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J. Biol. Chem., Vol. 279, Issue 40, 41960-41965, October 1, 2004

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Residues Lys-149 and Glu-153 Switch the Aminoacylation of tRNA^Trp in Bacillus subtilis^*

Jie Jia, Xiang-Long Chen, Li-Tao Guo, Ya-Dong Yu¶, Jian-Ping Ding¶, and You-Xin Jin||

From the State Key Laboratory of Molecular Biology, ^¶Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai 200031, China

Received for publication, February 22, 2004 , and in revised form, July 7, 2004.

	ABSTRACT

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Tryptophanyl-tRNA synthetase (TrpRS) consists of two identical subunits that induce the cross-subunit binding mode of tRNA^Trp. It has been shown that eubacterial and eukaryotic TrpRSs cannot efficiently cross-aminoacylate the corresponding tRNA^Trp. Although the identity elements in tRNA^Trp that confer the species-specific recognition have been identified, the corresponding elements in TrpRS have not yet been reported. In this study two residues, Lys-149 and Glu-153, were identified as being crucial for the accurate recognition of tRNA^Trp. These residues reside adjacent to the binding pocket for Trp-AMP and show phylogenic diversities in the charge on their side chains between eubacteria and eukaryotes. Single mutagenesis at Lys-149 or Glu-153 reduced the activity of TrpRS in the activation of Trp. The reduction was less than that caused by the double mutant WBHA (K149D/E153R). It is unusual that E153G had no detectable activity in the activation of Trp unless tRNA^Trp was added to the reaction. In addition, we successfully switched the species specificity of Bacillus subtilis TrpRS recognition of tRNA^Trp. The affinity of WBHA, K149E and E153K to human tRNA^Trp was 31-, 13.5-, and 12.9-fold greater than that of wild type B. subtilis TrpRS, respectively. Indeed WBHA and E153K were found to prefer genuine human tRNA^Trp to their cognate eubacteria tRNA^Trp.

	INTRODUCTION

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The evolutionary independence of eubacteria and eukaryotes ensured the diversity of these kingdoms. Each kingdom had its own synthetic and metabolic systems that precluded its invasion by the other life form. The process of protein synthesis emerged early in evolution. It translates the genetic information of a species and is an ideal system for investigating phylogenic diversity. Aminoacyl-tRNA synthetase, one of the most important enzymes in protein synthesis, catalyzes aminoacylation of its cognate tRNA. The activated tRNA carries its amino acid to the ribosome where it is added to the growing peptide chain. Most eubacterial and eukaryotic aminoacyl-tRNA synthetases cannot efficiently cross-aminoacylate the tRNA from the other species. The substrates in an aminoacylation reaction include amino acids, ATP, and tRNA. Because the 20 amino acids are identical in eubacteria and eukaryotes, the species specificity of the aminoacylation reaction is ensured and regulated by specificities residing in tRNA and/or the aminoacyl-tRNA synthetase. The identity elements in tRNA have been determined to be crucial for species-specific recognition of tRNA (1–4). Based on their contributions to aminoacylation, they have been divided into major and minor elements (5–7).

Previous work established detailed kinetic data for the identity elements in tRNA^Trp.In Bacillus subtilis tRNA^Trp, discriminator G73 and anticodon CCA are major elements whereas A1-U72, G5-C68, and A9 are minor elements. When G73 was mutated to A73, the mutant tRNA^Trp preferred yeast TrpRS¹ to its cognate eubacterial TrpRS (8). Because the identity elements are found mainly in the acceptor-stem of the tRNA, this implies that the acceptor-stem is an operational "RNA code" even older than the anticodon. Exchanges of identity elements between B. subtilis tRNA^Trp and human tRNA^Trp showed that all of the identity elements contribute to the species-specific aminoacylation of tRNA^Trp by TrpRS (9). Eubacterial TrpRS seems to be more sensitive than eukaryotic TrpRS to changes in the identity elements of tRNA^Trp. In a recent SELEX experiment, an aptamer containing three G-C base pairs mimicking G2-C71, G3-C70, and G4-C69 in B. subtilis tRNA^Trp showed high affinity to B. subtilis TrpRS (10). This result supports the hypothesis that the sequences and structures of the tRNA acceptor-stem were crucial for the operational RNA (4). These data also suggest that TrpRS may co-evolve with tRNA^Trp to adapt itself to changes in the operational RNA code for tryptophan. There is little enzymatic data available to support the suggestion that TrpRS contains the corresponding residues that recognize the identity elements in tRNA^Trp. However, this suggestion is supported and promoted by the recently disclosed crystal structure of the TyrRS-tRNA complexes (11–14).

