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J. Biol. Chem., Vol. 279, Issue 33, 34931-34937, August 13, 2004

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tRNA Recognition by Glutamyl-tRNA Reductase^*

Lennart Randau, Stefan Schauer, Alexandre Ambrogelly, Juan Carlos Salazar, Jürgen Moser, Shun-ichi Sekine¶, Shigeyuki Yokoyama¶, Dieter Söll||, and Dieter Jahn**

From the Institut für Mikrobiologie, Technical University Braunschweig, P. O. Box 3329, D-38023 Braunschweig, Germany, the Departments of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, and the ^¶Cellular Signaling Laboratory and Structurome Group, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan

Received for publication, February 11, 2004 , and in revised form, June 2, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
During the first step of porphyrin biosynthesis in Archaea, most bacteria, and in chloroplasts glutamyl-tRNA reductase (GluTR) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde. Elements in tRNA^Glu important for utilization by Escherichia coli GluTR were determined by kinetic analysis of 51 variant transcripts of E. coli Glu-tRNA^Glu. Base U8, the U13^*G22^**A46 base triple, the tertiary Watson-Crick base pair 19^*56, and the lack of residue 47 are required for GluTR recognition. All of these bases contribute to the formation of the unique tertiary core of E. coli tRNA-^Glu. Two tRNA^Glu molecules lacking the entire anticodon stem/loop but retaining the tertiary core structure remained substrates for GluTR, while further decreasing tRNA size toward a minihelix abolished GluTR activity. RNA footprinting experiments revealed the physical interaction of GluTR with the tertiary core of Glu-tRNA^Glu. E. coli GluTR showed clear selectivity against mischarged Glu-tRNA^Gln. We concluded that the unique tertiary core structure of E. coli tRNA^Glu was sufficient for E. coli GluTR to distinguish specifically its glutamyl-tRNA substrate.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Glutamyl-tRNA synthetase (GluRS)¹ esterifies the cognate tRNA^Glu with glutamate to form glutamyl-tRNA (Glu-tRNA^Glu), an aminoacyl-tRNA that possesses a dual function in the metabolism of most organisms. Apart from its well studied role in protein biosynthesis (1), Glu-tRNA^Glu is the initial precursor for the synthesis of tetrapyrroles, e.g. chlorophyll, heme, and vitamin B₁₂ (2, 3). In plants, Archaea, and most bacteria the common precursor of all tetrapyrroles, 5-aminolevulinic acid, is formed in a two-step reaction from the five-carbon chain of glutamate. Glutamyl-tRNA reductase (GluTR) catalyzes the NADPH-dependent reduction of glutamate to glutamate-1-semialdehyde (GSA), which is subsequently converted to 5-aminolevulinic acid by glutamate-1-semialdehyde-2,1-aminomutase (GSA-AM) (4, 5). To maintain the fidelity of protein biosynthesis and, at the same time, an adequate metabolite flux for 5-aminolevulinic acid formation, accurate recognition of various cellular tRNAs by their cognate aminoacyl-tRNA synthetase and of Glu-tRNA by GluTR must be secured. The characteristic nucleotides in tRNAs (identity elements) needed for recognition by their cognate aminoacyl-tRNA synthetases have been characterized for many systems (6). For the Escherichia coli GluRS·tRNA^Glu system, 5-methylaminomethyl-2-thiouridine in position 34, U35, C36, A37 in the anticodon loop, G1^*C72, U2^*A71 in the acceptor stem, the U11^*A24 base pair, the U13^*G22^**A46 base triple, and the lack of residue 47 (47) determine the charging identity (7, 8, 33). The crystal structure of the GluRS·tRNA^Glu complex (9, 10) provided further insight to tRNA^Glu recognition by GluRS. In contrast, there is little information on the recognition of Glu-tRNA^Glu by GluTR. Specific sequence information is required, as 5-aminolevulinic acid synthesis in barley chloroplasts demands chloroplast tRNA^Glu and not cytoplasmic tRNA^Glu (11). Specificity of GluTR for tRNA^Glu was also shown for the enzyme from Chlorella vulgaris (31), Chlorobium vibrioforme (32), Chlamydomonas reinhardtii (13), and Synechocystis 6803 (12). Based on a sequence comparison of tRNA^Glu species that are substrates for various GluTRs with tRNA species which are not utilized, putative identity elements were postulated (14). A point mutation (C56–U56) in Euglena gracilis chloroplast tRNA^Glu was reported to uncouple protein and chlorophyll biosynthesis. This mutant tRNA was still aminoacylated by chloroplast GluRS and utilized in protein biosynthesis but was not a substrate for GluTR and therefore did not support tetrapyrrole biosynthesis (15). The solution of the long sought-after crystal structure of GluTR (30) and the biochemical characterization of E. coli GluTR (16, 17) generated much interest in the details of tRNA recognition by this protein. The role of the glutamate part of the substrate was elucidated by site-directed mutagenesis of the E. coli GluTR substrate binding pocket and the co-crystallization of glutamycin, a glutamate analogue, with the Methanopyrus kandleri enzyme (29). Glutamycin was found tightly coordinated in the highly conserved catalytic pocket, which also includes the cysteine residue (Cys-50) known to form an intermediate thioester bond between the enzyme and glutamate (16). In contrast, much less is known about the contribution of the tRNA portion of the substrate to recognition and catalysis. Here we present an analysis of tRNA identity in GluTR recognition.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Production of Recombinant E. coli GluTR, GluRS, GSA-AM, and T7 RNAP—E. coli GluTR was produced as a six-histidine N-terminal fusion protein, renatured from inclusion bodies and purified as described (17). Recombinant E. coli GluRS and GSA-AM were purified to apparent homogeneity according to published procedures (18, 25). Bacteriophage T7 RNAP was purified from an overproducing E. coli strain harboring the plasmid pAR1219 as outlined previously (19).

