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
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ABSTRACT |
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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 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 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 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.
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
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
The N-terminally truncated derivative (LysRS-
Protein concentration was determined by using calculated absorption
coefficients of 0.547 and 0.600 A280 units × mg Gel Retardation Assay--
Plasmids
ptRNA
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
[
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 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 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.
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-
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- 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 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
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
To investigate this possibility, we prepared an in vitro
transcribed, 32P-labeled amino acid acceptor minihelix
(AccLys) of tRNA 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
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- 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
tRNAC stem-loop of tRNA
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of T. thermophilus PheRS,
or the C terminus of T. thermophilus ValRS interact with the
variable arm and T
C loop of tRNASer or with the
dihydrouridine and T
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).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol. Fractions containing LysRS were
dialyzed (30 mM potassium phosphate, pH 7.5, 1 mM EDTA, 10% glycerol, and 10 mM
-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
-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.
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).
1 × cm2, respectively for LysRS (and
LysRS mutants) and LysRS-
N.
-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.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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N, displayed a 50-fold lower apparent affinity for
tRNA
1) were
identical. This result extends our earlier observation showing that
LysRS and LysRS-
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.
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.
1 as compared with 4.85 s
1 for wild-type, moderate but reliable variations of the
Km values for
tRNA
Kinetic constants of wild-type and mutant LysRS in
tRNA
N did not (26). Mutants K8A, K10A, and K16A, which
exhibited kinetic parameters similar to wild-type, interacted with
tRNA
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Fig. 2.
Binding of mutant LysRSs to
tRNA
-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-
N, binds and aminoacylates RNA minihelices
derived from the acceptor-T
C stem-loop of
tRNA
C stem-loop structure of
tRNA
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Fig. 3.
Binding of mutant LysRSs to the
acceptor-T C stem-loop of
tRNA
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-
N (Fig. 4).
LysRS-
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-
N with N-LysRS was not
significantly improved as compared with LysRS-
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-
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 N), the isolated N-domain (N-LysRS), or an equimolar mixture
of N-LysRS and LysRS-
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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
N. The
results of tRNA
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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 C stem-loop
of tRNA
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 -sheet and contains the three canonical sequence motifs
that typify the class-II aminoacyl-tRNA synthetases. The N-terminal
-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
-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
-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
-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
-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
-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
-helix (Fig. 6), two features that support the
possibility of an interaction with the acceptor stem of
tRNALys.
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Apart from the tRNA binding residues identified in this work (K19, K23,
R24, and K27), the predicted -helix also contains basic residues
R25, K30, K31, and K35. Among these residues, the substitution of
lysine 31 into alanine increased Km for tRNA
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-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
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ACKNOWLEDGEMENTS |
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We are very grateful to Dr.
Frédéric Dardel for providing plasmid pBSTK3 for the
expression of human tRNA
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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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-N, N-terminally truncated derivative of LysRS.
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