From the Laboratoire d'Enzymologie et Biochimie
Structurales, CNRS, 91190 Gif-sur-Yvette, France and the
§ Institute of Molecular Biology and Genetics, National
Academy of Sciences of Ukraine, Kiev 232143, Ukraine
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ABSTRACT |
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In mammalian cells valyl-tRNA synthetase (ValRS)
forms a high Mr complex with the four subunits
of elongation factor EF-1H. The Aminoacyl-tRNA is the donor of amino acid in ribosomal protein
synthesis. The tRNA molecule is aminoacylated with the corresponding amino acid by an aminoacyl-tRNA synthetase, the aminoacyl-tRNA is
converted to a ternary complex with elongation factor 1 Channeling, or direct transfer of metabolites from one enzyme to
another in a metabolic pathway, is believed to increase significantly the efficiency of the overall reaction (12). For sequential metabolic
enzymes, the stimulation of activity of the first enzyme induced by a
protein-protein interaction with the second enzyme provides a
structural basis for a channeling mechanism. As far as the protein
biosynthesis machinery is concerned, the possible regulation of
aspartyl- (13) and phenylalanyl- (14) tRNA synthetase activities by
elongation factor EF-1 The only stable macromolecular assemblage that involves an
aminoacyl-tRNA synthetase and elongation factor 1 Protein Purification--
The ValRS·EF-1H complex was isolated
from rabbit liver according to a procedure adapted from that used to
isolate the multisynthetase complex (18). Briefly, livers (1 kg) from
13 rabbits were homogenized in three portions (w/v; total volume 2 liters) of extraction buffer (50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.1 mM EDTA, 10%
glycerol, 1 mM DTE (1,4-dithioerythritol)) containing 1 mM diisopropyl fluorophosphate. Postmitochondrial
supernatant was adjusted to 2% polyethylene glycol-6000 by addition of
a 50% stock solution in extraction buffer and stirred for 30 min at
4 °C. After centrifugation at 10,000 × g for 30 min, the supernatant was recovered, adjusted to 5% polyethylene
glycol-6000, and stirred for 30 min at 4 °C. After centrifugation,
the precipitate was dissolved in 200 ml of 25 mM potassium
phosphate (pH 7.6), 5% glycerol, and 10 mM 2-mercaptoethanol. The homogeneous solution was applied to a Bio-Gel A-5m column (Bio-Rad; 90 × 910 mm) equilibrated with the same buffer containing 10% glycerol. Fractions containing valyl-tRNA synthetase activity were combined and loaded on a tRNA-Sepharose column
(50 × 315 mm) equilibrated with the same buffer. The enzyme was
eluted by a linear gradient of 25-400 mM potassium
phosphate buffer (pH 7.6). Fractions with valyl-tRNA synthetase
activity were pooled and applied to a Source 15Q column (Amersham
Pharmacia Biotech; 20 × 95 mm) equilibrated in 25 mM
Tris-HCl (pH 7.5), 50 mM KCl, 10% glycerol, and 10 mM 2-mercaptoethanol and developed with a 50-1000
mM KCl gradient in the same buffer. Fractions with ValRS
activity were pooled, concentrated by vacuum dialysis, and stored at
Dissociation of the ValRS·EF-1H complex and isolation of its ValRS,
EF-1
The free form of elongation factor 1
ValRS from Saccharomyces cerevisiae, phenylalanyl-tRNA
synthetase, and the multisynthetase complex from rabbit liver were purified as described previously (14, 18, 19). Homogeneous EF-Tu from
Escherichia coli was generously provided by Dr. Andrea Parmeggiani (Ecole Polytechnique, Palaiseau, France).
Enzymatic Assays--
Initial rates of tRNA aminoacylation were
measured at 25 °C in 0.1 ml of 20 mM imidazole-HCl
buffer (pH 7.5), 3 mM KCl, 15% glycerol, 1 mg/ml bovine
serum albumin, 0.5 mM DTE, 5 mM
MgCl2, 3 mM ATP, 60 µM
[14C]valine (NEN Life Science Products, 50 Ci/mol), and
saturating amounts (5-10 µM) of partially purified beef
liver tRNA (valine acceptance of 325 pmol/A260).