Construction of Expression Vectors for tRNA^Glu Variants—The majority of expression vectors for the production of E. coli tRNA^Glu variants were described before (7). The plasmid pKR320 (20) contains the E. coli tRNA^Glu gene cloned downstream of a T7 RNA polymerase promoter. The tRNA^Glu variants encoding tRNAs harboring the point mutations C56U, G19A, C56U/G19A, U8C, C56G, minimalist tDNA constructs AC1 and AC2, and the minihelix (see Fig. 1) were generated by site-directed mutagenesis of the plasmid pKR320 using the QuikChange^TM site-directed mutagenesis kit (Stratagene, Heidelberg, Germany). To obtain the minimalist tDNA constructs an expression vector encoding a tRNA^Glu transcript that lacks the entire anticodon stem/loop (pKR320AC) was generated by deletion of 22 nucleotides using the oligonucleotide 5'-CTAGAGGCCCAGGACAGGGGTTCGAATCC-3'. The following oligonucleotides were employed to insert the linker region in plasmid pKR320AC underlined, 5'-CTAGAGGCCCAGGACAAATATAACAGGGGTTCGAATCC-3' (AC1) and 5'-CTAGAGGCCCAGGACAATTAACAGGGGTTCGAATCC-3' (AC2). The minihelix was constructed by the deletion of 19 nucleotides of plasmid pKR-320AC using the oligonucleotide 5'-CACTATAGTCCCCTAGGGGTTCGAATC-3'. All introduced mutations were verified by complete DNA sequence determination.

View larger version (27K): [in this window] [in a new window]   FIG. 1. Two-dimensional representation of the tertiary structure of the tRNA molecules employed in this study. A, E. coli tRNAGlu, tertiary base pairs, and base triplets (taken from Ref. 7) are indicated by dotted lines. The distinct domains of the tRNA are indicated. Numbering of nucleotides in tRNAs is according to Sprinzl et al. (45) B, two-dimensional model of the truncated tRNA structure of AC2. The linker AAUA was designed to facilitate folding of the tertiary core comprising interactions of the D-loop and T-loop. C, structure and sequence of the investigated minihelix comprising the T-stem/loop and the acceptor stem.

