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J. Biol. Chem., Vol. 277, Issue 38, 34743-34748, September 20, 2002

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Cysteine Activation Is an Inherent in Vitro Property of Prolyl-tRNA Synthetases^*

Ivan Ahel, Constantinos Stathopoulos, Alexandre Ambrogelly, Anselm Sauerwald, Helen Toogood, Thomas Hartsch^§¶, and Dieter Söll^¶

From the Departments of  Molecular Biophysics and Biochemistry and ^¶ Chemistry, Yale University, New Haven, Connecticut 06520-8114 and the ^§ Institut für Mikrobiologie und Genetik der Universität Göttingen, D-37077 Göttingen, Germany

Received for publication, July 11, 2002, and in revised form, July 16, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 tRNA^Pro, and the misacylated Cys-tRNA^Pro was not edited by ProRS. Therefore, ProRS exhibits a natural level of mischarging that is to date unequalled among the aminoacyl-tRNA synthetases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Aminoacyl-tRNA synthetases (AARSs)¹ ensure accuracy in the translation of the genetic code by precisely selecting and attaching their cognate amino acids to the corresponding tRNA species (1). 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 (1). 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 (2). A well described editing model is the "double-sieve" mechanism (3, 4). 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 (5-7), most recently also for prolyl-tRNA synthetase (ProRS) (8).

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 (9-12). 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 -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 the Thermus-Deinococcus group, the Cytophaga-Flexibacter-Bacteroides group, Borrelia, some Clostridium species, and some -proteobacteria.

Although the ProRS enzymes from Escherichia coli and Thermus 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 (13, 14). Thus, it was surprising that archaeal ProRS enzymes were shown to be capable of activating cysteine in addition to the cognate proline (15, 16). This appeared to be a desirable property as the methanogenic archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus lack a canonical cysteinyl-tRNA synthetase in their genomes (17, 18), and Cys-tRNA^Cys formation was reported to be carried out by a dual specificity ProRS (15, 16). Furthermore, the binding sites for proline and cysteine greatly overlap in M. jannaschii ProRS (19). Recently, however, cysteine activation was also detected for human (20), Giardia lamblia (21), Thermus thermophilus (22), and even the bacterial-type E. coli ProRS (20, 23). 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

General-- 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 (PP_i) (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 from Amersham 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-CodonPlus^TM 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.

Preparation of Native tRNAs-- Unfractionated mature tRNA from M. jannaschii was prepared as described previously (24). EF1A-purified E. coli tRNA^Cys and T. thermophilus tRNA^Cys were prepared as described (see Ref. 45).

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 (25). 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 tRNA^Pro and tRNA^Cys by charging with E. coli ProRS and cysteinyl-tRNA synthetase. A fraction enriched in tRNA^Pro (~150 pmol of proline/A₂₆₀) and devoid of tRNA^Cys was used in this study.

Preparation of tRNA Genes and Transcripts-- E. coli (CGG) and M. jannaschii (UGG) tRNA^Pro genes were synthesized as described previously (15, 19) and then transcribed from NsiI or BstNI-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 (26). 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.

Cloning and Site-directed Mutagenesis of ProRSs-- ProS clones of M. jannaschii, M. thermautotrophicus (15), G. lamblia (21), and T. thermophilus HB8 (22) were described. A strain of Novosphingobium aromaticivorans was obtained from M. F. Romine (Pacific Northwest National Laboratory, Richland, WA). Cells were grown, and DNA was extracted by standard procedures. Chromosomal R. 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 sticklandii DNA was from A. Pich (Universität Halle-Wittenberg, Germany) (27), 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 the E. 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, the E. 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.

Overexpression and Purification of His₆-tagged Enzymes-- 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 mM isopropyl-1-thio--D-galactopyranoside before cell harvesting. Expression of the ProRS deletion enzymes was induced with 0.2 mM isopropyl-1-thio--D-galactopyranoside at 28 °C for 1 h.

The enzymes were purified by nickel-nitrilotriacetic acid chromatography as described previously (22). 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. coli cysteinyl-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-- 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.