Because of similar conformational and catalytic characteristics, both TrpRS and TyrRS were assigned to aminoacyl-tRNA synthetase class Ic (15). Despite considerable differences in their amino acid sequences, the tertiary structures of TrpRS and TyrRS exhibit unexpected homologies in their catalytic domains and substrate binding pockets (16). Both TrpRS and TyrRS consist of two identical subunits. When the acceptorstem of the tRNA binds to the catalytic center in one subunit, the anticodon loop simultaneously binds to the C-terminal domain in the other subunit (17–20). The acceptor-stem binding domain is near the aminoacyl-AMP pocket (13, 14). Therefore, the activated amino acid is carried conveniently to the 2'-OH of adenosine in the CCA terminus in tRNA. Those residues that interact with the identity elements of tRNA^Tyr have also been identified in TyrRSs. They belong to two helices that are located adjacent to the tyrosine pocket. The helices form a groove to accept the acceptor-stem of the tRNA^Tyr. These findings have promoted ongoing research on B. subtilis TrpRS and are in agreement with our findings (20).

B. subtilis TrpRS is a small protein with a molecular weight of 77,000 (21). As is true of TyrRS, a Rossmann fold containing the conserved sequences HIGH and KMSKS forms its catalytic domain. A series of crystal structures of TrpRS have been solved recently, but the data for the structure of TrpRS-tRNA^Trp remains unavailable (22–25). Our previous study identified residues 108–122 and 234–238 as being crucial for recognition of the acceptor-stem and the anticodon loop of tRNA^Trp, respectively (20). In this research, we have further investigated the recognition mode of the acceptor-stem by B. subtilis TrpRS. By sequence and structural homology analysis, residues Lys-149 and Glu-153 exhibit phylogenic diversities that might have co-evolved with the identity elements in tRNA^Trp in both eubacteria and eukaryotes. Kinetic data obtained with mutant enzymes also revealed the importance of these two residues for species-specific recognition of tRNA^Trp.

	MATERIALS AND METHODS

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Strains, Plasmids, and Reagents—Escherichia coli strain JM109 and BL21-CodonPlus (DE3)-RIL were purchased from Promega (Madison, WI) and Stratagene (La Jolla, CA), respectively. Both the plasmid pKSW1 containing the B. subtilis trpS gene and the plasmid pTrc-hTrpRS containing the human trpS gene were provided by Dr. Xue's laboratory at the Hongkong University of Science and Technology. The expression vector pET24a(+) and pKK223–3 came from Novagen and Amersham Biosciences. Restriction endonucleases, T4 DNA ligase, T-vector, and Large-scale RNA production system were purchased from Promega. Ex Taq and DNA marker DL-2,000 were from Takara Biotech. A low M_r protein marker was produced by Shanghai Lizhu Dongfeng Biotechnology Company. Isopropyl-1-thio--D-galactopyranoside and imidazole was from Sangon, nickel-nitrilotriacetic acid-agarose was from Qiagen (Chatsworth, CA), and DEAE-Sepharose Fast-Flow and L-[5-³H]tryptophan were from Amersham Biosciences.

Sequence Homology Analysis—The protein sequences of TrpRSs were downloaded from the aminoacyl-tRNA synthetases data base (www.pozman.edu.pl/aars) (26). The selected TrpRSs were representative of both eubacteria and eukaryotes. Abbreviations of the species were as follows: A-Af, Archaeoglobus fulgidus; A-Hm, Archaea halobacterium; A-Mj, Archaea mjannaschii; B-Bs, B. subtilis; B-Ec, E. coli; B-Hi, Haemophilus influenzae; B-Rp, Rickettsia prowazekii; E-At, Arabidopsis thaliana; E-Bo, bovine; E-Hs, Homo sapiens; E-Sc, Saccharomyces cerevisiae; EM-Hs, H. sapiens mitochondria; EM-Sc, S. cerevisiae mitochondria. Multiple sequence alignments were carried out with Clustal X1.8 software (ftp-igbmc.u-strasbg.fr/pub/ClustalX/) (27). The phylogenic tree was illustrated with multiple alignment results as described previously (9).