Aminoacylation of tRNA^Glu—The GluTR substrate [¹⁴C]Glu-tRNA^Glu was prepared in 100 or 500 µl aminoacylation reaction mixtures containing 30 mM Na-HEPES, pH 7.5, 15 mM MgCl₂, 25 mM KCl, 3 mM dithiothreitol, 4 mM ATP, and 40 µM [¹⁴C]glutamate (10 µCi) with a specific activity of 284 mCi/mmol (10.5 GBq/mmol). The tRNA transcript concentrations ranged from 1 to 20 µM and GluRS concentrations varied from 400 nM to 8 µM depending on the specific tRNA variant. Minimalist tRNA substrates were denatured at 90 °C for 3 min and quickly cooled on ice to facilitate RNA folding.

GluTR Assay—As GSA, the product of GluTR catalysis, is highly unstable under physiological conditions and difficult to isolate in reproducible quantities, GluTR activity was measured by the substrate depletion assay, which is currently the most reliable method of analysis (24). This assay determines the decrease of [¹⁴C]Glu-tRNA^Glu substrate during GluTR catalysis and identifies the formed GSA product via high performance liquid chromatography (HPLC) analysis. The GluTR assay mix (100 µl) contained 50 nM purified recombinant E. coli GluTR in 30 mM Na-HEPES, pH 8.1, 10 mM MgCl₂, 15 mM KCl, 20% (v/v) glycerol, 1 mM dithiothreitol, 100 µg of bovine serum albumin, 2 mM NADPH, and 2 µM [¹⁴C]Glu-tRNA^Glu. Duplicate reactions were performed using 100 and 200 nM final GluTR concentrations. The reactions were started by the addition of [¹⁴C]Glu-tRNA^Glu, incubated at 37 °C, and stopped by pipetting aliquots of 20 µl from the assay mixture onto Whatman 3MM filters at fixed time points of 15-, 30-, 45-, 60-, and 180-s reaction times. The tRNA was precipitated, washed, and quantified as described previously (27). Reaction assays without GluTR for each employed condition served as background controls for spontaneous substrate hydrolysis (5% after a 3-min reaction time). As some of the mutations in tRNA^Glu also affected glutamylation, the amounts of the GluTR substrate, Glu-tRNA^Glu, were limited and did not permit use of the appropriate excess of substrate required for classical Michaelis-Menten kinetics. Therefore, GluTR specific activity was deduced from first-order equation by a method described previously (44).

HPLC Analysis of GluTR Product Formation—Reaction products were analyzed on a Waters Bondapack^TM C18 reversed phase column (3.9 x 150 mm, 125-Å pore size, 10-µm particle diameter) as described previously (24). The reaction product GSA was identified by its specific conversion into 5-aminolevulinic acid using purified E. coli GSA-AM (25).

tRNA Footprinting Using RNase Digestion—The E. coli tRNA^Glu transcript was 3'-end-labeled using published procedures (26) and charged with glutamate as described above. RNase footprintings were performed according to the manufacturer's instruction using RNase V1, RNase T1, and Nuclease S1 purchased from Ambion (Austin, TX). Prior to the reaction 2–3 µg of end-labeled Glu-tRNA^Glu, 0.1–10 µM GluTR, and 10 mM of the GluTR inhibitor -nicotinamide-mononucleotide (reduced form) (Sigma) were incubated at 4 °C for 3 min to allow the formation of the GluTR·tRNA complex. Control experiments omitted either RNase or GluTR. The cleavage pattern of the footprinting reactions was analyzed by electrophoresis on denaturing 15% polyacrylamide gels. The gels were exposed to a Fuji Bas-III imaging screen for 16 h at -80 °C.

Circular Dichroism (CD) and UV Absorption Measurements—CD and UV molecular absorption spectra (195–320 nm) were recorded on a Jasco J-810 spectropolarimeter equipped with Neslab RET-110 temperature control unit. Each tRNA sample (0.3 mg/ml) was initially stabilized at 27 °C for 15 min. Melting experiments were performed at 1 °C increments using a temperature rate of 30 °C/h in the presence of either 10 mM EDTA or 10 mM MgCl₂.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Because both GluTR and GluRS recognize the same tRNA^Glu, we wanted to compare which nucleotides are crucial for recognition. The earlier studies on GluRS identity not only presented a basis for comparison but also provided the large set of in vitro synthesized Glu-tRNA^Glu variants required to identify residues in E. coli tRNA^Glu that are important for E. coli GluTR activity.

