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J. Biol. Chem., Vol. 279, Issue 31, 32151-32158, July 30, 2004

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Leucyl-tRNA Synthetase from the Hyperthermophilic Bacterium Aquifex aeolicus Recognizes Minihelices^*

Min-Gang Xu, Ming-Wei Zhao, and En-Duo Wang

From the State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China

Received for publication, March 18, 2004 , and in revised form, May 10, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Aminoacylation of the minihelix mimicking the amino acid acceptor arm of tRNA has been demonstrated in more than 10 aminoacyl-tRNA synthetase systems. Although Escherichia coli or Homo sapiens cytoplasmic leucyl-tRNA synthetase (LeuRS) is unable to charge the cognate minihelix or microhelix, we show here that minihelix^Leu is efficiently charged by Aquifex aeolicus synthetase, the only known heterodimeric LeuRS (-LeuRS). Aminoacylation of minihelices is strongly dependent on the presence of the A73 identity nucleotide and greatly stimulated by destabilization of the first base pair as reported for the E. coli isoleucyl-tRNA synthetase and methionyl-tRNA synthetase systems. In the E. coli LeuRS system, the anticodon of tRNA^Leu is not important for recognition by the synthetase. However, the addition of RNA helices that mimic the anticodon domain stimulates minihelix^Leu charging by -LeuRS, indicating possible domain-domain communication within -LeuRS. The leucine-specific domain of -LeuRS is responsible for minihelix recognition. To ensure accurate translation of the genetic code, LeuRS functions to hydrolyze misactivated amino acids (pretransfer editing) and misaminoacylated tRNA (posttransfer editing). In contrast to tRNA^Leu, minihelix^Leu is unable to induce posttransfer editing even upon the addition of the anticodon domain of tRNA. Therefore, the context of tRNA is crucial for the editing of mischarged products. However, the minihelix^Leu cannot be misaminoacylated, perhaps because of the tRNA-independent pretransfer editing activity of -LeuRS.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Aminoacyl-tRNA synthetases (aaRSs)¹ establish the genetic code by catalyzing the esterification of cognate amino acids to their specific transfer RNAs (tRNA) that bear the corresponding anticodons, which are defined by the genetic code (1). Aminoacylation of tRNA catalyzed by aaRSs is a two-step reaction: (a) activation of amino acids with ATP by formation of aminoacyl adenylate and (b) transfer of the aminoacyl moiety from the aminoacyl adenylate to the cognate tRNA substrate. On the basis of the architecture of the conserved active site domain, aaRSs are divided into two groups (2). Class I enzymes have an active site based on the Rossmann fold, whereas the conserved active core of class II enzymes contains an antiparallel -sheet flanked by -helices.

One hypothesis about the assembly of aminoacyl-tRNA synthetases is that the enzymes are assembled in a modular fashion, incorporating new domains around the conserved active site representing ancestral aaRS such as the tRNA-binding domain and editing domain (3). Interestingly, two domains of tRNA (specifically the amino acid-accepting stem (minihelix) and anticodon stem biloop (SBL)), which interact with the aminoacylation active site and tRNA-binding domain of aaRS, respectively, may have arisen independently (4). Chargeable minihelices and smaller helices (e.g. microhelices of 7 bp) mimicking the acceptor stem have been identified in more than 10 aaRS systems (5). Thus, whereas it is recognized that aaRS is in contact with the tRNA anticodon domain containing the genetic code, the acceptor stem often contains determinants sufficient for specific aminoacylation and constitutes an operational RNA code for amino acids (5).

To maintain the fidelity of protein biosynthesis, aaRSs must develop mechanisms to distinguish between amino acids that are structurally similar with reasonable accuracy. Fersht (6) proposed a "double sieve" model for two-step amino acid selection composed of a "coarse sieve" excluding amino acids with side chains larger than those of the cognate one and a second "fine sieve" hydrolyzing misactivated amino acids (pretransfer editing) or misaminoacylated tRNA (posttransfer editing). The net result of either editing pathway is an abortive cycle of non-cognate amino acid activation followed by tRNA-dependent ATP hydrolysis. A number of aaRSs have evolved proofreading mechanisms, including the class I enzymes (isoleucyl-tRNA synthetase (IleRS), valyl-tRNA synthetase (ValRS), and leucyl-tRNA synthetase (LeuRS)) and class II enzymes (threonyl-tRNA synthetase (ThrRS), alanyl-tRNA synthetase (AlaRS), and prolyl-tRNA synthetase (ProRS)) (7). The class Ia enzymes (IleRS, ValRS, and LeuRS) are partially related and possess similar editing domains, specifically, an unusually large insertion splitting the ATP-binding fold (termed connective peptide 1) (8–10).

