Functional Dissection of the Eukaryotic-specific tRNA-interacting Factor of Lysyl-tRNA Synthetase*

Mathilde FrancinDagger and Marc Mirande§

From the Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 1 Avenue de la Terrasse, 91190 Gif-sur-Yvette, France

Received for publication, August 28, 2002, and in revised form, October 31, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the cytoplasm of higher eukaryotic cells, aminoacyl-tRNA synthetases (aaRSs) have polypeptide chain extensions appended to conventional prokaryotic-like synthetase domains. The supplementary domains, refered to as tRNA-interacting factors (tIFs), provide the core synthetases with potent tRNA-binding capacities, a functional requirement related to the low concentration of free tRNA prevailing in the cytoplasm of eukaryotic cells. Lysyl-tRNA synthetase is a component of the multi-tRNA synthetase complex. It exhibits a lysine-rich N-terminal polypeptide extension that increases its catalytic efficiency. The functional characterization of this new type of tRNA-interacting factor has been conducted. Here we describe the systematic substitution of the 13 lysine or arginine residues located within the general RNA-binding domain of hamster LysRS made of 70 residues. Our data show that three lysine and one arginine residues are major building blocks of the tRNA-binding site. Their mutation into alanine led to a reduced affinity for tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> or minimalized tRNA mimicking the acceptor-TPsi C stem-loop of tRNA<UP><SUB><RM>3</RM></SUB><SUP><RM>Lys</RM></SUP></UP> and a decrease in catalytic efficiency similar to that observed after a complete deletion of the N-terminal domain. Moreover, covalent continuity between the tRNA-binding and core domain is a prerequisite for providing LysRS with a tRNA binding capacity. Thus, our results suggest that the ability of LysRS to promote tRNALys networking during translation or to convey tRNA<UP><SUB><RM>3</RM></SUB><SUP><RM>Lys</RM></SUP></UP> into the human immunodeficiency virus type 1 viral particles rests on the addition in evolution of this tRNA-interacting factor.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The aminoacyl-tRNA synthetases (aaRSs)1 are responsible for the interpretation of the genetic code in terms of amino acids. This family of 20 enzymes aminoacylates the corresponding tRNA species and thus provides the essential link between anticodons and amino acids (1). The rules that govern the accurate pairing between tRNAs and aminoacyl-tRNA synthetases have been especially scrutinized in bacterial systems (reviewed in Refs. 2-4). Numerous crystal structures of tRNA synthetase complexes have now been described and contribute to our understanding of the RNA-protein recognition problem (5). Although the mode of tRNA binding differs from one synthetase to another, nucleotides from the acceptor stem, the anticodon stem-loop domain, and the dihydrouridine stem located in the inner, concave side of the L-shaped tRNA molecule generally provide the basis for cognate tRNA synthetase interaction. On particular occasions, a non-canonical appended domain of the synthetase makes contacts with outer regions of the convex side of tRNA. The coiled-coil domains appended at the N terminus of Thermus thermophilus SerRS, the N terminus of the alpha -subunit of T. thermophilus PheRS, or the C terminus of T. thermophilus ValRS interact with the variable arm and TPsi C loop of tRNASer or with the dihydrouridine and TPsi C loop of tRNAPhe or tRNAVal (6-8). The N-terminal domain of Saccharomyces cerevisiae or T. thermophilus ArgRS recognizes the D-loop of tRNA (9, 10). The C-terminal domain of T. thermophilus TyrRS makes contact with the anticodon stem and the long variable arm of tRNATyr (11). The C-terminal junction domain of Staphylococcus aureus IleRS contacts the anticodon stem of tRNA (12).

One of the major differences that characterizes aminoacyl-tRNA synthetases from higher eukaryotes (from Drosophila to human) as compared with their prokaryotic homologues is the presence of polypeptide chain extensions appended to the N or C terminus of the protein (13). MetRS, GlyRS, HisRS, TrpRS, and bifunctional GluProRS share a motif of ~50 amino acid residues (14) that folds into a coiled-coil conformation (15, 16). This polypeptide extension is a tRNA-interacting factor (tIF) that acts as a cis-acting factor for aminoacylation (17). One of the components of the multi-synthetase complex, the protein p43, has the potential to bind tRNA non-specifically (18, 19) and to play the role of a trans-acting tIF (20). The crystal structure of the C-terminal moiety of p43 identified a putative oligonucleotide binding-fold-based tRNA-binding site (21). An homologous domain is appended to the C terminus of human TyrRS (22), but its involvement in tRNA binding has not yet been established. The N-terminal eukaryotic-specific domains of ~70 amino acid residues appended to mammalian AspRS, AsnRS, and LysRS share sequence similarities (23) and participate in tRNA binding (24-26). These supplementary tRNA binding modules appended to eukaryotic aminoacyl-tRNA synthetases decrease dissociation constants for their cognate tRNAs. Because of the scarcity of non-acylated tRNA in the cytoplasm of higher eukaryotic cells (discussed in Ref. 26), these tIFs are thought to be required for tRNA cycling during translation (27).