Construction and Purification of the Mutant TrpRSs—Seven B. subtilis TrpRS mutants (WBHA, K149R, K149E, K149G, E153D, E153K, E153G) were constructed to investigate the importance of residues Lys-149 and Glu-153. Mutagenesis was carried out by PCR as described previously (20). After confirmation by DNA sequencing, the correctly mutated trpS genes were cloned into a high level expression system (28). The constructed strains were induced by isopropyl-1-thio--D-galactopyranoside at 0.1 mM to overexpress TrpRS proteins in a soluble form. Purification of these eubacteria TrpRS mutants was carried out with DEAE-Sepharose Fast-Flow according to the method described previously (28). Human TrpRS was purified with nickel-nitrilotriacetic acid-agarose (29). Concentrations of purified proteins were determined with the Bradford reagent (30). Purified TrpRSs were stored at –80 °C until use.

Purification of the tRNA^Trp Transcripts in Vitro—Genes for B. subtilis tRNA^Trp and human tRNA^Trp were synthesized with a Beckmann DNA synthesizer in our laboratory. The transcription template was amplified by PCR and then digested with BstOI to yield the CCA end (20). Subsequent transcription was performed according to the instructions of the RiboMAX Large-scale RNA production system-T7. The tRNA^Trp transcript was purified by 10% PAGE containing 8 M urea. Before the aminoacylation assay, tRNA^Trp was denatured in boiling water for 5 min and slowly cooled to room temperature.

Kinetic Analysis of Trp Activation—The assay was carried out as reported previously (31). The final reaction concentration of TrpRS was 0.1 µM. Other components included 4 mM ATP, 0.02 µCi of [-³²P]ATP, 50 mM Tris-HCl, pH 9.0, 1 mM KF, 0.02% gelatin, 10 mM MgCl₂, 0.1 mM L-Trp, and 4 mM pyrophosphate. Pyrophosphate and TrpRS were added last to initiate the reaction. The sample was incubated at 25 °C for 30 min and rapidly cooled to 0 °C to stop the reaction. 3 µl of the reaction mixture was spotted on polyethyleneimine and developed with 1 M KH₂PO₄ for the final calculation. When the concentration of Trp was changed, ATP was kept at 4 mM. Conversely, when the concentration of ATP was changed, Trp was held constant at 2 mM. For all kinetic assays, the concentration of substrate was changed at least 10-fold. Each reaction was repeated at least four times under the same conditions. The data were fitted to a hyperbola by non-linear regression to yield K_m and k_cat.

Kinetic Determination of Trp Activation in the Presence of tRNA^Trp— Because the catalytic behaviors of WBHA, E153K, and E153G seemed different to that of wild type TrpRS in Trp activation, the kinetic data for these enzymes were further investigated in the presence of tRNA^Trp. The assay and components for the reaction were identical to those mentioned above, except for inactivated tRNA^Trp. Inactivated tRNA^Trp transcripts were produced by oxidation with NaIO₄ for 1 h. The oxidization blocked both the 2'-OH and 3'-OH of adenosine in the CCA terminus, and the inactivated tRNA^Trp was unable to accept the activated Trp. The inactivated tRNA^Trp was incubated with TrpRS before it was added to the reaction. The concentration of the inactivated tRNA^Trp at 8 µM was 80-fold higher than that of TrpRS. The determined data were obtained for Trp activation but not for tRNA^Trp aminoacylation in the presence of the inactivated tRNA^Trp.

