroeluted from the gel using a Schleicher & Schuell Biotrap. The transcripts were extracted with phenol and chloroform, precipitated with ethanol, resuspended in sterile water, and stored at −20 °C. ProS clones of M. jannaschii, M. thermautotrophicus(15Stathopoulos C., Li, T. Longman R. Vothknecht U.C. Becker H.D. Ibba M. Söll D. Science. 2000; 287: 479-482Crossref PubMed Scopus (64) Google Scholar), G. lamblia (21Bunjun S. Stathopoulos C. Graham D. Min B. Kitabatake M. Wang A.L. Wang C.C. Vivares C.P. Weiss L.M. Söll D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12997-13002Crossref PubMed Scopus (39) Google Scholar), and T. thermophilus HB8 (22Feng L. Stathopoulos C. Ahel I. Mitra A. Tumbula-Hansen D. Hartsch T. Söll D. Extremophiles. 2002; 6: 167-174Crossref PubMed Scopus (3) Google Scholar) were described. A strain ofNovosphingobium aromaticivorans was obtained from M. F. Romine (Pacific Northwest National Laboratory, Richland, WA). Cells were grown, and DNA was extracted by standard procedures. ChromosomalR. palustris DNA was from C. S. Harwood (Iowa University, Iowa City, IA), Cytophaga hutchinsonii DNA was from M. J. McBride (University of Wisconsin-Milwaukee, Milwaukee, WI), Borrelia burgdorferi DNA was from P. Rosa (National Institutes of Health, Hamilton, MT), Clostridium sticklandiiDNA was from A. Pich (Universität Halle-Wittenberg, Germany) (27Kabisch U.C. Grantzdorffer A. Schierhorn A. Rucknagel K.P. Andreesen J.R. Pich A. J. Biol. Chem. 1999; 274: 8445-8454Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), Magnetospirillum magnetotacticum DNA was from E. L. Bertani (California Institute of Technology, Pasadena, CA),Deinococcus radiodurans DNA was from D. Tumbula-Hansen (Yale University, New Haven, CT), and A. aeolicus DNA was from T. Steitz (Yale University, New Haven, CT). The coding sequences of the proS genes were amplified by PCR from genomic DNA and cloned into the pCR2.1 TOPO vector. Correct sequences were subsequently recloned into pET15b (Invitrogen) for expression of the N-terminally His6-tagged protein in theE. coli BL21-Codon Plus (DE3)-RIL strain. Deletions in the insertion sequence of the E. coli proS from nucleotide (amino acid) positions 559–1128 (196–376) [ECdel1], 634–1182 (211–394) [ECdel2], 691–1050 (230–350) [ECdel3] (see Fig. 1), and 652–1218 (217–406) [ECdel4] were prepared by site-directed mutagenesis, and the genes were subcloned into pET20b for expression of C-terminally His6-tagged proteins. The resulting deletion genes encoded the extra 16 amino acids (LKGEFCRTPSHWRPLE) originating from the TOPO vector on the C terminus just before the His6-tag sequence. For comparison, theE. coli wild-type proS gene was also cloned in the same fashion. Deletion of nucleotides (amino acids) 691-1041 (230–347) of the A. aeolicus proS gene [AAdel1], corresponding to the ECdel3 mutant of E. coli, was also made. The mutant gene was cloned into the pET15b vector. Cultures were grown at 37 °C in Luria-Bertani (LB) medium supplemented with 100 μg/ml ampicillin and 34 μg/ml chloramphenicol. Expression of the His6-tagged protein was induced for 2–3 h at 30 °C with the addition of 1 mmisopropyl-1-thio-β-d-galactopyranoside before cell harvesting. Expression of the ProRS deletion enzymes was induced with 0.2 mm isopropyl-1-thio-β-d-galactopyranoside at 28 °C for 1 h. The enzymes were purified by nickel-nitrilotriacetic acid chromatography as described previously (22Feng L. Stathopoulos C. Ahel I. Mitra A. Tumbula-Hansen D. Hartsch T. Söll D. Extremophiles. 2002; 6: 167-174Crossref PubMed Scopus (3) Google Scholar). The His6-ProRSs were >95% pure as judged by Coomassie Brilliant Blue staining after SDS-PAGE. Active fractions were pooled and dialyzed against reaction buffer (50 mm HEPES-KOH, pH 7.2, 50 mm KCl, 15 mm MgCl2, 5 mm 2-mercaptoethanol, 1 mm benzamidine) containing 40% glycerol and stored at −20 °C. The E. coli ProRS enzyme was further purified on DEAE-cellulose and Uno S columns to minimize the possibility of E. colicysteinyl-tRNA synthetase contamination. For the DEAE-cellulose column, 50 mm Tris-HCl, pH 8.0, and a gradient of KCl (0–200 mm) were used. For the Uno S column, a gradient of potassium phosphate (20–200 mm), pH 6.8, was used. Active site titration was performed to determine