We have shown previously that native mammalian LysRS has the ability to form a stable tRNA-protein complex with human tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP>, whereas a N-terminally truncated derivative has lost this property (26). The presence of this extension decreases the Kd and Km values for tRNA and therefore should facilitate tRNA aminoacylation under the conditions of the suboptimal tRNA concentration prevailing in vivo. These in vitro data provided a rational explanation to the phenotype of growth retardation observed for yeast cells that express an allele of the yeast KRS1 gene with a deletion of its eukaryotic-specific N-terminal extension (28). Other studies have also involved human LysRS as the possible vector of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> for packaging into the HIV viral particles. In retroviruses, the initiation of reverse transcription is primed by a cellular tRNA that is selectively encapsidated into the virion. In the case of HIV-I, the primer tRNA is tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP>. It forms an extended network of template/primer interactions with the viral primer-binding site but also with viral sequences located upstream of the primer-binding site (29). Accordingly, tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> is efficiently packaged into HIV-1 viral particles, albeit not selectively (30). The other major tRNA species is tRNA<UP><SUB>1,2</SUB><SUP>Lys</SUP></UP>. Primer/template annealing is not responsible for incorporation of tRNALys into the virion, which suggested that a viral protein may be involved in selection of the primer tRNA for packaging (31). Recent data have shown that LysRS or a proteolytically truncated derivative is selectively packaged in HIV-1 viral particles and may thus be the carrier of tRNALys (32).

To understand the role of the eukaryotic-specific N-domain of LysRS in tRNA cycling in translation and thereby in tRNA capture into the HIV-1 viral particle, we wanted to determine the molecular basis for the potent tRNA binding properties of mammalian LysRS. To evaluate the contribution of the many lysine residues present in the tIF of LysRS, we determined the kinetic and tRNA binding capacities of a series of LysRS mutants with single lysine to alanine changes. We eventually delineated the residues that contribute to the function of the eukaryotic-specific tIF of class-II aminoacyl-tRNA synthetases.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutant Construction-- Plasmid pYeDP10/CKRS (2µ, URA3) contains the wild-type hamster LysRS cDNA expressed under the control of the PGK promoter (33). The lysine-rich region of the N-domain of LysRS, from Met-1 to Lys-40, is encoded by a BglII-SacI fragment of 135 nucleotides. The 13 lysine or arginine codons encoding residues at positions 8, 10, 16, 19, 23, 24, 25, 27, 30, 31, 35, 38, and 40 were substituted with GCU codons corresponding to the major tRNAAla species in yeast, tRNA<UP><SUB>1</SUB><SUP>Ala</SUP></UP>. After removal of the wild-type BglII-SacI fragment, cDNAs carrying a single one of these mutations were constructed by assembling six overlapping oligonucleotides. The ligation mixture was used to transform Escherichia coli HB101 cells. The sequence of the constructs was checked by DNA sequencing.

The diploid yeast strain CCdYK01 (his3/his3, leu2/leu2, ura3/ura3, trp1/trp1, KRS1/krs1::TRP1) (33) was transformed to Ura+ with plasmid pYeDP10 encoding the various hamster LysRS mutants by the lithium chloride method (34). Sporulation of diploid cells in the nitrogen-deficient starvation medium (1% potassium acetate, 0.1% yeast extract, 0.05% dextrose) and random spore analysis were performed according to standard procedures (35). Briefly, sporulated cells were incubated overnight at 28 °C with lyticase at 750 units/ml (Sigma) supplemented with 1.5% IGEPAL (Sigma), incubated for 15 min on ice, and subjected to sonication (2-fold, 30 s) to release spores from their asci. After washing and resuspension in H2O, spore colonies were grown on selective medium. Haploid cells Trp+ and Ura+ in phenotype were selected and then analyzed by Western blotting for the lack of the endogenous yeast LysRS and the presence of plasmid-encoded hamster LysRS as described (33).

Protein Overexpression and Purification-- Wild-type and mutant LysRS was expressed in yeast and purified according to a purification scheme adapted from Ref. 33. All purification steps were conducted at 4 °C with a BioCAD work station (Applied Biosystems) except where otherwise stated. Briefly, recombinant yeast cells were grown at 28 °C in 1.5 liters of YPG medium (0.5% yeast extract, 0.5% Bacto Peptone, and 3% glucose) to an A600 of 8. Cells were washed, resuspended in extraction buffer (1 ml/g of cells), and lysed in an Eaton press. After a 2-fold dilution with extraction buffer containing protease inhibitors (5 mM diisopropyl fluorophosphate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml chymostatine, and 2 µg/ml antipaine), cell debris were removed by centrifugation. Nucleic acids were removed by precipitation with Polymin P at 0.2%, and the clear supernatant was applied to a 50 ml S-Sepharose FF column (Amersham Biosciences). LysRS was eluted by a linear gradient (20-column volume) of potassium phosphate from 50 to 300 mM (pH 7.5) containing 1 mM EDTA, 10% glycerol, and 10 mM beta -mercaptoethanol. Fractions containing LysRS were dialyzed (30 mM potassium phosphate, pH 7.5, 1 mM EDTA, 10% glycerol, and 10 mM beta -mercaptoethanol), applied to a Mono Q HR 5/5 column equilibrated in 50 mM potassium phosphate, pH 7.5, 1 mM EDTA, 10% glycerol, and 10 mM beta -mercaptoethanol and washed with 5-column volumes of the same buffer containing protease inhibitors (5 mM diisopropyl fluorophosphate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml chymostatine, and 2 µg/ml antipaine), and eluted at room temperature by a linear gradient (40-column volume) of potassium phosphate from 50 to 150 mM. Fractions containing LysRS were dialyzed against 25 mM potassium phosphate, pH 7.5, 2 mM dithiothreitol, 55% glycerol, and stored at -20 °C at a protein concentration of ~2-10 mg/ml.