Kinetic Analysis of tRNA^Glu Variants—The recognition of Glu-tRNA^Glu by GluTR was examined by the kinetic analysis of unmodified wild type tRNA^Glu (Fig. 1) and 51 tRNA^Glu variants that were prepared by in vitro run-off transcription. All transcripts were aminoacylated with [¹⁴C]glutamate using E. coli GluRS and tested as GluTR substrates in the depletion assay by monitoring the loss of charged tRNA^Glu (24). The formation of the reaction product GSA was verified by HPLC analysis. It was shown previously that E. coli GluTR utilizes fully modified E. coli Glu-tRNA^Glu and unmodified Glu-tRNA^Glu transcripts at comparable catalytic rates (16). The specific activity for this process was measured as 0.47 µmol min^-1 mg^-1 (k_cat = 0.38 s^-1) and was set to 100%. All other values obtained in this study were related to this value. The majority of charged tRNA^Glu transcripts harboring single, double, or triple mutations were utilized by E. coli GluTR without any significant reduction of the catalytic activity (Table I). Interestingly, this set also includes tRNA^Glu transcripts carrying the mutations of bases that are known to be identity elements in tRNA^Glu for recognition by E. coli GluRS (7, 8).

The Unique Tertiary Core Structure of tRNA^Glu Is Recognized by GluTR—The tRNA^Glu variants that led to significantly reduced GluTR activity are summarized in Table II. A set of tRNA^Glu variants harboring mutations in the tertiary core (D-stem/loop, variable loop, and T-stem/loop) was synthesized by T7 RNAP transcription. The U13^*G22^**A46 base triple is formed via a tertiary base pairing between the phylogenetically conserved pyrimidine 13-purine 22 motif (U13^*G22 in tRNA-^Glu) in the D-stem and base 46 (A46) in the variable loop (36) (see Fig. 2C). A tRNA^Glu transcript carrying a mutation of U13–C13, which allows the formation of the Watson-Crick base pair C13^*G22, was still a reasonable substrate for GluTR but led to significantly reduced GluRS activity (7). However, for both GluRS and GluTR mutations of the bases G22 and A46 (to A22, G46, U46) almost abolished substrate utilization. Furthermore the tertiary core of tRNA^Glu is characterized by the lack of residue 47 in the four nucleotide short variable loop. The insertion of U into position 47 decreased the catalytic activity of GluTR as well as of GluRS. As the base triple 13^*22^**46 was shown to play an important role in maintaining the overall folding of the tRNA (34), it seems likely that GluTR recognition strongly depends on the unique core structure of tRNA^Glu. In addition, the lack of residue 47 is thought to stabilize the mentioned base triple via stable positioning of base A46 (35). Furthermore the strictly conserved tertiary base pair U8^*A14, which forms a reverse Hoogsteen pairing configuration, was disrupted in a tRNA^Glu variant, which harbored a mutation of the base U8–C8. The transcript, although charged by GluRS, was only a weak substrate for GluTR (Table II). The finding that the tertiary core structure of tRNA^Glu is recognized by GluTR was supported by in vivo data from E. gracilis. It was shown that a point mutation of tRNA^Glu led to a chlorophyll-deficient phenotype, with a tRNA^Glu variant still participating in chloroplast protein biosynthesis but no longer able to be utilized by GluTR (15). The E. gracilis tRNA^Glu harbored the single mutation of C56–U56. As this base forms a tertiary Watson-Crick base pair with the base G19 it was interesting to test the consequences of this mutation in the E. coli system. We verified that the E. coli tRNA^Glu variant transcript carrying a mutation of the base C56–U56 was efficiently aminoacylated by E. coli GluRS. However, it was a poor substrate for E. coli GluTR. A single mutation of base G19–U19 also prevented utilization by GluTR. Interestingly, the double mutant restoring this tertiary base pair (A19^**U56–G19^**C56) led to nearly full GluTR utilization. Thus the base exchanges C56–U56 and G19–U19 destroy another important feature of the tertiary core of tRNA^Glu, the G19^**C56 base pair (Fig. 2). The most crucial determinant for tRNA^Glu recognition by GluRS (7), the U11^*A24 base pair, which does not form any tertiary interactions, was found to be unimportant for GluTR activity.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Nucleotides of E. coli tRNAGlu involved in E. coli GluTR recognition. A, nucleotides of tRNAGlu transcript affecting GluTR activity (see Table II) are indicated in the L-shaped representation. B, three-dimensional presentation of tRNAGlu highlighting the bases required for GluTR recognition. The bases are positioned according to the crystal structure of Thermus thermophilus tRNAGlu (9). C, the tertiary triple contact U13*G22**A46 present in E. coli tRNAGlu.