the amount of active enzyme in the preparation. The formation of ProRS·Pro-AMP complexes took place in 100 μl of the 0.5× EAP buffer (50 mm Tris-HCl, pH 7.5, 5 mm KCl, 5 mm MgCl2) containing 0.5 units of inorganic pyrophosphatase and 20 μm[14C]proline (500 cpm/pmol) in the presence of varying concentrations of ProRSs (0.2–5 μm). After 1–10-min incubation, aliquots of 30 μl were spotted onto nitrocellulose filters, filtered, and washed twice with 5 ml of 0.5× EAP buffer. The filters were dried, and the radioactivity was measured by liquid scintillation counting. The reaction mixture (200 μl) contained 50 mm HEPES-NaOH, pH 7.2, 15 mm MgCl2, 50 mm KCl, 5 mm dithiothreitol, 1 mm potassium fluoride, 2 mm ATP, l-proline, orl-cysteine varying from 5–500 μm forK m determinations, 2 mm[32P]pyrophosphate (2 cpm/pmol) and, when indicated, 1 mg/ml of suitable unfractionated tRNA (E. coliin the case of bacterial enzymes, M. jannaschii for archaeal ProRs, and yeast for eukaryotic ProRSs). ProRS concentrations ranged from 2–10 nm for proline activation and from 200–4000 nm for cysteine activation. After various incubation times, the [32P]ATP present in 40-μl aliquots of the reaction mixture was specifically adsorbed on acid-washed Norit (200 μl of a 1% suspension (w/v) of Norit in 0.4 m sodium pyrophosphate solution containing 15% (v/v) perchloric acid) rinsed with 25 ml of water and 10 ml of ethanol on Whatman GF/C fiberglass filter disks, dried, and with the radioactivity determined by liquid scintillation counting. Reactions were performed at 37 °C with the exception ofT. thermophilus, A. aeolicus, M. jannaschii, and M. thermautotrophicus ProRSs where the reaction temperature was 60 °C. K m and k cat values were determined using Hanes-Wolf plots. The standard reaction mixture (100 μl) contained 50 mm HEPES-KOH, pH 7.2, 50 mmKCl, 15 mm MgCl2, 5 mmdithiothreitol, 10 mm ATP, 50 μm[3H]proline (200 cpm/pmol) or 50 μm[35S]cysteine (500 cpm/pmol), 1 mg/ml of suitable unfractionated tRNA, and 1–10 nm purified recombinant ProRSs for the proline charging or 40–2000 nm for cysteine. Radioactive aminoacyl-tRNAs synthesized after 1–30 min were quantified in 20-μl aliquots as described previously (15Stathopoulos C., Li, T. Longman R. Vothknecht U.C. Becker H.D. Ibba M. Söll D. Science. 2000; 287: 479-482Crossref PubMed Scopus (64) Google Scholar). Cys-tRNAProwas generated by charging of unfractionated E. coli tRNA with C. sticklandii ProRS. Mischarged E. coliAla-tRNAPro was generated by charging either theE. coli tRNAPro transcript or unfractionated E. coli tRNA with D. radioduransProRS. Charging reactions were performed in standard reaction buffer using 50 μm [35S]cysteine or 500 μm [3H]alanine. After phenol and chloroform extraction, the tRNA was precipitated with ethanol and the precipitate was washed and dried. 0.5–2 μm of charged tRNA were used in the 60-μl reaction. The deacylation assay was performed as described previously (8Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8920Crossref PubMed Scopus (121) Google Scholar). The reaction was initiated with 20 nm to 2 μm enzyme, and at each time point, 13-μl aliquots were removed. As a positive control, 0.1 mNaOH was used instead of the enzyme. Reactions were quenched on Whatman 3MM filters pre-soaked with 10% trichloroacetic acid, washed, and quantified as described for aminoacylation assays. Because archaeal ProRS enzymes charge cysteine in addition to proline onto tRNA (15Stathopoulos C., Li, T. Longman R. Vothknecht U.C. Becker H.D. Ibba M. Söll D. Science. 2000; 287: 479-482Crossref PubMed Scopus (64) Google Scholar), we wanted to determine whether this property is widespread among this family of enzymes. Therefore, we selected proS genes from a number of organisms so that enzymes from all domains of life, as well as from the two different structural classes, were represented. The archaeal examples were M. jannaschii and M. thermautotrophicus, whereas Saccharomyces cerevisiaerepresented the eukarya. We used a larger number of bacterial enzymes as both archaeal-type and bacterial-type ProRS proteins are represented here. For the archaeal-type proS, T. thermophilus, D. radiodurans, C. sticklandii, N. aromaticivorans, C. hutchinsonii, B. burgdorferi, and M. magnetotacticum were selected, whereas the bacterial-type ProRS was cloned from E. coli and the hyperthermophileA. aeolicus. An atypical bacterial-type ProRS lacking the insertion domain was cloned from R. palustris. Because the insertion domain appears to be involved in editing (28Wong F.C. Beuning P.J. Nagan M. Shiba K. Musier-Forsyth K. Biochemistry. 