The N-terminally truncated derivative (LysRS-Delta N) was obtained by elastase treatment of the native enzyme (36). The N-terminal polypeptide extension of LysRS (N-LysRS) was expressed in E. coli and purified as described (26).

Protein concentration was determined by using calculated absorption coefficients of 0.547 and 0.600 A280 units × mg-1 × cm2, respectively for LysRS (and LysRS mutants) and LysRS-Delta N.

Gel Retardation Assay-- Plasmids ptRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> and pAcctRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> (26) were linearized with FokI and BstNI, respectively, and subjected to in vitro transcription with T7 RNA polymerase purified from the strain BL21/pAR1219 generously provided by Prof. W. Studier (Brookhaven National Laboratory).

32P-labeled tRNAs were synthesized in a reaction mixture (50 µl) containing 1 µg of template DNA, 40 mM Tris-HCl, pH 8.0, 6 mM MgCl2, 1 mM spermidine, 5 mM dithiothreitol, 0.01% Triton X-100, 1 mM each CTP, UTP, and GTP, 10 µM [alpha -32P]ATP (200 Ci/mmol), and 50 units of T7 RNA polymerase. After incubation at 37 °C for 1 h, the transcripts were purified by electrophoresis on a denaturing 12% polyacrylamide gel (mono/bis, 19:1), recovered from the gel by soaking in H2O, precipitated with ethanol, and resuspended in H2O. The RNA transcripts were renatured by heating at 90 °C for 2 min and cooling at room temperature for 20 min in the presence of 5 mM MgCl2.

Protein-RNA interactions were analyzed using a band shift assay. Homogeneous wild-type LysRS and LysRS mutants were incubated at increasing concentrations with radiolabeled RNA (25,000 cpm per point) in a 11-µl volume containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 10% glycerol, and bovine serum albumin at 0.1 mg/ml. After incubation at 25 °C for 20 min, the mixture was placed on ice and loaded on a 6% polyacrylamide gel (mono/bis, 29:1) containing 5% glycerol in 0.5× TBE, pH 8.0, at 4 °C. After electrophoresis, the gel was fixed, dried, and subjected to autoradiography. Free and bound tRNA was quantified by densitometry measurements.

tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> Expression and Purification-- For aminoacylation assays, human tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> was produced in E. coli JM101Tr transformed with the plasmid pBSTK3 (37). Cells were grown in 1.5 liters of 2× tryptone/yeast extract medium supplemented with ampicillin. Total tRNA fraction (500 A260 units; 300 pmol tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP>/A260 unit) was prepared as described by Meinnel et al. (38), and tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> was partially purified on a SOURCE 15Q column (160 A260 units; 600 pmol tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP>/A260 unit).

tRNA Aminoacylation Assay-- Initial rates of tRNA aminoacylation were measured at 25 °C in 0.1 ml of 20 mM imidazole-HCl buffer, pH 7.5, 100 mM KCl, 0.5 mM dithiothreitol, 12 mM MgCl2, 2 mM ATP, 180 µM 14C-labeled lysine (PerkinElmer Life Sciences; 16.66 Ci/mol) and saturating amounts of tRNA (26). Human tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> expressed in E. coli (lysine acceptance of 600 pmol/A260) was used as tRNA substrate. The incubation mixture contained catalytic amounts (1-2 nM) of enzymes appropriately diluted in 10 mM Tris-HCl, pH 7.5, and 10 mM 2-mercaptoethanol containing bovine serum albumin at 4 mg/ml. One unit of activity is the amount of enzyme producing 1 nmol of lysine-tRNALys/min at 25 °C. For the determination of Km values for tRNA, tRNALys concentrations of 0.2-50 µM were used. Michaelian parameters were obtained by non-linear regression of the theoretical Michaelis-Menten equation to the experimental curve using the KaleidaGraph 3.0.8 software (Abelbeck Software).