LeuRS from the deep-rooted bacterium Aquifex aeolicus is the only known heterodimeric synthetase with a 634-amino acid -chain and 289-amino acid -subunit (thus denoted -LeuRS) (11). All other known LeuRSs are monomeric in structure. Despite the relatively high sequence identity between A. aeolicus and Escherichia coli LeuRSs, significant differences are observed at the insertion domain (leucine-specific domain) corresponding to a split domain in -LeuRS (see Fig. 1). Genes encoding -LeuRS and the -subunit alone were expressed in E. coli, and the -subunit bound cognate or non-cognate tRNAs (11). Data from another study further indicate that the nonconserved leucine-specific domain of -LeuRS may interact with the acceptor stem of tRNA^Leu (12).

View larger version (30K): [in this window] [in a new window]   FIG. 1. Schematic diagram of the domain structures of E. coli LeuRS, -LeuRS, SLeuRS, SLeuRS', and DLeuRS'. The diagram is based on sequence alignments and the three-dimensional structure of Thermus thermophilius LeuRS. The different structural domains are indicated as reported previously (11, 12).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—L-Leucine, L-isoleucine, L-norvaline, 5'-GMP, ATP, GTP, CTP, UTP, tetrasodium pyrophosphate, inorganic pyrophosphatase, and dithiothreitol were purchased from Sigma. DE81 filters were obtained from Whatman. L-[³H]Leucine (1 mCi/ml), L-[³H]isoleucine (1mCi [PDB] /ml), [-³²P]ATP (10 mCi/ml, 3000 Ci/mmol), and [³²P]tetrasodium pyrophosphate are products of Amersham Biosciences. Kinase, ligase, ribonuclease inhibitor, and restriction endonucleases were obtained from Sangon Company. T7 RNA polymerase was purified from an E. coli overproducing strain in our laboratory (15). A. aeolicus tRNA^Leu was isolated from an overproducing strain constructed in our group (11). E. coli LeuRS, -LeuRS, -subunit, SLeuRS (the fusion of the two and peptides from A. aeolicus as a single chain analogous to canonical LeuRS), SLeuRS' (a fusion protein with an -like subunit from E. coli LeuRS and the -subunit of -LeuRS), and DLeuRS' (a mixed heterodimer with an -like subunit from E. coli LeuRS and the -subunit of -LeuRS) were purified to homogeneity in SDS-PAGE by two-step chromatography in our laboratory (12). Their domain structures are shown in Fig. 1.

RNA Substrate Preparation—Plasmids containing A. aeolicus and E. coli tRNA^Leu genes were prepared using procedures described previously (11). T7 transcripts were generated in a reaction mixture containing 40 mM Tris-HCl, pH 8.0, 5 mM dithiothreitol, 10 mM MgCl_2, 2 mM nucleotide triphosphates, 15 mM 5'-GMP, 0.5 unit/ml inorganic pyrophosphatase, and 2 mg/ml pure T7 RNA polymerase. Transcripts were purified by 15% (w/v) denaturing PAGE, annealed by heating for 2 min in 5 mM MgCl₂ at 80 °C, and allowed to cool slowly to 30 °C.

Minihelices derived from E. coli or A. aeolicus tRNA^Leu were synthesized by in vitro transcription of single-stranded synthetic templates (16, 17). The transcription system was similar to that described above, except that 4 mM nucleotide triphosphates and 2 mM spermidine were added. RNA helices were purified by 20% (w/v) denaturing PAGE, annealed by heating for 3 min in 2 mM MgCl₂ at 85 °C, and allowed to cool slowly to 30 °C. The final concentration of each RNA product was calculated by UV spectroscopy at 260 nm using the appropriate extinction coefficients (18).

Thermal melting curves for minihelices in 25 mM Tris-HCl, pH 6.8, and 10 mM MgCl₂ were obtained by monitoring the change in absorbance at 260 nm over a temperature range of 30–90 °C. This was performed using a Beckman DU 7400 UV-visible spectrophotometer equipped with a temperature controller. The thermal transition midpoint (T_m) was estimated from the first derivative of the melting profile.