Aminoacylation Assay—Recognition of tRNA^Trp by B. subtilis TrpRS or its mutants was determined at 22 °C (20). The reaction buffer consisted of 8 mM ATP, 0.8 mM dithiothreitol, 1.5 µCi of _l-[5-³H]Trp, 8 mM MgCl₂, 120 mM Tris-HCl, pH 7.5, and 0.02 µM tRNA^Trp in a total volume of 50 µl. After 30 min, the reaction was quenched in ice. 20-µl aliquots were spotted onto Whatmann 3-mm filter discs. These discs were washed three times with ice-cold 5% trichloroacetic acid containing 0.05% tryptophan and then with 95% ethanol; then the discs were dried and transferred to vials for determination of radioactive counts. One unit of aminoacylation activity was defined as the amount of enzyme needed to charge 1 pmol of tRNA^Trp per minute under the assay conditions. For all kinetic assays, the concentration of tRNA^Trp varied from 0.02 µM to 1.28 µM; [Trp] was constant at 8 µM. Each reaction was repeated at least four times under the same conditions. k_cat/K_m for aminoacylation was calculated from Eadie-Hofstee plots. All data were fitted to the Michaelis-Menten equation.

	RESULTS

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Charges on Two Residues Display Phylogenic Diversities— Two residues in the E helix of B. subtilis TrpRS have charges opposite to those on the corresponding TrpRS residues in Archaea and eukaryotes (Fig. 1). Lys-149 is a positively charged residue, whereas the corresponding residue in Archaea and eukaryotes is aspartic acid, a negatively charged residue. The residue in both Archaea and eukaryotes corresponding to Glu-153 is arginine, a negatively charged residue. Although the first site showed some changes in mitochondria, the second site in mitochondria was identical to that in eubacteria. Mitochondrial TrpRS is more homologous to eubacteria TrpRS than to eukaryotic TrpRS. Such an evolutionary relationship is also found in other conserved regions, such as HXGH and KMSKS (31).

View larger version (118K): [in this window] [in a new window]   FIG. 1. Multiple sequence alignment of 13 TrpRSs from eubacteria, Archaea, mitochondria, and eukaryotes. Abbreviations are as follows: A-Af, A. fulgidus; A-Hm, A. halobacterium; A-Mj, A. mjannaschii; B-Bs, B. subtilis; B-Ec, E. coli; B-Hi, H. influenzae; B-Rp, R. prowazekii; E-At, A. thaliana; E-Bo, bovine; E-Hs, H. sapiens; E-Sc, S. cerevisiae; EM-Hs, H. sapiens mitochondria; EM-Sc, S. cerevisiae mitochondria. Residues Lys-149 and Glu-153 investigated in this study are marked with red arrows. The predicted tRNA binding helices that are highlighted with pink helix marks are the D helix and the E helix. Sequence alignments were carried out with the software Clustal X1.8, *,: and showed the conservative degree and were automatically added in the output result.

Kinetic Parameters for TrpRS Mutants in Trp Activation—To understand the importance of Lys-149 and Glu-153 in the first-step reaction, K_m and k_cat values were determined for 7 TrpRSs mutants (Table I). WBHA was a double mutant in which Lys-149 and Glu-153 were substituted by Asp-149 and Arg-153, the corresponding residues in human TrpRS. This mutant was designed to insert eukaryotic genetic information into eubacterial TrpRS. Although the conformation of the Trp binding pockets seems similar in different TrpRSs, recent data show that the residues in the Trp pocket vary somewhat between eubacteria and eukaryotes (23–25, 32). When the affinity of TrpRSs to Trp and ATP was determined, k_cat/K_m ratios of WBHA were about 10- and 3.4-fold less than that of wild type B. subtilis TrpRS, respectively. The activity of WBHA was the least among all those determined. K_m showed the largest change among the kinetic constants for Trp. The increased K_m for Trp confirmed that this change resulted in less efficient Trp binding. On the other hand, it supported the idea expressed previously that recognition of Trp differs from eubacteria to eukaryotes.