Lysine Activation Assay-- The isotopic [32P]PPi-ATP exchange reaction was conducted as described previously (39). The assay mixture contained, in a final volume of 0.1 ml, 20 mM imidazole-HCl, pH 7.5, 10 mM MgCl2, 0.1 mM EDTA, and 2 mM each of ATP, [32P]pyrophosphate (2.5 Ci/mol), and lysine. The reaction was started by the addition of limiting amounts of enzymes (4 nM) appropriately diluted in 10 mM Tris-HCl, pH 7.5, containing 10 mM 2-mercaptoethanol and bovine serum albumin at 4 mg/ml. After 10 min at 25 °C, the reaction was stopped by the addition of 2.5 ml of a solution containing 100 mM pyrophosphate, 50 mM sodium acetate, pH 4.5, 0.35% perchloric acid, and 0.4% Norit. Samples were filtered through Whatmann no. 1 filters, washed extensively with water, and counted in a liquid scintillator. A unit of enzyme activity is defined as the amount of enzyme required to form 1 nmol of [32P]ATP/min.

For the determination of kinetic parameters in the PPi-ATP exchange reaction, the concentration of ATP in the assay was varied from 25 µM to 5 mM, and that of lysine was varied from 10 µM to 2 mM. Michaelian parameters were deduced as described above.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Rationale for Mutations in the Eukaryote-specific N-domain of LysRS-- Mammalian LysRS is a dimer of 2 × 68 kDa. An N-terminal deletion of 50 amino acid residues removes the eukaryotic-specific appended domain and results in a dimer of 2 × 62 kDa. This polypeptide extension precedes helix H1 from the anticodon-binding domain of bacterial LysRS (Fig. 1). The truncated derivative of LysRS, LysRS-Delta N, displayed a 50-fold lower apparent affinity for tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> and a 4-fold increase in Km for tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> in the aminoacylation reaction, as compared with the native enzyme (26). To ascertain that the removal of the N-domain of LysRS did not perturb the catalytic center of the enzyme, kinetic parameters for lysine and ATP in the ATP-PPi exchange reaction were determined. The Km values of the wild-type and N-terminally truncated LysRS for ATP (384 ± 20 and 371 ± 12 µM) and lysine (157 ± 9 and 152 ± 7 µM) and their kcat values of ATP formation (26 ± 1 and 27 ± 2 s-1) were identical. This result extends our earlier observation showing that LysRS and LysRS-Delta N also display identical kinetic parameters for ATP and lysine in the tRNALys aminoacylation reaction (26).


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Fig. 1.   Sequence alignments of the N-terminal regions of LysRS. The N terminus of hamster (Cricetulus longicaudatus) LysRS (Cl) from amino acid residues 1 to 90 is aligned with the corresponding LysRS sequences from Homo sapiens (Hs), Mus musculus (Mm), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), Lycopersicon esculentum (Le); S. cerevisiae (Sc), T. thermophilus (Tt) and E. coli (lysS (EcS) and lysU (EcU) gene products). Helix H1 from the anticodon-binding domain of LysRS-S (44) is indicated. The 14 lysine or arginine residues from the eukaryotic-specific N-domain of hamster LysRS and the corresponding conserved residues are highlighted in black boxes with white lettering. The five strictly conserved (K/R) residues are marked with an asterisk.

We have shown previously that interactions between tRNA and the N-domain of LysRS are essentially nonspecific; similar band shifts have been observed with cognate or noncognate tRNAs. This result has suggested that the formation of RNA-protein complexes involves electrostatic interactions between side chains of basic amino acid residues from the N-domain of LysRS and the tRNA phosphate-sugar backbone. In the N-domain of hamster LysRS, 12 lysine or 2 arginine residues are located between positions 8 to 45 (Fig. 1) and are good candidates for building the RNA-protein interface. Because no structural data is available for this domain, we sought to identify positions in this RNA binding motif that would influence its function. For this purpose, we performed systematic substitution of these basic residues with alanines. These substitutions were K8A, K10A, K16A, K19A, K23A, R24A, R25A, K27A, K30A, K31A, K35A, K38A, and K40A.

Expression and Purification of LysRS Variants-- Incorporation of additional sequences such as a His-tag in LysRS impairs its catalytic parameters (26). Thus, wild-type and mutant LysRS were isolated by standard chromatography procedures. Proteins were expressed in yeast from a multicopy plasmid (2 µ, URA3) that encoded each of the cDNAs under the control of a phosphoglycerate kinase (PGK) promoter. The endogenous yeast LysRS possesses an N-domain related to that of the hamster enzyme (Fig. 1) and efficiently aminoacylates mammalian tRNALys. We showed that expression of hamster LysRS in S. cerevisiae functionally replaces a null allele of the yeast KRS1 gene (33). Thus, wild-type and mutant LysRS were expressed in yeast in a null background. The diploid yeast strain CCdYK01 (KRS1/krs1::TRP1) (33) was transformed with plasmids encoding the various hamster LysRSs. After sporulation and random spore analysis, haploid strains (Trp+ and Ura+ in phenotype) bearing a disrupted yeast KRS1 chromosomal allele and rescued by plasmid-encoded hamster LysRSs were selected. The expression of the mammalian enzymes and the lack of the yeast enzyme in these strains was verified by Western blot analysis using antibodies directed to the mammalian or yeast LysRS (result not shown). This observation already showed that the 13 LysRS variants are functional and are able to rescue yeast cells lacking endogenous LysRS.