ATP-PP_i Exchange Assay-- 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, or L-cysteine varying from 5-500 µM for K_m determinations, 2 mM [³²P]pyrophosphate (2 cpm/pmol) and, when indicated, 1 mg/ml of suitable unfractionated tRNA (E. coli in 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 of T. 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.

Aminoacylation of tRNA-- The standard reaction mixture (100 µl) contained 50 mM HEPES-KOH, pH 7.2, 50 mM KCl, 15 mM MgCl₂, 5 mM dithiothreitol, 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 (15).

Assays for Post-transfer Editing-- Cys-tRNA^Pro was generated by charging of unfractionated E. coli tRNA with C. sticklandii ProRS. Mischarged E. coli Ala-tRNA^Pro was generated by charging either the E. coli tRNA^Pro transcript or unfractionated E. coli tRNA with D. radiodurans ProRS. 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 (8). 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 M NaOH 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Selection of ProRS Enzymes for Study-- Because archaeal ProRS enzymes charge cysteine in addition to proline onto tRNA (15), 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 cerevisiae represented 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 hyperthermophile A. 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 (28), 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.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   A schematic representation of the two ProRS clusters. The shaded area in the insert illustrates the part that was successfully deleted in the ECdel3 enzyme.

Cysteine Is a Substrate of ProRS-- Kinetic parameters of cysteine and proline activation were determined by ATP-PP_i exchange (Table I). It is clear that all of the ProRSs tested are able to use cysteine as substrate. Cysteine activation (expressed as k_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 (20). 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-PP_i exchange 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 (23, 29). 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 with M. jannaschii tRNA and S. cerevisiae ProRS 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 versus cysteine 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 eukaryote G. 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.

View larger version (51K): [in this window] [in a new window]   Fig. 2.   Ratio of initial rates of proline acylation/cysteine acylation by various ProRSs. Enzymes from the following organisms were used: AA, Aquifex aeolicus; BB, Borrelia burgdorferi; CS, Clostridium sticklandii; CH, Cytophaga hutchinsonii; DR, Deinococcus radiodurans; EC, Escherichia coli; ECdel3, Escherichia coli with partially deleted insert region (see Text); GL, Giardia lamblia; MM, Magnetospirillum magnetotacticum; MJ, Methanocaldococcus jannaschii; MT, Methanothermobacter thermautotrophicus; NA, Novosphingobium aromaticivorans; RP, Rhodopseudomonas palustris; SC, Saccharomyces cerevisiae; TT, Thermus thermophilus. Bars representing E. coli wild-type and deletion proteins are striped for easier comparison.

ProRS Forms Cys-tRNA ^Pro-- To determine tRNA specificity of the ProRS enzymes, transcripts of E. coli tRNA^Pro(CGG) or M. jannaschii tRNA^Pro(UGG) (for the archaeal ProRSs), an E. coli tRNA preparation devoid of tRNA^Cys (prepared by benzoylated DEAE-cellulose chromatography), and EF1A-purified E. coli tRNA^Cys and T. thermophilus tRNA^Cys were used. Because the results with the different ProRS enzymes were essentially the same, only the T. thermophilus ProRS acylation data are presented. The tRNA^Pro transcript (Fig. 3A) or the tRNA^Pro column fraction (Fig. 3B) was efficiently charged with both proline and cysteine. This indicates that cysteine charging of unfractionated tRNA is the result of Cys-tRNA^Pro formation. In agreement with this finding, the pure E. coli tRNA^Cys and T. thermophilus tRNA^Cys preparations could not be cysteinylated by the ProRS enzymes (Fig. 3B). Therefore, we conclude that tRNA^Pro both as transcript and fully modified can be readily mischarged with cysteine by all of the ProRSs tested.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Proline and cysteine charging by T. thermophilus ProRS. A, charging of E. coli tRNAPro transcript (1.5 µM) with 50 µM proline and 50 nM enzyme (), or 50 µM cysteine and 750 nM enzyme (). B, partially purified mature E. coli tRNA enriched in tRNAPro (2 µM) with 50 µM proline, and 50 nM enzyme (), or 50 µM cysteine, and 750 nM enzyme (); T. thermophilus tRNACys (5 µM) with 50 µM cysteine, and 750 nM enzyme ().