Glu-153 is located in the second circle of the E helix. Although this location is farther from GXDQ than the distance between Lys-149 and GXDQ, Glu-153 seemed to have a greater effect on Trp activation. Kinetic data show that changes in charge on Glu-153 induced notable increases in V_max and reduced the affinity of TrpRS for the substrates Trp and ATP. The V_max of E153K was 19-fold higher, and the K_m values of E153K for Trp and ATP were 27- and 24-fold higher, respectively, than those of wild type B. subtilis TrpRS. The resulting effect caused by changes of both V_max and K_m was indeed a partial reduction in the activities of E153K. It is shown that activation of Trp must depend on the polarity of the side chain of Glu-153. When Glu-153 was replaced by Gly-153, the ATP-pyrophosphate exchange reaction was too slow to be detected by polyethyleneimine-cellulose chromatography even if the concentration of E153G TrpRS was increased 1000-fold above the normal range. E153G, however, had aminoacylation activity in the recognition of tRNA^Trp (Table II), and it is possible that binding of tRNA helped induce the conformation required for Trp recognition. These observations were consistent with the findings in other class I aminoacyl-tRNA synthetases in which activation of the amino acid did not occur without the help of tRNA (33–35).

tRNA^Trp Molecule Is Helpful for TrpRS to Catalyze Trp Activation—Can the activities of WBHA, E153K, and E153G for Trp activation be recovered with the help of tRNA^Trp? The contribution of the inactivated tRNA^Trp to Trp activation supported our above hypothesis (Table III). The inactivated tRNA^Trp oxidized with NaIO₄ has the structure of tRNA but cannot accept the activated amino acid. When the inactivated tRNA^Trp was added to the reaction, it only affected the reaction by conformational changes to itself and TrpRS. The determined data is limited for Trp activation but not for tRNA aminoacylation. With the help of tRNA^Trp, the affinities of the mutant TrpRSs approach those of wild type TrpRS to Trp and ATP. k_cat/K_m of WBHA to ATP and Trp increased 4.6- and 16.8-fold, respectively. V_max of E153K in the presence of the inactivated tRNA^Trp was 3-fold lower than that determined in the absence of tRNA^Trp, but it was still 2.7-fold higher than that of wild type TrpRS. This result implies that the longer side chain of Lys-153 might interact with other critical residues in TrpRS. The most exciting result is that E153G has detectable activities for Trp activation in the presence of the inactivated tRNA^Trp. The k_cat/K_m values for E153G to ATP and Trp were 0.46 and 7.77 µM^–1 s^–1.

	DISCUSSION

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The switched specificity in the recognition of tRNA^Trp and the null catalysis of E153G in the activation of Trp confirm that Glu-153 contributed significantly to the activity of TrpRS in the two-step reaction. Changing the charge on Glu-153 had a great effect on TrpRS kinetic behavior. In the absence of its cognate tRNA, E153G showed little activity in the activation of Trp. Similar phenomena have been reported only in three Class I aminoacyl-tRNA synthetases, GluRS, GlnRS, ArgRS, and some LysRS-I (33–36). The reason for this had not been identified until Sekine et al. (37) found that the interaction of tRNA^Glu with GluRS caused conformational changes around the ATP-binding site that allowed ATP to bind to the "productive" subsite. Because TrpRS also changes conformation during the aminoacylation reaction, we believe that the reaction was shut down by the negative conformation change of TrpRS during transfer of ATP or Trp (22).

Based on their high homology, B. subtilis TrpRS is likely to be identical to Bacillus stearothermophilus TrpRS in structure and mode of substrate binding. Glu-153 in our study corresponded to Glu-152 in B. stearothermophilus TrpRS. Glu-152 is close to Met-129 and Gln-107 and offered H-bonds to the latter in the B. stearothermophilus Trp-5'AMP complex. If Glu-152 was mutated to Gly-152, the loss of a H-bond promoted the rotation of Met-129 and Gln-107 that subsequently resulted in the mouth of the Trp pocket becoming too narrow for Trp to gain access. In addition, insertion of Gly-152, which made the E helix become flexible, increased the flexibility of the E helix. Gly-144 and Asp-146 are so close to ATP that the transfer of pyrophosphate might be limited. Because the conformation of TrpRS turned into a closed state, E153G could not catalyze activation of Trp as usual. Once tRNA^Trp was added to the reaction, TrpRS could change to an open conformation. Glu-152 interacts more favorably with tRNA^Trp than to Met-129 and the substrate Trp. Here, E153G had a detectable activity for Trp activation again in the presence of the inactivated tRNA^Trp. This partially confirms that the conformation of E153G is induced by the substrate. The long and positively charged lysine side chain of E153K induced an increased V_max and K_m. This can also be explained in the B. stearothermophilus Trp-5'AMP complex. Lysine has two more -CH₂-groups than glutamic acid and a shorter distance between Glu-152, Met-129, and Gln-107. Interactions between residues were enhanced so that the mouth of the Trp pocket remained preferentially in the open status. Therefore, the V_max of E153K was 19-fold higher than that of wild type B. subtilis TrpRS.