Wild-type and mutant LysRS were purified to homogeneity using a standardized isolation scheme on S-Sepharose and Mono-Q columns. The binding of wild-type LysRS on S-Sepharose essentially rests on its N-domain; it is eluted at a potassium phosphate concentration of 140 mM, whereas LysRS-Delta N does not bind at a salt concentration of 25 mM. Among the different mutants, the six LysRS derivatives, K16A, K19A, K23A, R24A, K27A, and K31A, were eluted early in the gradient (95-110 mM), whereas other mutants were eluted at salt concentrations ranging from 120 to 140 mM. This observation suggested that the six lysine residues listed above are especially exposed to the solvent and are good candidates for interacting with tRNA.

Single Mutations Have Km Effects for tRNA Aminoacylation-- Because the N-domain of LysRS is a key element to tRNA binding, its removal is accompanied by a 3-fold decrease in catalytic efficiency (kcat/Km) in the aminoacylation reaction of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP>, resulting essentially from a 4-fold increase in Km (Table I). The change in kinetic parameters for tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> aminoacylation triggered by the 13 mutant enzymes was investigated. Partially purified human tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> (lysine acceptance of 600 pmol/A260) expressed in E. coli was used to determine the steady-state kinetic parameters in the aminoacylation reaction. Whereas all mutant enzymes exhibited similar kcat values of Lys-tRNALys formation comprised between 3.76 and 4.83 s-1 as compared with 4.85 s-1 for wild-type, moderate but reliable variations of the Km values for tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> were determined (Table I). Mutant K23A had a Km value that increased more than 3-fold as compared with wild-type. Thus, it is worth noting that the single mutation of lysine-23 into alanine induced kinetic changes comparable with that observed for a complete deletion of the N-domain. Similarly, the four single mutants K19A, R24A, R25A, and K27A also displayed a significant increase in Km (2.1-3.6-fold). Conversely, the Km and kcat values for the eight other mutants (lysine into alanine at positions 8, 10, 16, 30, 31, 35, 38, or 40) were not significantly affected. These results suggested that the tRNA binding motif of the tIF of LysRS may be built around the conserved basic residues located between positions 19 to 27 (Fig. 1).

                              
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Table I
Kinetic constants of wild-type and mutant LysRS in tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> aminoacylation

tRNA Binding Is Significantly Perturbed by Single Mutations-- To determine the effect of single mutations in the N-domain of LysRS on the binding of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP>, we measured the dissociation constant Kd in a gel mobility shift assay using radiolabeled human tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> obtained by in vitro transcription. Free and bound tRNA species were quantified by densitometry measurements. Earlier, we showed that wild-type LysRS forms a stable complex with tRNALys, whereas LysRS-Delta N did not (26). Mutants K8A, K10A, and K16A, which exhibited kinetic parameters similar to wild-type, interacted with tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> with an apparent dissociation constant of 100 ± 10 nM (Fig. 2), a value very similar to that determined for wild-type LysRS (75 nM) under the same assay conditions. By contrast, the three mutants K23A, R24A, and K27A, which exhibited the largest increases in Km values for tRNALys, also displayed a significant 2-fold decrease in affinity toward tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> (Kd ~200 nM). An exception is mutant R25A, which showed a 2.2-fold increase in Km for tRNA but a Kd similar to wild-type. Its behavior will be further addressed under "Discussion." Other mutants displayed intermediate Kd values.


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Fig. 2.   Binding of mutant LysRSs to tRNA<UP><SUB><B>3</B></SUB><SUP><B>Lys</B></SUP></UP>. In vitro transcribed 32P-labeled tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> was incubated with site-directed mutants of hamster LysRS at different concentrations (0-0.5 µM, expressed as dimer concentrations). After electrophoresis at 4 °C on a 6% native polyacrylamide gel, the mobility shift of tRNA was visualized by autoradiography. In each assay, the bottom band corresponds to the free tRNA species. Concentrations of LysRS at which half of tRNA forms a complex are marked by boxes; apparent Kd values are indicated at right.

Because the effect of single mutations on the Kd values for tRNA were of modest amplitude, we sought to compare the RNA binding potentials of wild-type and mutant LysRS toward a minimized substrate. Indeed, in the lysine system, the anticodon is a major if not a unique identity determinant for aminoacylation of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> by LysRS from mammalian origin, either in vitro (40) or in vivo (41). Assuming that the mode of tRNA binding in mammals is similar to that described in bacteria (42, 43), anticodon interacts with the beta -barrel N-terminal domain of LysRS, a domain that is present in all of our mutant enzymes. In contrast, we reported previously that wild-type LysRS, but not LysRS-Delta N, binds and aminoacylates RNA minihelices derived from the acceptor-TPsi C stem-loop of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP>. Thus, assuming that the N-domain of LysRS preferentially interacts with the acceptor arm of tRNALys, we expected that mutations in the N-domain of LysRS would reveal sharper differences when focusing on the binding of the acceptor arm alone.