Aminoacylation Assays—ATP-PP_i exchange activities of LeuRS were measured at 37 °C as described (11). The aminoacylation activity for tRNA was determined at 37 °C in a reaction mixture containing 100 mM Tris-HCl, pH 7.8, 30 mM KCl, 12 mM MgCl₂, 4 mM ATP, 0.5 mM dithiothreitol, 10 µM tRNA^Leu, 70 µM ¹⁴C-labeled amino acids, and enzyme (11). Enzyme kinetic parameters were determined in the presence of 40 nM -LeuRS and tRNA transcripts concentrations from 0.1 to 80 µM. The kinetic constants were derived from a Lineweaver-Burk plot and were the average data of at least two independent experiments.

Aminoacylation assays for minihelices were performed in a 70-µl reaction mixture containing 25 mM Tris-HCl, pH 6.8, 10 mM MgCl₂, 1 mM ATP, 1.3 µM L-[³H]leucine (1 mCi/ml) or 1.2 µM L-[³H]isoleucine (1 mCi/ml), 15 µM minihelix, 0.05 unit/ml inorganic pyrophosphatase, and 2 µM LeuRS. A low concentration of amino acid was used for maximum sensitivity as reported (19). The aminoacylation of minihelices depended on the synthetase concentration, ionic strength, and pH value (data not shown). Under the optimized conditions, the aminoacylation of minihelices was determined. At time intervals, 9-µl aliquots were spotted on a Whatman DE81 filter for 5 min before washing with 6% trichloroacetic acid as described previously (20). Plateau experiments were performed at 37 °C with transcripts at 5 and 1 µM -LeuRS. No RNA controls were employed to correct for background rates (less than 5% of the Aa-minihelix^Leu charging).

Measurement of ATP Consumption in Editing—Assays measuring overall tRNA-dependent editing were essentially performed as reported previously (21). Assays with minihelices or tRNA were performed at 60 °Cina70-µl reaction mixture containing 30 mM Tris-HCl, pH 6.8, 12 mM MgCl₂, 5 mM dithiothreitol, 1 unit/µl ribonuclease inhibitor, 2 units/ml inorganic pyrophosphatase, 2 mM [-³²P]ATP (50–70 cpm/pmol), 15 mM norvaline, 15 µM Aa-minihelix or A. aeolicus tRNA^Leu, and 1 µM -LeuRS. Aliquots (10 µl) of the editing reaction mixture were mixed with 350 µl of quenching liquid containing 6% activated charcoal, 7% HClO₄, and 10 mM tetrasodium pyrophosphate. After centrifugation, the amount of inorganic phosphate (³²P) in 50 µl of supernatant was quantified by scintillation counting. The amount of ATP hydrolysis was calculated from the inorganic phosphate (³²P) released in 50 µl of supernatant; the present values were the average from three independent determinations. Background ATP hydrolysis in the absence of RNA was performed for each determination.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Design and Construction of RNA Substrates—The L-shaped tRNA comprises two domains, specifically, the acceptor-TC minihelix with an amino acid attachment site at the 3' terminus and the anticodon stem biloop with one of the loops encoding the anticodon triplets. The secondary structures of whole E. coli, A. aeolicus tRNA^Leu, and RNA molecules designed as minimalist substrates are shown in Fig. 2. The SBLs of E. coli and A. aeolicus tRNA^Leu are presented in Fig. 2, A and B. In Fig. 2B, AL and DL are the hairpin helices corresponding to the anticodon stem loop and D-stem loop of A. aeolicus tRNA^Leu, respectively. Mutations in tRNA and RNA molecules are specified by arrows. Eight minihelices were derived from the amino acid-accepting branches of E. coli and A. aeolicus tRNA^Leu and E. coli tRNA^Ser (Fig. 2C). Of these, the first five were derived from A. aeolicus (denoted Aa-), and the last three were derived from E. coli (designated Ec-). These include Aa-minihelix^Leu, Aa-G1-U72 minihelix^Leu withaCtoU mutation at position 72, Aa-1-C72 minihelix^Leu missing the 5'-terminal guanine, Aa-A73G minihelix^Leu with an A to G mutation at discriminator position 73, Aa-1-C72 DNA minihelix^Leu, Ec-minihelix^Leu, Ec-minihelix^Ser derived from E. coli tRNA^Ser (CAA), and Ec-G73A minihelix^Ser with a G to A mutation at discriminator position 73. Minihelices were prepared by in vitro transcription.