Because of high structural homology to TyrRS, the crystal structure of the T. thermophilus TyrRS-tRNA^Tyr complex was used as a comparison to our model of the TrpRS-tRNA^Trp complex. In the model tRNA^Trp is recognized by two helices, D and E (Fig. 2). tRNA^Tyr is also recognized by two helices in TyrRS in which residue Glu-154 is in the 8 helix and residues Arg-198 and Leu-202 are in the 11 helix. Arg-105 in the D helix in TrpRS corresponds to Glu-154 in TyrRS, and the E helix is superimposed favorably onto the 11 helix in TyrRS. In our K149D/E153R construct, two -NH₂-groups in Arg-153 attract the negatively charged tRNA to the groove. Asp-149 partially pushed G73 close to the D helix and still offered H-bonds to tRNA. This moved tRNA in the groove back a little from its original location. This also slows the transfer of acyl to tRNA, and the k_cat of TrpRS decreased 44 times in the aminoacylation of tRNA by WBHA. In all mutants, K149R was the only one with a lower K_m than that of wild type B. subtilis TrpRS. Substitution by arginine attracts tRNA more closely to the Trp pocket; however, it also prevents tryptophanyl-tRNA from being released. The net result is that the k_cat/K_m value of K149R is 50% less than that of wild type TrpRS.

View larger version (66K): [in this window] [in a new window]   FIG. 2. Modeling structure of the acceptor-stem of tRNA bound to the groove adjacent to GXDQ. tRNATrp is shown in stick mode. The D helix and the E helix are highlighted in yellow and green, respectively. The side chain of crucial residues Arg-105, Lys-149, and Glu-153 are shown and titled in the structure. Glu-153 interacted with both A1-U72 and G73. Lys-149 offered H-bond only to G73.

Because the CP1 domain in TrpRS was too short to have an editing function found in other aminoacyl-tRNA synthetases (38, 39), species-specific recognition of tRNA^Trp became more important than ever. Without a structure for the TrpRS-tRNA^Trp complex, we cannot directly observe which residues interact with tRNA and confer species-specific recognition in TrpRS. The biochemical data presented in this study offer evidence that Lys-149 and Glu-153 correspond to the identity elements in tRNA^Trp and function as species-specific residues in B. subtilis TrpRS. Changing these residues would lead to a switch in the species-specific recognition of tRNA^Trp. In addition, phylogenic diversities in TrpRS imply that the co-evolution with its cognate tRNA^Trp was important for maintaining the fidelity of aminoacylation of tRNA.

	FOOTNOTES

 
^* This work is supported by Grant 2002CB512803 from State Key Programs Basic Research of China, Grant 30370325 from the National Natural Science Foundation of China, and Grant 02DJ14004 from the Shanghai Council of Science and Technology. 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.

These authors contributed equally to this work.

^|| To whom correspondence should be addressed: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Yueyang Road No. 320, Shanghai 200031, China. Tel.: 86-21-5492-1122; Fax: 86-21-5492-1011; E-mail: yxjin{at}sunm.shcnc.ac.cn.

¹ The abbreviation used is: RS, tRNA synthetase.

² J. Jia, L.-T. Guo, Y. Shi, X.-L. Chen, H. Xue, and Y.-X. Jin, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Dr. Tze-Fei Wong and Dr. Hong Xue for their comments. The plasmid pKSW1 and pTrc-hTrpRS are gifts from Dr. Hong Xue. We thank Dr. M. Steel and Dr. Sheng-Xiang Lin for modifying the manuscript in English.

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