To investigate this possibility, we prepared an in vitro transcribed, 32P-labeled amino acid acceptor minihelix (AccLys) of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP>. This minimalized substrate was used in a gel-mobility shift assay to examine its association with mutant enzymes (Fig. 3). Wild-type LysRS is known to bind this minimalized substrate with an apparent Kd of 0.50 ± 0.05 µM (26). Mutants K8A, K10A, K16A, and K40A bound this stem-loop structure with a Kd similar to wild-type. In contrast, the four mutants K19A, K23A, R24A, and K27A produced a much weaker complex, with Kd increasing more than 5-fold (Fig. 3). Thus, mutations within the N-domain of LysRS mainly alter interactions of wild-type LysRS with the acceptor-TPsi C stem-loop structure of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP>.


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Fig. 3.   Binding of mutant LysRSs to the acceptor-TPsi C stem-loop of tRNA<UP><SUB><B>3</B></SUB><SUP><B>Lys</B></SUP></UP>. An in vitro transcribed 32P-labeled RNA minihelix mimicking the acceptor arm of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> was incubated with site-directed mutants of hamster LysRS at different concentrations (0-4 µM, expressed as dimer concentrations). After electrophoresis at 4 °C on a 6% native polyacrylamide gel, the mobility shift of RNA was visualized by autoradiography. In each assay, the bottom band corresponds to the free RNA species. Concentrations of LysRS at which half of RNA forms a complex are marked by boxes; apparent Kd values are indicated at right.

The N-domain of LysRS Is Not Intimately Related to the Core Enzyme-- Having shown that the tRNA-binding site of the eukaryotic-specific N-domain of LysRS accommodates the four basic residues K19, K23, R24, and K27, we asked whether the N-domain and the core enzyme had to be physically linked to provide wild-type LysRS with RNA-binding properties. Previously, we found that the isolated core and N-domains of LysRS bind tRNA weakly but act synergistically in the wild-type protein to build a high affinity binding site for tRNA (26). We reasoned that the sum of three weak interactions between the acceptor arm of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> and the N-domain of LysRS, the anticodon arm of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> and the core domain of LysRS, and the N-domain and core domain of LysRS might be sufficient to produce a stable complex. Thus, provided that the two domains of LysRS interact, we expected that the tRNA binding capacity of the core enzyme might be enhanced after mixing with its N-domain. Alternatively, if covalent continuity is required to associate the two domains of LysRS, no enhancement of tRNA binding should be observed in the presence of the two separate domains.

To test this possibility, we compared the tRNA-binding potential of wild-type dimeric LysRS (Kd of about 150 nM, expressed as monomer concentration), N-terminally truncated dimeric LysRS (LysRS-Delta N; Kd of about 6 µM, expressed as monomer concentration), and the monomeric N-domain of LysRS (N-LysRS; Kd of about 20 µM) with that obtained with a 1:1 mixture of N-LysRS and LysRS-Delta N (Fig. 4). LysRS-Delta N (1 µM, dimer concentration) was preincubated with N-LysRS (2 µM, monomer concentration) in the tRNA binding assay buffer for 30 min at 4 °C. The mixture was serially diluted, immediately incubated with tRNA, and subjected to gel mobility shift assay. The tRNA-binding capacity revealed by the mixture of LysRS-Delta N with N-LysRS was not significantly improved as compared with LysRS-Delta N alone (Fig. 4). Increasing the concentration of N-LysRS up to 10 µM in the preincubation mixture did not cause a greater effect. Therefore, the covalent link between N-LysRS and LysRS-Delta N is required to confer on the native enzyme a robust tRNA binding propensity.


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Fig. 4.   The covalent continuity between the core and N-domain of LysRS is required for binding tRNA. 32P-labeled in vitro transcribed tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> was incubated with the wild-type enzyme (LysRS), the N-terminally truncated enzyme (LysRS-Delta N), the isolated N-domain (N-LysRS), or an equimolar mixture of N-LysRS and LysRS-Delta N at protein concentrations of 0.03-2 µM (expressed as monomer concentrations). The mobility shift of tRNA was visualized by autoradiography. In each assay, the bottom band corresponds to the free tRNA species.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The eukaryote-specific N-terminal domain of LysRS is an RNA-binding domain that acts as a cis-acting tIF (26). The removal of the N-domain of LysRS is accompanied by a 3.9-fold increase in the Km value for tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP>, as compared with wild-type LysRS, and a two order-of-magnitude increase in Kd. Basically, the four mutants K19A, K23A, R24A, and K27A recapitulated the aminoacylation properties of the deletion mutant LysRS-Delta N. The results of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> and Acc-tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> binding assays together with the effect of single substitutions on the retention on Mono-Q columns and the aminoacylation of tRNALys (Fig. 5) collectively demonstrate that the tRNA-binding site of the tIF of LysRS is likely to accommodate these four basic residues.


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Fig. 5.   Comparative bar graph representation of the properties of wild-type and mutant LysRS. The affinity of LysRS for the S-Sepharose matrix (represented by the molarity of elution, top panel), the Km value for tRNA in the aminoacylation reaction (represented as 1/Km, second panel from top), and the affinity for tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> or the acceptor-TPsi C stem-loop of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> (represented as 1/Kd, bottom two panels) are indicated for the wild-type enzyme (WT), the N-terminally truncated enzyme (Delta N), or mutant species (identified by the corresponding sites of the mutations).