View larger version (33K): [in this window] [in a new window]   FIG. 2. Sequences of RNA substrates for aminoacylation and editing assays. A, the structure of E. coli tRNALeu consists of two helical domains arranged in an L shape. The dotted lines denote tertiary interactions. SBL contains a D-loop, anticodon loops, and the anticodon stem of E. coli tRNALeu. Mutations in the D-loop of SBL that enhance tertiary interactions with the TC loop are indicated by an arrow. B, structures of A. aeolicus tRNALeu, SBL fragment, anticodon stem/loop of A. aeolicus tRNALeu (AL), and D-stem/loop of A. aeolicus tRNALeu (DL) loop. Mutations in whole tRNA or the SBL fragment are signified by arrows. C, minihelices derived from tRNAs as indicated. Aa- and Ec-are the abbreviated forms of A. aeolicus and E. coli.

View larger version (21K): [in this window] [in a new window]   FIG. 3. -LeuRS recognizes minihelices derived from E. coli or A. aeolicus tRNALeu. Under the same conditions, E. coli LeuRS does not recognize these minihelices. The reactions were assayed at 25 °C with 15 µM minihelix and 2 µM -LeuRS.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Enhancement of minihelixLeu aminoacylation by destabilization of the first base pair. The figure shows the time course of aminoacylation of the Aa-minihelixLeu, Aa-G1-U72 minihelixLeu, Aa-1-C72 minihelixLeu, and Ec-minihelixLeu. The reactions were performed at 25 °C with a 15 µM concentration of various minihelices and 2 µM -LeuRS.

View larger version (15K): [in this window] [in a new window]   FIG. 5. Temperature profile for aminoacylation of Aa-minihelixLeu. The time course of leucine incorporation was performed at 25, 40, and 65 °C with 15 µM Aa-minihelixLeu and 2 µM -LeuRS.

Aminoacylation of Minihelices Is Dependent on the Determinant Base A73—To assess the specificity of leucylation of the minihelix^Leu, we synthesized Aa-A73G minihelix^Leu (Fig. 2C) in which the base at the discriminator position was altered and subjected it to an aminoacylation assay. The Aa-A73G minihelix^Leu was not charged at a detectable level even at 60 °C (Fig. 6). The same substitution in native tRNA^Leu led to an over 80-fold decrease in the k_cat/K_m value (Table I), suggesting similar recognition mechanisms between minihelices and tRNA.

View larger version (19K): [in this window] [in a new window]   FIG. 6. Aminoacylation of minihelices is strongly dependent on the presence of the A73 "discriminator" identity nucleotide. The replacement of the A73 with G73 totally abolishes aminoacylation of Aa-minihelixLeu even at 60 °C. The Ec-minihelixSer derived from E. coli tRNASer is not a substrate for -LeuRS. However the mutant Ec-G73A minihelixSer is a substrate at 60 °C. The reaction was performed at 60 °C with 15 µM minihelix and 2 µM -LeuRS.

DNA analogues serve as active substrates for RNase P or tRNA synthetases (26, 27). Moreover, the tDNA minihelix is recognized by a CCA-adding enzyme (28). Accordingly, we speculated whether -LeuRS recognizes the tDNA minihelix. To investigate this possibility, we constructed the DNA form of Aa-1-C72 minihelix^Leu (the best substrate among the minihelix variants) (Fig. 2C) and examined for aminoacylation. -LeuRS did not aminoacylate the Aa-1-C72 DNA minihelix^Leu (data not shown). The data indicate that the A form of the helix is important for aminoacylation.