Where indicated, the incubation mixture also contained GDP, GTP, or
GMP-PNP, with or without EF-1
Saturation kinetics of Val-tRNAVal synthesis by ValRS (2 nM) from the ValRS·EF-1H complex in the presence or in
the absence of an excess of free EF-1
For measuring the activity of EF-1
To obtain the GTP form of bacterial EF-Tu, purified EF-Tu was incubated
with 100 µM GTP in an incubation mixture containing 25 mM Tris-HCl (pH 7.5), 50 mM NH4Cl,
10 mM MgCl2, 1 mM DTE, 0.5 mM EDTA, in the presence of pyruvate kinase (30 µg/ml) and phosphoenolpyruvate (1 mM) to remove traces of
GDP. Incubation was conducted at 30 °C for 15 min, and the
EF-Tu·GTP preparation was used immediately.
EF-1
Therefore, we devised an aminoacylation assay designated to test a
putative effect of EF-1 EF-1 Bacterial EF-Tu Cannot Replace Eukaryotic EF-1
Since EF-Tu has a much lower affinity for GTP than for GDP (24) and
cannot be charged with GTP by the nucleotide exchange subunits of EF-1H
(unpublished observation), nucleotide exchange was performed by
preincubation of the factor with an excess of GTP in the presence of
phosphoenolpyruvate and pyruvate kinase. When EF-1 EF-1
These two monomeric ValRS species were assayed for their potential to
be stimulated by EF-1
In the case of yeast ValRS, for which dissociation of the
aminoacyl-tRNA was shown to be the rate-limiting step in the
aminoacylation reaction (25), addition of EF-1 EF-1 The EF-1 Mammalian ValRS is a 1265-amino acid protein (27). As compared with its
bacterial counterpart, the human enzyme displays a large
NH2-terminal polypeptide extension dispensable for its activity (20). In vitro reconstitution experiments have
shown that this domain interacts with the The EF-1,
, and
subunits, that
contribute the guanine nucleotide exchange activity of EF-1H, are
tightly associated with the NH2-terminal polypeptide
extension of valyl-tRNA synthetase. In this study, we have examined the
possibility that the functioning of the companion enzyme EF-1
could
regulate valyl-tRNA synthetase activity. We show here that the addition
of EF-1
and GTP in excess in the aminoacylation mixture is
accompanied by a 2-fold stimulation of valyl-tRNAVal
synthesis catalyzed by the valyl-tRNA synthetase component of the
ValRS·EF-1H complex. This effect is not observed in the presence of
EF-1
and GDP or EF-Tu·GTP and requires association of valyl-tRNA synthetase within the ValRS·EF-1H complex. Since valyl-tRNA
synthetase and elongation factor EF-1
catalyze two consecutive steps
of the in vivo tRNA cycle, aminoacylation and formation of
the ternary complex EF-1
·GTP·Val-tRNAVal that serves
as a vector of tRNA from the synthetase to the ribosome, the data
suggest a coordinate regulation of these two successive reactions. The
EF-1
·GTP-dependent stimulation of valyl-tRNA
synthetase activity provides further evidence for tRNA channeling
during protein synthesis in mammalian cells.
INTRODUCTION
Top
Abstract
Introduction
References
, to give the
immediate precursor of amino acid for protein synthesis: EF-1
·GTP·aminoacyl-tRNA. Several lines of evidence have
suggested that in mammalian cells the translational apparatus is highly organized. In particular, association of the protein vectors of tRNA,
aminoacyl-tRNA synthetases and elongation factors, with the
cytoskeletal framework has been reported (1-3) and colocalization of
these components described (4). The isolation and characterization of
supramolecular assemblies of aminoacyl-tRNA synthetases and elongation
factors (5-7) have provided structural evidence for the subcellular
organization of the protein synthesis machinery. The existence of a
channeled tRNA cycle during mammalian protein synthesis provided
functional evidence for cellular compartmentalization of translation
(8-10). According to the proposed channeling scheme, aminoacyl-tRNAs
are vectorially transferred from the aminoacyl-tRNA synthetases to the
ribosomes as ternary complexes EF-1
·GTP·aminoacyl-tRNA (8, 10).