The three-dimensional structure of the core domain of mammalian LysRS is likely to be a close structural homologue of T. thermophilus (45% identities) or E. coli (43% identities) LysRS. The level of sequence identity is similar between the human LysRS and the bacterial enzymes or, on the other hand, between the T. thermophilus and E. coli LysRS (46% identities), two enzymes that display an analogous fold. In the crystal structure of the two bacterial enzymes (42, 44) there are two major domains. The C-terminal catalytic domain is built around an antiparallel beta -sheet and contains the three canonical sequence motifs that typify the class-II aminoacyl-tRNA synthetases. The N-terminal beta -barrel domain interacts with the anticodon stem-loop region of tRNA. Base-specific contacts involve the three anticodon bases. Helix H1 establishes water-mediated interactions with the tRNA backbone in the anticodon stem region. The extra N-terminal domain specific to eukaryotic LysRS, although appended to the anticodon binding domain of the synthetase, improves docking of the CCA end of tRNA in the active site of the enzyme (26). Hydrodynamic properties of the isolated N-domain are characteristic of an elongated molecule. In the crystal structure of bacterial LysRSs, the N-terminal residue is separated by ~50 Å from the entrance of the active site crevice of the same monomer but also by only ~60 Å from the active site of the other monomer. Thus, stabilization of the binding of the tRNA acceptor arm on one monomer of the dimeric synthetase may result from an additional interaction with the N-domain of the same monomer or from cross-subunit interaction with the N-domain from the other monomer. Future experiments can address this conundrum.

The three-dimensional structure of the N-domain of LysRS has not yet been solved, either as an isolated domain or when it is associated within the wild-type protein. The PSIRED method of secondary structure prediction (45) suggests that amino acid residues 19-36 of the N-domain of LysRS have a high probability to fold into an alpha -helical conformation. This hypothesis is further supported by the observation that a synthetic tricosapeptide representing residues 30-52 of the N-domain of yeast AspRS forms an alpha -helix in solution in the presence of equimolar amounts of octadecaphosphate (46). As shown in Fig. 6, the sequence of the tricosapeptide contains the conserved sequence motif KXXXK(K/R)XXK identified in this work as the tRNA-binding site of the N-domain of LysRS. The finding that an octadecaphosphate induced the alpha -helical conformation of the tricosapeptide has suggested that the lysine-rich region of the extension of yeast AspRS can provide a structural motif involved in nonspecific nucleic acid/protein interactions. The sequence SKXXLKKXXK is the most conserved signature sequence of N-terminal extensions of class IIb synthetases (23). This observation indicated that this motif may be responsible for the strong tRNA-binding capacity of wild-type AspRS from yeast. Collectively, these data support the conclusion according to which the sequence-motif KXXXK(K/R)XXK has an alpha -helical conformation and builds the nonspecific tRNA-binding site of mammalian LysRS and other eukaryotic synthetases that possess it. It is worth noting that the four basic residues K19, K23, R24, and K27, identified in this work as the major components of the eukaryote-specific RNA binding motif of hamster LysRS, are clustered on one side and at one extremity of the predicted alpha -helix (Fig. 6). Thus, the RNA-binding motif of mammalian LysRS is separated by 44 residues from the N terminus of helix H1, located in the anticodon binding domain of the synthetase, and is carried by a long alpha -helix (Fig. 6), two features that support the possibility of an interaction with the acceptor stem of tRNALys.


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Fig. 6.   Secondary structure prediction and three-dimensional model of the RNA-binding motif of LysRS. The sequence of the N-domain of hamster LysRS, from residues 1 to 60, is indicated with predicted secondary structure elements (E for extended or beta -strands; H for alpha -helices). The sequence of the tricosapeptide of the N-terminal extension of yeast AspRS (Drs-Sc) that folds into an alpha -helical conformation is shown at the top. The conserved basic residues are boxed; those building the tRNA binding-motif of hamster LysRS are presented with a white background. An axial (left) or longitudinale (right) view of the putative alpha -helix is represented.

Apart from the tRNA binding residues identified in this work (K19, K23, R24, and K27), the predicted alpha -helix also contains basic residues R25, K30, K31, and K35. Among these residues, the substitution of lysine 31 into alanine increased Km for tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> and Kd for native tRNA but caused a weaker increase in Kd for the minimalized substrate (Fig. 5). In yeast LysRS, lysine 31 is replaced by a valine (Fig. 1). Thus, although located at the border of tRNA-binding site (Fig. 6), we chose not to include this residue in the RNA binding motif, strictly speaking. Concerning arginine 25, although its replacement into alanine had no effect on RNA binding (either tRNA or AcctRNA), the increase in Km value was similar to that observed for the K19A and K27A mutants (Fig. 5). These data can be explained by taking into account its location on the opposite side of the putative helix as compared with K19, K23, R24, and K27 (Fig. 6). In accordance with the finding that mutation R25A did not change the molarity of elution of LysRS on S-Sepharose (Fig. 5), we may assume that the side of the helix that contains arginine 25 is turned toward the core of the protein. Thus, although arginine 25 is not included in the tRNA-binding motif, an incorrect positioning of the N-domain of LysRS relative to the catalytic domain may account for the Km effect observed for the R25A mutant.