Aminoacylation of tRNA^Leu Acceptor Minihelix by -LeuRS in the Presence of Small RNA Helices—For ValRS and IleRS, which directly interact with the anticodon domain of tRNA, a weak stimulatory effect on minihelix charging was observed when an anticodon stem loop was added to the minihelix reaction (20, 29) as small RNA helices may induce the active conformation of enzymes. In contrast, E. coli LeuRS does not recognize the anticodon loop of tRNA^Leu, and the minimal RNA molecule required for leucylation is the minihelix structure with the D-stem loop (13). Therefore, we examined whether the addition of the isolated SBL domain of tRNA^Leu into the reaction stimulated leucylation of the acceptor minihelix. Charging of the Ec-minihelix^Leu by E. coli LeuRS was not detected even following the addition of Ec-SBL or its derivate containing mutations in the D-loop to enhance the hydrogen bond between TC in the minihelix and D-loop in SBL (Fig. 2A and data not shown). However, the addition of the Aa-SBL (Fig. 2B) fragment of A. aeolicus tRNA^Leu slightly enhanced the charging of the Aa-minihelix^Leu by -LeuRS (Fig. 7). Leucylation of the acceptor minihelix was additionally stimulated after the addition of a hairpin helix mimicking the anticodon arm and loop (Aa-AL) (Fig. 2B) of A. aeolicus tRNA^Leu to the aminoacylation reaction (Fig. 7). This stimulatory effect seems to be sequence-specific because the replacement of anticodon nucleotide 35A with G (Aa-A35G SBL, Fig. 2B) eliminated the stimulatory effect of Aa-SBL (Fig. 7). Previous footprinting analyses using cytoplasmic LeuRS with Phaseolus vulgaris, yeast, and E. coli tRNA^Leu revealed that the D-arm and hinge region are in contact with the enzyme (30). However, our present data indicate that the D-loop alone (Aa-DL) is an inhibitor of minihelix charging (Fig. 7).

View larger version (21K): [in this window] [in a new window]   FIG. 7. Aminoacylation of the minihelices in the absence and presence of small RNA helices. Aminoacylation was performed at 25 °C using a 15 µM concentration of Aa-minihelixLeu, 2 µM -LeuRS, and 15 µM Aa-SBL, Aa-A35G SBL, anticodon stem/loop, or D-stem/loop as indicated.

A Possible Role of the Leucine-specific Domain of -LeuRS in Minihelix Recognition—According to the sequence alignment, one significant difference between the A. aeolicus and E. coli LeuRSs is an insertion domain designated "leucine-specific domain," which corresponds to the split domain in A. aeolicus LeuRS (Fig. 1). Our previous data suggest that the leucine-specific domain interacts with the acceptor stem of tRNA^Leu at the aminoacylation step (12). Therefore, three enzymes were assembled with aminoacylation activity from -LeuRS and E. coli LeuRS. Specifically, SLeuRS, SLeuRS', and DLeuRS' were tested for the ability to charge the Aa-1-C72 minihelix^Leu (the best substrate among the minihelices). Although SLeuRS' and DLeuRS' maintained 8.4 and 4.2% aminoacylation activity of the native enzyme using native tRNA^Leu (12), the two assembled enzymes failed to charge the minihelix (Fig. 8). The single peptide, SLeuRS, displayed slightly higher (110%) tRNA aminoacylation activity and more resistance to heat denaturation than the original -LeuRS (12). However, its ability to charge the minihelix was weakened (Fig. 8), and a relative lower plateau value of minihelix charging was observed as compared with that of -LeuRS (data not shown). The possible minor conformation change by the linkage of split leucine-specific domain seems to be responsible for the relatively poor recognition of minihelix. Thus the exact conformation of the leucine-specific domain is important for charging the minihelix. Although the -subunit with a partial leucine-specific domain binds tRNA^Leu (11), the addition of excess free -subunit (>10-fold more than -LeuRS) into the above reaction did not affect aminoacylation of the minihelix (data not shown), suggesting that the partial leucine-specific domain on the -subunit does not interact with the acceptor stem of tRNA^Leu and the -subunit cannot induce a conformation of -LeuRS to recognize the minihelix more efficiently. Additionally, the -subunit alone is unable to catalyze the aminoacylation reaction (31). Thus, the -or -subunit alone is not sufficient for minihelix recognition.

View larger version (17K): [in this window] [in a new window]   FIG. 8. The effect of disorder of the leucine-specific domain on aminoacylation of the minihelix. This figure shows a comparison of aminoacylation of minihelixLeu with -LeuRS, SLeuRS', DLeuRS', and SLeuRS. Reactions were assayed at 25 °C with 15 µM Aa-1-C72 minihelixLeu and a 2 µM concentration of various enzymes.