Moreover, the GDP form of EF-1
could be involved in the capture of
deacylated tRNA at the exit site of the ribosome and its delivery to
the synthetase (11).
has been reported. In both cases, no stable
protein-protein interaction between the synthetase and the elongation
factor was detected. In connection with tRNA channeling from
aminoacyl-tRNA synthetase to EF-1
, two enzymes ensuring consecutive
steps of the tRNA cycle, the multienzyme complex containing valyl-tRNA
synthetase and EF-1H deserves special mention.
is the
ValRS1·EF-1H complex (15,
16). EF-1H, the "heavy" form of the translation elongation factor
1, is a pentameric complex of the four subunits
,
,
, and
in molar ratio 2:1:1:1 (17). EF-1
forms a ternary complex with
aminoacyl-tRNA and GTP to give the active species of amino acid for
protein synthesis and delivers aminoacyl-tRNA to the A site of the
ribosome. The EF-1
subunits contribute the guanine nucleotide
exchange activity to regenerate EF-1
·GTP from EF-1
·GDP. The
finding that in mammalian cells valyl-tRNA synthetase is exclusively
found as a complex with EF-1H has suggested that this association might
contribute an essential function in vivo. Here we show that
EF-1
controls the aminoacylation reaction catalyzed by ValRS. Our
results demonstrate that association of ValRS with EF-1H is absolutely
required for the stimulation in trans by EF-1
. The
ValRS·EF-1H complex provides a structural basis for the functional
interaction between ValRS and EF-1
. The coupling of these two
consecutive reactions during protein biosynthesis could have a
regulatory function.
EXPERIMENTAL PROCEDURES
20 °C after dialysis against 25 mM potassium phosphate (pH 7.6), 50% glycerol, and 2 mM DTE.
, and EF-1
subunits in their native state was conducted in
the presence of 0.5 M NaSCN as reported previously (19). The free truncated ValRS species deprived of its
NH2-terminal extension was obtained by controlled elastase
treatment, as described (20).
was purified to homogeneity
from rabbit liver as described in (14) and stored at
80 °C in 25 mM Tris-HCl (pH 7.5), 25 mM NH4Cl,
2 mM MgCl2, 2 mM DTE, and 25% glycerol.
. Catalytic amounts of ValRS were added
(1-5 nM) after appropriate dilution in 20 mM
Tris-HCl (pH 7.5), 10% glycerol, 0.2 mM DTE, and 4 mg/ml
bovine serum albumin. All dilutions and incubations were conducted in
plastic tubes. The reaction mixture was preincubated at 25 °C for 2 min and the aminoacylation reaction was started by addition of tRNA.
Incubation was conducted at 25 °C. Aliquots were withdrawn at times
indicated and immediately quenched in 5% trichloroacetic acid.
Precipitated tRNA was collected on GF/C filters (Whatman). Filters were
dried and counted in Lipoluma scintillation fluid (Lumac LSC). One unit
of activity is the amount of enzyme producing one nmol of
aminoacyl-tRNA/min, at 25 °C.
(500 nM) were
obtained in the presence of 100 µM GTP in the incubation
mixture. Michaelian parameters Km and
kcat were determined by nonlinear regression of the theoretical Michaelis equation to the experimental curves using the
KaleidaGraph 3.0.4 software (Abelbeck Software).
, the guanine nucleotide exchange
assay was carried out as described previously (20). The
EF-1
·[3H]GDP complex was prepared following
incubation of 6 µM EF-1
with 4 µM
[3H]GDP (Amersham Pharmacia Biotech; 1500 Ci/mol) in 80 µl of 35 mM Tris-HCl (pH 7.5) containing 0.5 mM DTE, 8.6 mM magnesium acetate, 100 mM NH4Cl, 1 mg/ml bovine serum albumin, and
18% glycerol for 5 min at 37 °C. The reaction mixture was put on
ice and diluted by addition of 800 µl of ice-cold exchange buffer (20 mM Tris-HCl (pH 7.5), 10 mM magnesium acetate,
50 mM NH4Cl, 0.1 mg/ml bovine serum albumin).
The exchange reaction was conducted at 0 °C after addition of
exchange buffer containing nucleotide and specified exchange factors.
Aliquots were taken at times indicated and immediately filtered through
nitrocellulose filters (Sartorius; pore size 0.45 µm). Filters were
washed three times with 1 ml of ice-cold washing buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl2,
100 mM NH4Cl, 0.1 mg/ml bovine serum albumin),
dried, and counted in Lipoluma scintillation fluid.
RESULTS
·GTP Stimulates ValRS Activity in the ValRS·EF-1H
Complex--
Earlier studies suggested that tRNA aminoacylation
catalyzed by ValRS is independent of its association with EF-1
.