Post-transfer Editing by ProRS-- Because all of the ProRSs tested misacylated tRNA^Pro, we examined their post-transfer editing capacity using E. coli Cys-tRNA^Pro. ProRSs from C. sticklandii, M. magnetotacticum, E. coli, A. aeolicus, and R. palustris were tested, but even at high enzyme concentrations (1 µM), none showed any significant deacylation of cysteine (Fig. 4A). Because E. coli ProRS is known to edit Ala-tRNA^Pro (8, 28), we tested the alanine editing activity of our ProRS enzymes. Only bacterial-type ProRS enzymes with the intact insertion domain deacylate Ala-tRNA^Pro (Fig. 4B). Even the R. palustris ProRS, which has a high similarity to the E. coli protein but lacks the insertion domain, did not show any editing. As a further confirmation to the reported involvement of the insertion domain in alanine editing (28), we showed that the ProRS AAdel1 enzyme did not deacylate Ala-tRNA^Pro (Fig. 4B).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Deacylation of mischarged E. coli Cys-tRNAPro and Ala-tRNAPro. A, deacylation of Cys-tRNAPro with: 1 µM C. sticklandii ProRS (), 1 µM M. magnetotacticum ProRS (), 1 µM E. coli ProRS (), 1 µM A. aeolicus ProRS (), 1 µM R. palustris ProRS (), 0.1 M NaOH (), or no enzyme (). B, deacylation of Ala-tRNAPro: 1 µM C. sticklandii ProRS (), 1 µM M. magnetotacticum ProRS (), 40 nM E. coli ProRS (), 200 nM A. aeolicus ProRS (), 1 µM R. palustris ProRS (), 200 nM AAdel1 ProRS (), or no enzyme ().

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mischarging of non-cognate amino acids by AARSs has been observed previously (2). However, the levels of mischarging have been relatively insignificant compared with the charging of the cognate amino acid, and many AARSs are able to efficiently hydrolyze misactivated or mischarged amino acids through their editing activities. The exceptions are mutant AARSs that are unable to perform their editing function (7, 30-32). It has been estimated that editing would be necessary for cell viability if the discrimination factor between cognate and non-cognate amino acids is <3000 (33). Wild-type ProRSs may present the only example where lower discrimination factors naturally exist. ProRSs efficiently misactivate cysteine and form Cys-tRNA^Pro, which appears resistant to the editing functions of enzymes in vitro. Although it is unclear whether archaeal-type ProRSs possess an editing activity for Ala-tRNA^Pro (Ref. 20 and this study), the bacterial-type enzymes certainly do (Refs. 8, 28, and this work). Therefore, it is really puzzling why the bacterial-type ProRS does not edit cysteine when it acts efficiently on alanine. This fact might be explained if bacterial-type ProRSs were to use a double sieve mechanism (3, 4), because the molecular volume of alanine is smaller than the very similar volumes of proline and cysteine. As such, alanine would be able to enter the active site of the enzyme where it could be subsequently activated and even transferred onto tRNA. However, its ability to be edited suggests that alanine fits also to the distinct editing site, which was recently shown to be in the insertion domain of bacterial-type ProRSs (Ref. 28 and this work). In contrast, having a similar molecular volume to proline, cysteine cannot be edited without putting at risk hydrolysis of the cognate amino acid. Hence, it seems that cysteine evades efficient discrimination by both sieves so that mischarging of cysteine remains the same problem for both the bacterial and archaeal types of ProRS.

The highest rate of cysteine mischarging compared with that of the cognate proline was shown by the archaeal ProRSs, where the high cysteine acylation was initially observed (15). Looking at our data, it appears that for most ProRSs, the rate of cysteine charging correlates with that of activation. There probably is no significant mechanism in place to prevent the transfer of activated cysteine onto tRNA in vitro. However, S. cerevisiae, C. sticklandii, and T. thermophilus ProRSs show obviously poorer cysteine mischarging compared with the activation data. This may be attributed to a better induced fit mechanism in these enzymes. This mechanism, recently proposed for T. thermophilus ProRS (34), envisages the formation of prolyl-adenylate as a prerequisite for a conformational change, enabling correct binding of the 3' terminus of tRNA.