View larger version (22K): [in this window] [in a new window]   FIG. 3. CD spectra of unfolding tRNAGlu transcripts. A, the concentration of employed wild type E. coli tRNAGlu transcript was 0.3 mg/ml in distilled water. The CD spectra in the presence of 10 mM EDTA were measured in 1 °C increments, using a temperature increase of 30 °C/h. B, the differential representation of the melting curves for wild type tRNAGlu transcript (solid line) in comparison to the tRNA variant harboring the mutation C56–U56 (dashed line) are based on changes in the ellipticity at the single wavelength 210 nm. The vertical line indicates the melting point of 33 °C of unmodified E. coli tRNAGlu in the presence of 10 mM EDTA.

RNase Footprinting Reveals Glu-tRNA^Glu·GluTR Interactions at the Tertiary Core—RNase footprinting was employed to investigate GluTR·Glu-tRNA^Glu interactions. Initial experiments were hampered by substrate destruction caused by GluTR. In the absence of NADPH GluTR possesses an esterase activity that hydrolyzes the Glu-tRNA^Glu substrate into tRNA^Glu and glutamate. To stabilize the GluTR-bound substrate GluTR esterase activity was inhibited by the NADPH analogue -nicotinamide-mononucleotide (reduced form) as described before (24). We used RNases V1, T1, and nuclease S1 to digest Glu-tRNA^Glu alone and in complex with GluTR to investigate changes in the protection pattern. During the precharging reaction nonspecific cleavage of the transcript at certain weak points was inevitable and might be induced by GluRS (28). These positions were omitted from further analysis. Surprisingly, the addition of up to 100 nM GluTR reproducibly led to increased RNase V1 digestion at the T-stem, D-stem, and parts of the anticodon stem. These findings might indicate conformational changes of the tRNA structure induced by docking to GluTR or alternatively the occurrence of GluTR RNase V1 protein-protein interactions. Thus, changes in the protection pattern of tRNA^Glu were only observed by RNase T1 and nuclease S1 treatment. Protection of the base G10 in the D-stem and the bases G53, U54, and U55 in the T-loop from RNase digestion by GluTR was observed (Fig. 4A). The protection of these positions was verified by digestion of phosphorothioate containing tRNA transcripts (data not shown). Therefore, the direct interaction of GluTR with the tertiary core of tRNA^Glu was demonstrated. In the current model of Glu-tRNA^Glu·GluTR interactions (29), based on the position of glutamycin, which mimics the 3'-end of Glu-tRNA^Glu in the active site pocket, the tRNA^Glu is bound from the inside of the L-shape of the molecule, similar to its binding to GluRS. It is in precisely this region of the tRNA that the protected bases are localized (Fig. 4). The Glu-tRNA^Glu·GluTR model is based on the M. kandleri GluTR structure, which reveals significant catalytic and structural similarities to the E. coli enzyme (16) so that an analogue tRNA orientation is proposed.