2002; 41: 7108-7115Crossref PubMed Scopus (64) Google Scholar), we wanted to generate forms of the E. coli and A. aeolicus enzymes that lack partially or completely the 180-amino acid insertion domain. Not unexpectedly, only two of the enzymes could be expressed in a stable form. This was the ProRS ECdel3 enzyme (see Fig. 1) with 452 amino acids compared with 572 amino acids in wild-type E. coli ProRS. Similarly, the A. aeolicus ProRS AAdel1 lacked 117 amino acids from the wild-type open reading frame of 570. In both of these enzyme constructs, only 65% of the insertion domain was deleted; however, they were significantly less stable than the wild-type enzymes. Kinetic parameters of cysteine and proline activation were determined by ATP-PPiexchange (Table I). It is clear that all of the ProRSs tested are able to use cysteine as substrate. Cysteine activation (expressed ask cat/K m) by these ProRSs was 30–1400-fold lower than the activation of the cognate proline. The "affinities" of ProRSs for cysteine were surprisingly high (K m values ranging from 10–260 μm), which was in most cases even higher than those for proline (K m values ranging from 50–290 μm). However, turnover numbers for cysteine activation were severely decreased compared with proline (k cat dropped 150–2300-fold). Thus, cysteine activation is still considerably poorer than proline activation. Alanine for comparison was also detectably activated by ProRSs (20Beuning P.J. Musier-Forsyth K. J. Biol. Chem. 2001; 276: 30779-30785Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). It has a profoundly poorer affinity than cysteine or proline (K m values between 31–500 mm); however the k cat values are 10-fold higher than those for cysteine (data not shown). Thus, cysteine is still a significantly better substrate in ATP-PPiexchange than alanine. Although the addition of unfractionated tRNA significantly improved cysteine activation in most cases, cysteine activation can always be observed in the absence of tRNA (23Jacquin-Becker C. Ahel I. Ambrogelly A. Ruan B. Söll D. Stathopoulos C. FEBS Lett. 2002; 514: 34-36Crossref PubMed Scopus (8) Google Scholar, 29Lipman R.S.A. Beuning P.J. Musier-Forsyth K. Hou Y.M. J. Mol. Biol. 2002; 316: 421-427Crossref PubMed Scopus (9) Google Scholar). tRNA-dependent cysteine activation is a property common to both archaeal-type and bacterial-type ProRS families. The activation of cysteine by M. jannaschii ProRS, for instance, was found to be stimulated up to 4-fold in the presence of unfractionated tRNA, whereas a 2-fold stimulation was observed for A. aeolicus ProRS (data not shown). The ability of all ProRS enzymes to charge cysteine onto unfractionated tRNA was tested by the standard aminoacylation assay and compared to the reaction with proline. In this assay, the source of tRNA for most ProRSs was E. coli. The archaeal enzymes were tested withM. jannaschii tRNA and S. cerevisiaeProRS with homologous tRNA. Most of the enzymes charged tRNA with cysteine to a similar plateau level as with proline (data not shown). The ratio of the initial velocities of proline versuscysteine charging is plotted in Fig. 2. As can be seen, the values greatly vary from 7 to 4900. The archaeal enzymes and the closely related ProRS from the primitive eukaryoteG. lamblia showed the best cysteine charging, whereas the eukaryotic S. cerevisiae enzyme showed the lowest. However, even the bacterial-type A. aeolicus ProRS charges cysteine very well. The effect of the insertion domain on cysteine charging was investigated with the E. coli enzyme. The partial lack of this domain in the bacterial-type ProRS ECdel3 showed a 2.3-fold increase in the relative cysteine charging when compared with the intact E. coli ProRS. To determine tRNA specificity of the ProRS enzymes, transcripts of E. colitRNAPro(CGG) or M. jannaschiitRNAPro(UGG) (for the archaeal ProRSs), an E. coli tRNA
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