Although there are no data on the in vivo formation of Cys-tRNA^Pro, it is difficult to imagine that the high level of mischarging observed in vitro would be tolerated by living cells. Careful regulation of intracellular proline and cysteine levels of an organism or the presence of additional factors (protein or RNA) may be required in vivo to prevent the formation of Cys-tRNA^Pro or induce its hydrolysis. Yet, cysteine may present a special challenge for fidelity in protein biosynthesis. An analysis of the accuracy of charging of non-cognate amino acids by seven yeast AARSs revealed poor specificity in discrimination of cysteine by these enzymes (35). Furthermore, based on a number of studies it was suggested that cysteine, possibly because of its small size and amphiphilic character, is an acceptable replacement at most protein positions without deleterious effect on cell viability (32, 36, 37).

ProRS is the only aminoacyl-tRNA synthetase that charges two canonical amino acids at similar levels without subsequent error correction. The implications of this finding for AARS evolution are discussed below. The co-evolution theory of the genetic code (38, 39) proposes cysteine as a late addition to the amino acids used in encoded protein synthesis. Furthermore, analysis of factors which might have contributed to the temporal order of the amino acid recruitment in the genetic code also proposed cysteine to have arrived late (40). Lastly, a recent proteome analysis that potentially reveals how amino acid frequencies in proteins evolved in biological time showed cysteine to be a late addition to the protein amino acids (41). By the same criteria proline is an `early' amino acid (38-41). To guarantee efficiency in protein synthesis, aminoacyl-tRNA synthetases may have evolved their particular specificities in response to the appearance of new amino acids. Thus, if cysteine was added to the pool of protein amino acids later than proline, an existing ProRS would not be expected to have evolved means to discriminate against cysteine. In this context, the difficulty of discriminating against the `newcomer' amino acid cysteine is not surprising, especially if the new amino acid has similar properties (molecular volume) to the cognate proline which may make its editing mechanisms less efficient. Cysteinyl-tRNA synthetase, on the other hand, arguably a much newer enzyme that did not yet reach some methanogens (17, 18, 42), evolved as a highly discriminating enzyme employing a metal ion (Zn²⁺) for specific cysteine recognition (43) and not requiring editing (44). This leaves unresolved the question as to why ProRS did not subsequently evolve a means to discriminate against cysteine; the misreading of proline codons as cysteine would surely have provided a powerful selective pressure. This may readily be explained by the observation that the accuracy of ProRS product formation may be more dependent on intracellular amino acid levels than that of any other aminoacyl-tRNA synthetase, providing a highly effective example of kinetic discrimination.

	ACKNOWLEDGEMENTS

We thank M. Ibba, S. Bunjun-Srihari, and B. Ruan for many discussions. We are grateful to Drs. E. L. Bertani, C. S. Harwood, M. McBride, A. Pich, M. Romine, P. Rosa, T. Steitz, and D. Tumbula-Hansen for gifts of strains, DNA, and enzymes.

	FOOTNOTES

^* This work was supported by grants from the NIGMS, National Institutes of Health, the Department of Energy, and the National Aeronautics and Space Administration.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: Integrated Genomics, Winzlauer Strasse 2a, D-07745 Jena, Germany.

To whom correspondence should be addressed: Dept. of Molecular Biophysics and Biochemistry, Yale University, P. O. Box 208114, 266 Whitney Ave., New Haven, CT 06520-8114. Tel.: 203-432-6200; Fax: 203-432-6202; E-mail: soll@trna.chem.yale.edu.

Published, JBC Papers in Press, July 18, 2002, DOI 10.1074/jbc.M206928200

	ABBREVIATIONS

The abbreviations used are: AARS, aminoacyl-tRNA synthetase; ProRS, prolyl-tRNA synthetase; MOPS, 4-morpholinepropanesulfonic acid; ATP-PP_i, ATP-pyrophosphate.

	REFERENCES

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