Because all of the known editing reaction by LeuRSs was trigged in the presence of tRNA^Leu, reactions without RNA were used as a background control reaction. The addition of A. aeolicus tRNA^Leu to a reaction mixture of LeuRS, ATP, and norvaline led to ATP consumption. However, minihelix^Leu was unable to stimulate the hydrolytic editing of misactivated norvaline under similar conditions as shown in Fig. 9. The addition of an isolated Aa-SBL did not enhance editing (Fig. 9). Covalent bonds between the domains of tRNA should be important for editing.

View larger version (17K): [in this window] [in a new window]   FIG. 9. Aa-minihelixLeu does not induce an editing response, even upon addition of the SBL fragment. Assays were performed with 15 mM norvaline, 2 mM [-32P]ATP, 1 µM -LeuRS, and a 15 µM concentration of various RNAs at 60 °C, pH 6.8. Reactions with no RNA were used as background controls.

View larger version (14K): [in this window] [in a new window]   FIG. 10. RNA-independent pretransfer editing of -LeuRS. The non-cognate amino acid, norvaline, stimulates ATP hydrolysis by -LeuRS. ATP hydrolysis activity of -LeuRS (1 µM) was assayed in the presence of 2 mM [-32P]ATP, 15 mM norvaline or leucine without RNA at 60 °C. Reactions with no RNA and amino acid were used as background controls.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In more than 10 minihelix aminoacylation systems, efficient charging often occurs when major identity elements are included mainly within the minihelix itself (e.g. in AlaRS, glycyl-tRNA synthetase (GlyRS), histidyl-tRNA synthetase (HisRS), and tyrosyl-tRNA synthetase (TyrRS) systems) (16, 19, 37, 38). In contrast, for tRNAs with identity elements distributed over the whole structure (especially the anticodon position), aminoacylation of the minihelix is generally weak as shown for the IleRS, ValRS, and methionyl-tRNA synthetase (MetRS) systems (20, 29, 39). A. aeolicus tRNA^Leu belongs to the type II tRNA group with an extra variable loop, and its anticodon loop is not necessary for aminoacylation as E. coli tRNA^Leu (40, 41). However, the aminoacylation level of minihelix^Leu is low. In the leucine system studied here, the most efficient Aa-1-C72 minihelix^Leu is estimated to be 10⁶-fold less active than A. aeolicus tRNA^Leu at 37 °C (data not shown). The plateaus of minihelices charging are not more than 5% (Table I). It has been suggested that the tertiary structure of tRNA^Leu may be important for correct positioning of the acceptor stem for entering the aminoacylation pocket (25), which is deficient in the interaction between the minihelix and LeuRS.

For the E. coli MetRS and IleRS systems (which are also grouped into aaRS class Ia), destabilization of the first 1:72 base pair activates aminoacylation of the minihelices (42). In view of the importance of the anticodon for efficient aminoacylation of IleRS or MetRS systems, built-in helix destabilization in the minihelix may compensate in part for the lack of presumptive anticodon-induced acceptor stem distortion. For tRNA^Val, the most efficient identity determinants are located within the tRNA anticodon loop (41). In accordance with this finding, the addition of a hairpin helix that mimics the anticodon arm may stimulate aminoacylation of the acceptor minihelix (20). Our results indicate that minihelix^Leu aminoacylation is additionally activated by the destabilization of the first base pair. Significantly, unlike E. coli LeuRS, the binding of the anticodon domain of A. aeolicus tRNA may induce domaindomain communication between the aminoacylation domain and tRNA-binding domain within -LeuRS, which stimulates the minihelix charging, although this is effective only under specific conditions, and the anticodon loop may not be the identity element for whole tRNA^Leu. The donation of the domain-domain communication can be replaced by raising the temperature or artificial modification of the first base pair of minihelices, both of which induce destabilization of the first base pair. Therefore, despite the observed communication, -LeuRS has developed a mechanism to recognize the tRNA; this mechanism depends on the tertiary structure of the tRNA.

A structural comparison of the E. coli and A. aeolicus LeuRS enzymes reveals different structures and sizes of the leucine-specific domain, which is crucial for the stable charge of tRNA possibly via interactions with the acceptor stem of tRNA (12). Although the fusion protein, SLeuRS, has similar tRNA-charging activity as heterodimeric -LeuRS at temperatures ranging from 30 to 60 °C (12), the minihelix charging ability is weakened. The small surrounding interference caused by linkage of the original split leucine-specific domain may account for the differences with wild-type -LeuRS in minihelix recognition. The similar tRNA charging activity between -LeuRS and SLeuRS suggests that interactions within the tertiary structure of tRNA and LeuRS may compensate for the relatively poor binding between the minihelix acceptor stem and the leucine-specific domain induced by the linkage of two split leucine-specific domains.