View larger version (74K): [in this window] [in a new window]   FIG. 4. RNase footprinting analysis of GluTR-Glu-tRNAGlu interactions. A, RNase footprinting analysis of GluTR·Glu-tRNAGlu interactions via enzymatic cleavage of E. coli tRNAGlu in the presence of various amounts of GluTR and 10 mM of -nicotinamide-mononucleotide. Lane AH, RNA ladder generated by alkaline hydrolysis of tRNAGlu transcript. Lane DM, decade marker (Ambion, Austin, TX) indicating 10 nucleotide intervals. The tRNA was 3'-labeled and digested with RNase V1, RNase T1, and nuclease S1. The numbering for each block of RNase treatment is -, no GluTR added; 1, 0.1 µM GluTR added; 2, 1 µM GluTR added; 3, 10 µM GluTR added. Positions of tRNAGlu protection by GluTR interaction are indicated by arrows. B, nucleotides of E. coli tRNAGlu protected by E. coli GluTR are indicated in the three-dimensional representation and positioned according to the crystal structure of T. thermophilus tRNAGlu (9).

General Discussion—GluTR is an enzyme that uses aminoacyl-tRNA as substrate. Specific recognition of Glu-tRNA by E. coli GluTR is brought about by the unique tertiary core of tRNA^Glu created by the U13^*G22^**A46 base triple, 47, and the tertiary base pair G19^*C56. Neither the anticodon nor acceptor stem are major identity elements, as they are for most aminoacyl-tRNA synthetases. The accuracy of this process is further sustained by the tight coordination of the glutamate part of the substrate in the active site pocket of GluTR.

Do other enzymes that utilize aminoacyl-tRNA as a substrate recognize tRNA in a similar fashion? For instance, E. coli methionyl-tRNA^fMet formyltransferase, the enzyme required for formylating the initiator tRNA, requires a cluster of determinants in the acceptor stem and the A11^*U24 base pair in the D-stem (37, 38). Furthermore, similar to GluTR, increased RNase V1 digestion was observed in the anticodon stem, although this region is located distal to the tRNA-protein contacts observed in the co-crystal structure (39). This observation was explained by conformational changes of the tRNA structure induced by Met-tRNA formyltransferase binding (40). In addition, enhanced RNase V1 cleavage in the anticodon stem was also described for the EF-Tu·Phe-tRNA^Phe·GTP and IF2·fMet-tRNA complexes suggesting changes in anticodon stem conformation even though these proteins interact with the distal acceptor stem (41, 42). It will be interesting to explore conformational changes in Glu-tRNA^Glu structure possibly induced by GluTR binding. Finally, selenocysteyl-tRNA^Sec is an aminoacyl-tRNA substrate specifically recognized by selenocysteine synthase and the SELB translation factor. The unusually long eight-base-pair acceptor stem of E. coli tRNA^Sec was found to be the major recognition determinant for SELB (43). The mechanisms of further enzymes utilizing an aminoacyl-tRNA substrate, e.g. Glu-tRNA^Gln amidotransferase, which transamidates misacylated Glu-tRNA^Gln, are still under investigation. These results suggest that enzymes utilizing aminoacyl-tRNA as substrate interact directly with the aminoacylated acceptor stem and the D-stem, whereas the anticodon domain serves as a major recognition element of aminoacyl tRNA synthetases.

	FOOTNOTES

 
^* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to D. J.) and from the National Institute of General Medical Sciences and the Department of Energy (to D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence may 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{at}trna.chem.yale.edu. ^** To whom correspondence may be addressed: Institute of Microbiology, Technical University Braunschweig, Spielmannstr 7, D-38106 Braunschweig, Germany. Tel.: 49-531-3915801; Fax: 49-531-3915854; E-mail: d.jahn{at}tu-bs.de.

¹ The abbreviations used are: GluRS, glutamyl-tRNA synthetase; CD, circular dichroism; GSA, glutamat-1-semialdehyde; GSA-AM, glutamate-1-semialdehyde-2,1-aminomutase; GluTR, glutamyl-tRNA reductase; HPLC, high performance liquid chromatography; T7 RNAP, T7 RNA polymerase; N^*N, secondary base pair; N^**N, tertiary base pair.

	ACKNOWLEDGMENTS

 
We thank G. Layer, C. Lüer, L. Feng, S. Herring, J. Sabina, J. Rinehart, and J. Yuan for discussions and the generous gift of reagents. We thank F. W. Studier (Brookhaven National Laboratory, Upton, NY) for providing plasmid pAR1219.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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