Indeed, the intrinsic specific activity of the enzyme, determined in
the aminoacylation reaction, was not affected following its
dissociation from the EF-1H complex (15, 19). However, whereas the
EF-1
, -
, and -
subunits are tightly bound to ValRS, the
EF-1
subunit can be easily depleted from the ValRS·EF-1H complex
(21). This finding suggested that the ternary complex
EF-1
·GTP·Val-tRNAVal is readily dissociated from the
ValRS·EF-1H complex following completion of a single aminoacylation
cycle. We reasoned that if valylation of tRNAVal by ValRS
is controlled by EF-1
, this effect should be detected only in the
presence of an excess of free EF-1
. If ValRS and EF-1
activities
are coupled, as expected if tRNA is channeled from the synthetase to
the elongation factor, addition in the reaction mixture of free EF-1
in excess, that could be effectively transformed into its EF-1
·GTP
species by the
,
, and
subunits of EF-1H in the presence of
GTP, should result in an enhanced rate of Val-tRNAVal
formation. When all EF-1
is transformed to a ternary complex with
the aminoacylated tRNA, this stimulation should ceased.
·GTP on the valylation efficiency catalyzed
by the ValRS component of the ValRS·EF-1H complex. In the assay
procedure used in our study, the reaction mixture containing the
ValRS·EF-1H complex and free EF-1
in excess, but deprived of tRNA,
was incubated 2 min at 25 °C before the aminoacylation reaction was
started by addition of the tRNA substrate. This preincubation was
necessary to avoid a lag in the time course experiments described below. Presumably, EF-1H is preloaded with EF-1
·GTP during this initial incubation. When catalytic amounts of the ValRS·EF-1H complex
(1.5 nM) is incubated in the presence of an excess of free
EF-1
(500 nM) and saturating amounts of the substrates
for the aminoacylation reaction, in the presence of GTP to produce the
active species EF-1
·GTP, the rate of valyl-tRNAVal
synthesis was 2-fold increased, raising from 1.2 pmol/min to 2.1 pmol/min (Fig. 1A). GTP alone
had no effect on the rate of Val-tRNAVal formation. This
increased aminoacylation rate was especially observed during the first
minutes of the incubation, when the free form of EF-1
is still in
excess. As exemplified in Fig. 1B with a higher amount of
the ValRS·EF-1H complex (3 nM), when consumption of free
EF-1
is faster, the time course of valyl-tRNAVal
formation is clearly biphasic. The initial rate in the presence of
EF-1
·GTP (4.7 pmol of valyl-tRNAVal formed/min) is
2-fold that observed in the absence of EF-1
(2.4 pmol/min). After
the reaction proceeded for about 9 min, producing 45 pmol of
valyl-tRNAVal, corresponding approximately to the amount of
EF-1
added in the incubation mixture (50 pmol), the time course of
aminoacylation returned to its control value determined in the absence
of EF-1
. As shown in the inset of Fig. 1A, in
the presence of GTP the duration of the stimulation and the apparent
initial rate of valyl-tRNAVal synthesis are functions of
the amount of free EF-1
added in the incubation mixture.
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Fig. 1.
Stimulation of [14C]valyl-tRNA
formation by valyl-tRNA synthetase in the presence of an excess of free
EF-1 . The time course of the
tRNAVal aminoacylation reaction catalyzed by ValRS from the
purified ValRS·EF-1H complex was determined under the standard
assay conditions, in 0.1 ml of the incubation mixture without additives
(
) or containing 100 µM GTP (
) or 100 µM GTP and 500 nM free homogenous EF-1
(
). The mean value of three independent experiments and the
associated S.E. values are indicated. A, the time course of
Val-tRNAVal formation by 1.5 nM ValRS. A
possible biphasic fitting of the experimental values is indicated by a
dotted line. Inset, dependence of the initial rates of
Val-tRNAVal formation on EF-1
concentration in the assay
mixture, in the presence of 100 µM GTP. B, the
time course of Val-tRNAVal synthesis catalyzed by 3 nM ValRS.
Stimulation of ValRS Activity Is
GTP-dependent--
The GTP form of elongation factor
EF-1
, and therefore its ability to contribute a ternary complex
EF-1
·GTP·Val-tRNAVal, is absolutely required to
stimulate the aminoacylation activity of ValRS. Indeed, in the presence
of GDP instead of GTP, the rate of valylation of tRNAVal by
the ValRS·EF-1H complex is not affected by the presence of a large
excess (200-fold) of free EF-1
(Fig.