To maintain a high accuracy of amino acid selection, some aaRSs have developed pretransfer editing and posttransfer editing pathways for hydrolyzing misactivated and misaminoacylated products, respectively. In contrast to tRNA^Ile, minihelix^Ile is unable to trigger the hydrolysis of misactivated valine and misaminoacylated with non-cognate amino acids, although the mischarged minihelix^Ile may be deacylated by E. coli IleRS (43, 44). Previous studies additionally demonstrate tertiary structure interactions between D- and TC-loops or crucial identities within these structures that play important roles in the editing of ValRS, LeuRS, or IleRS (21, 34, 45). Similar to class I IleRS, class II AlaRS needs the entire tRNA structure to induce editing (46). Our results show that minihelix^Leu cannot induce an editing response to non-cognate amino acids even following the addition of the SBL domain of tRNA, suggesting that continuity of the tRNA structure is also crucial for the tRNA-dependent posttransfer editing of -LeuRS. For -LeuRS, proof of tRNA-dependent posttransfer editing was obtained from tRNA-dependent ATP hydrolysis and direct hydrolysis of mischarged tRNA². However, minihelices^Leu cannot be mischarged, which may be explained by tRNA-independent pretransfer editing of -LeuRS. For most known aaRS editing systems, both pretransfer and posttransfer editing are induced by the binding of tRNA to facilitate an editing complex conformation for aaRS even for pretransfer editing, which has no covalent linkage with tRNA (8, 9, 47–49). However, some reports show that E. coli ProRS displays tRNA-independent pretransfer editing and yeast cytoplasmic LeuRS exhibits only pretransfer editing (36, 50).

Structural information from crystals of ValRS and IleRS indicates that in the tRNA-free forms of the above two enzymes, the editing pocket in the connective peptide 1 domain does not face toward the aminoacylation pocket. However, the binding of cognate tRNA induces rotation of the connective peptide 1 domain and formation of a narrow gap between the two active sites. Therefore, misactivated amino acids (pretransfer editing substrates) are translocated (8, 9, 51, 52). However, the Thermus thermophilius LeuRS structure reveals a different scene, where the editing pocket has faced toward the aminoacylation pocket even in the absence of tRNA (10, 53). In this case, at least in some species of LeuRSs the possibility for tRNA-independent pretransfer editing seems plausible.

Bearing in mind that the minihelix is the possible ancestor RNA for aminoacylation, and A. aeolicus is located near the root of the life tree, our first report of tRNA-independent editing response of -LeuRS suggests that pretransfer editing has occurred earlier than posttransfer editing, thus ensuring the relatively specific synthesis of the Leu-minihelix (at least for the LeuRS system). With the formation of whole tRNA significantly increasing aminoacylation efficiency, higher accuracy is required, which depends on the 3'-terminal translocation of tRNA (posttransfer editing).

	FOOTNOTES

 
^* This work was funded by Natural Science Foundation of China Grants 30170224, 30270310, and 30330180; Chinese Academy of Sciences Grant KSCX-2-2-04; and Shanghai Committee of Science and Technology Grant 02DJ140567. 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 should be addressed. Tel.: 86-21-54921241; Fax: 86-21-54921011; E-mail: edwang{at}sibs.ac.cn.

¹ The abbreviations used are: aaRS, aminoacyl-tRNA synthetase; SBL, anticodon stem biloop; IleRS, isoleucyl-tRNA synthetase; ValRS, valyl-tRNA synthetase; LeuRS, leucyl-tRNA synthetase; AlaRS, alanyl-tRNA synthetase; ProRS, prolyl-tRNA synthetase; MetRS, methionyl-tRNA synthetase; -LeuRS, Aquifex aeolicus leucyl-tRNA synthetase; ThrRS, threonyl-tRNA sythetase; TyrRS, tyrosyl-tRNA synthetase.

² M. W. Zhao, unpublished observation.

	ACKNOWLEDGMENTS

 
We thank Dr. Dieter Söll for carefully reading the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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