Several pieces of evidence suggest that the N-domain of LysRS is essentially structurally independent of the core enzyme. Indeed, upon mixing the N-domain with the core enzyme, no increase in RNA-binding capacity was observed (Fig. 4). Thus, if the helical region of the N-domain, including arginine 25, does interact with the core domain of LysRS, this association is likely to be weak. This result is in agreement with previous works showing that wild-type LysRS can be readily modified by controlled elastase treatment to give a truncated, yet active dimer that has lost polyanion-binding properties (36). Because the three-dimensional structure of the core domains of AspRS and LysRS are closely related (44, 47), and taking into account the similarities observed for the extensions of yeast AspRS and LysRS (23), the domain organization of the two class II enzymes of eukaryotic origin should be very similar. The finding that the N-domain could not be depicted in the electron density map of crystallized yeast AspRS (47) also suggests that the tertiary structure of the extension is not stabilized by protein-protein interactions with the catalytic domain.

The tIFs of eukaryotic aminoacyl-tRNA synthetases are believed to be involved in tRNA cycling during translation (26). We previously investigated the biological significance of the N-terminal polypeptide extension of LysRS from S. cerevisiae with in vivo approaches (28). A yeast KRS1 mutant allele with a deletion encompassing amino acid residues 11-68 is able to complement a deletion of the wild-type KRS1 allele, but a phenotype of growth retardation has been observed. This result established the biological relevance of this eukaryotic-specific domain, but the exact reason for defective growth was poorly understood. Our data now provide a rational explanation. The extension of that enzyme possesses the characteristic KXXXK(K/R)XXK sequence motif. Wild-type yeast LysRS is also able to form a stable complex with tRNALys (Kd of 75 nM), but a mutant with a N-terminal deletion of 68 amino acid residues binds tRNA with a very reduced affinity (Kd > 5 µM).2 Thus, yeast and mammalian LysRS acquired in evolution a supplementary domain that confers on them a potent RNA binding capacity by decreasing the dissociation constant for their cognate tRNA. The data obtained in vivo suggest that this property improves the translational efficiency of these enzymes.

In regard to the putative involvement of human LysRS in the selective packaging of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> in the HIV-1 viral particle, our data suggest that the removal of the 27 N-terminal residues of LysRS containing the tRNA-binding motif would impair its propensity to bind tRNALys and thus may preclude packaging of the primer RNA. Although a polypeptide with an apparent Mr of 63,000 has been identified in extracts of HIV-1 viral particles with antibodies directed to human LysRS, its size is significantly shorter than that of wild-type LysRS (apparent Mr of 70,000) (32). Because the capacity of LysRS to be the carrier of tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> into the virion is likely to be impaired by the loss of its N-domain, the polypeptide of Mr 63,000 might result from the loss of C-terminal sequences. However the last strand of the antiparallel beta -sheet that builds the active site of class IIb synthetases is located close to the C terminus. Accordingly, the removal of a few C-terminal residues in yeast LysRS (deletion of 15 residues) or AspRS (deletion of 10 residues), two class IIb enzymes closely related to human LysRS, is accompanied by their inactivation (28, 48). This is at odds with the observation that aminoacylation of tRNA is a prerequisite for packaging (41). The actual LysRS species packaged into the virion remains elusive, but its propensity to carry tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> into the viral particle should rest on the presence of the tRNA-binding residues identified in this work.

    ACKNOWLEDGEMENTS

We are very grateful to Dr. Frédéric Dardel for providing plasmid pBSTK3 for the expression of human tRNA<UP><SUB>3</SUB><SUP>Lys</SUP></UP> in E. coli. The excellent technical assistance of Françoise Triniolles is gratefully acknowledged.

    FOOTNOTES

* This work was supported by grants from the Agence Nationale de Recherche sur le Sida, the Association pour la Recherche sur le Cancer, and La Ligue.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.

Dagger Recipient of a fellowship from École Centrale Paris.

§ To whom correspondence should be addressed. Tel.: 33-1-6982-3505; Fax: 33-1-6982-3129; E-mail: mirande@lebs.cnrs-gif.fr.

Published, JBC Papers in Press, November 1, 2002, DOI 10.1074/jbc.M208802200

2 M. Francin, unpublished results.

    ABBREVIATIONS

The abbreviations used are: aaRS, aminoacyl-tRNA synthetase (standard amino acid abbreviations precede RS throughout); tIF, tRNA-interacting factor; HIV, human immunodeficiency virus; HIV-1, HIV type 1; N-LysRS, N-terminal polypeptide extension of LysRS; LysRS-Delta N, N-terminally truncated derivative of LysRS.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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