2). The nonhydrolyzable GTP analogue
GMP-PNP did produce a stimulation of ValRS activity in the presence of
EF-1
, albeit to a lesser extent, as compared with GTP (Fig. 2). This
lower efficiency could be due to a slower rate of GDP/GMP-PNP exchange,
as compared with the GDP/GTP exchange, leading to a decreased rate of
dissociation of EF-1
·GMP-PNP·Val-tRNAVal from the
ValRS·EF-1H complex, as compared with the regular ternary complex
EF-1
·GTP·Val-tRNAVal. Accordingly, the rate of
dissociation of GDP from the EF-1
·[3H]GDP complex in
the presence of an excess of free GMP-PNP is not as fast as that
observed in the presence of GTP (Fig. 3). Similarly, EF-1
·GMP-PCP has a stronger affinity for the EF-1
subunits than EF-1
·GTP, thus slowing down the exchange of GDP from
EF-1
·GDP (22). Alternatively, the inability of GMP-PNP to mimic
GTP for the stimulation of ValRS by EF-1
also suggests that GMP-PNP,
from which Pi cannot be released, has not the potential of
GTP to produce a conformational change in the ValRS·EF-1H complex that would favor the dissociation of the ternary complex
EF-1
·GTP·Val- tRNAVal.
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Fig. 2.
Nucleotide-dependent activation
of valyl-tRNA synthetase in the presence of
EF-1 . The time course of
valyl-tRNAVal synthesis by ValRS (2.5 nM) from
the purified ValRS·EF-1H complex was determined under the standard
assay conditions, in 0.1 ml of the incubation mixture supplemented with
140 µM GMP-PNP (
) or containing free EF-1
in excess
(500 nM) and 100 µM GDP (
), 140 µM GMP-PNP (
) or 100 µM GTP (
).
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Fig. 3.
Effect of nucleotide on the GDP exchange
reaction catalyzed by EF-1H from the ValRS·EF-1H complex. The
time course of GDP exchange from EF-1 ·[3H]GDP (400 nM) was determined at 0 °C in the absence (
) or in
the presence (
,
,
) of the EF-1H subunits (4 nM)
from the ValRS·EF-1H complex. The reaction was initiated by addition
of 100 µM GDP (
,
), 100 µM GTP (
),
or 140 µM GMP-PNP (
), as described under
"Experimental Procedures." Radioactivity of [3H]GDP
bound to EF-1
before addition of a chasing nucleotide was taken as
100%.
--
The
simplest mechanism that might account for the observed
EF-1
·GTP-dependent stimulation of ValRS activity would
merely imply that the Val-tRNAVal formed is sequestered by
EF-1
·GTP, thereby preventing product inhibition of ValRS activity.
We tested this possibility by adding to the aminoacylation mixture the
prokaryotic analogue of EF-1
, EF-Tu, that is known to interact, in
its GTP-bound form, with eukaryotic aminoacyl-tRNAs (23).
·GTP was
substituted by EF-Tu·GTP in the aminoacylation reaction, no
stimulatory effect of EF-Tu·GTP on the ValRS aminoacylation activity
was observed at different concentrations of the factor (0.5-2
µM) (Fig. 4). This result
suggested that the EF-1
-induced stimulation of Val-tRNA synthesis
catalyzed by the ValRS component of the ValRS·EF-1H complex is
contributed by a protein-protein interaction requiring cognate factors
from higher eukaryotic origin.
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Fig. 4.
Effect of bacterial EF-Tu on eukaryotic
valyl-tRNA synthetase activity. The time course of the
aminoacylation reaction catalyzed by ValRS (1.7 nM) from
the ValRS·EF-1H complex was followed in the presence of 100 µM GTP ( ) or 100 µM GTP and 0.5 µM (
) or 2 µM (
) EF-Tu·GTP in the
aminoacylation reaction. GTP-bound EF-Tu was obtained as described
under "Experimental Procedures."
·GTP Stimulation Requires a Native ValRS·EF-1H
Complex--
To test for the requirement of the native
ValRS·EF-1H assembly in the EF-1
·GTP stimulation of ValRS
activity, two ValRS derivatives that behave as free species were
isolated from the complex. It was shown previously that the ability of
ValRS to associate with EF-1H is lost upon conversion by elastase of
the native enzyme of 140 kDa to a fully active truncated form of 125 kDa (20). The 200-amino acid extension of ValRS lost upon elastase
treatment is involved in the assembly of the ValRS·EF-1H complex
through protein-protein interaction with the
subunit of EF-1H.
Furthermore, native ValRS can be dissociated from the EF-1H components
in the presence of 0.5 M NaSCN, a chaotropic salt (19). The
resulting monomeric ValRS species is isolated without loss of its
aminoacylation activity.
·GTP in conditions where the native ValRS·EF-1H species does. Neither the truncated free ValRS, nor the
native free ValRS, could be activated by the addition of preformed EF-1
·GTP in the incubation mixture (Fig.
5). Further addition of the EF-1
subunits in the assay had no effect on ValRS activity (Fig. 5). As
shown previously, the EF-1
and -
subunits form a stable complex
that efficiently exchanges the nucleotide from EF-1
·GDP but does
not associate to the isolated 140- or 125-kDa ValRS species (20). In
the absence of the EF-1
subunit the ValRS·EF-1H complex cannot be
reconstituted. On the other hand, the EF-1
subunit associates with
the native 140-kDa ValRS form (20), but in the absence of the
and
subunits, this nucleotide exchange factor alone did not confer on
EF-1
the ability to stimulate ValRS activity (not shown). Therefore,
the association of ValRS with the
,
, and
subunits of EF-1H
seems to be absolutely required to produce a functional interaction
between the synthetase and elongation factor EF-1
. This result
suggests that a proper positioning of EF-1
·GTP and
ValRS·Val-tRNAVal contributes an essential step for the
activation of the synthetase activity. The presence in solution of a
competitor protein (EF-1
·GTP or EF-Tu·GTP) with a high affinity
for the newly synthesized aminoacyl-tRNA cannot per se
explain the observed stimulation effect.
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Fig. 5.
Effect of EF-1 on
the activity of valyl-tRNA synthetase dissociated from the
ValRS·EF-1H complex. The aminoacylation activity of the
monomeric ValRS dissociated from the complex as a
NH2-terminally truncated form after elastase treatment
(ValRS-
N) or recovered in the native state after NaSCN
treatment of the complex (free ValRS) was assayed in the
presence (
) or in the absence (
) of EF-1
·GTP in the
incubation mixture. 100 µM GTP and 110 nM
EF-1
were also added in the aminoacylation mixtures to convert
EF-1
to its GTP-bound form. Enzyme concentrations were 3 nM for the two ValRS species and 500 nM for
EF-1
.
, EF-1
, and GTP
proved to have no stimulatory effect (result not shown). Similarly,
incubation of the multisynthetase complex from rabbit containing nine
aminoacyl-tRNA synthetases (18) in the presence of EF-1
·GTP
did not confer on methionyl-, lysyl-, or aspartyl-tRNA synthetases
increased aminoacylation rates (results not shown).
·GTP Increases the Apparent kcat of the
Aminoacylation Reaction Catalyzed by ValRS--
The Michaelian
parameters for tRNA in the aminoacylation reaction catalyzed by ValRS
from the ValRS·EF-1H complex were determined in the presence or in
the absence of EF-1
·GTP in the incubation mixture. Kinetic
parameters were determined using 0.05-3 µM beef liver
tRNA enriched in tRNAVal (325 pmol/A260). The apparent dissociation constant
(Km) for tRNA of 0.33 ± 0.08 µM
was not affected by the addition of EF-1
·GTP in the aminoacylation
reaction. By contrast, the catalytic constant
kcat was found to be increased 2-fold, raising
from 0.17 ± 0.03 s
1 to 0.32 ± 0.04 s
1 in the presence of EF-1
·GTP. Therefore, we
suggest that the major effect of the interaction of ValRS·EF-1H with
EF-1
·GTP is in increasing the turnover number of ValRS, through a
conformational change that would promote the release of
Val-tRNAVal.
DISCUSSION
-induced stimulation of ValRS can be detected in the
presence of GTP but not of GDP. This finding clearly demonstrates the
importance of the ternary complex
EF-1
·GTP·Val-tRNAVal for the stimulation mechanism.
As expected, activation is observed as long as some free EF-1
·GTP
is available in the incubation mixture. When all EF-1
is converted
to EF-1
·GTP·Val-tRNAVal, ValRS is no longer
stimulated, suggesting that EF-1
does not rapidly dissociate from
GTP and Val-tRNAVal. Although the intrinsic GTPase activity
of EF-1
is stimulated by the aminoacyl-tRNA (26), EF-1
does not
appear to be efficiently recycled in this assay. Accordingly, it is
possible to substitute GTP by GMP-PNP. This nonhydrolyzable GTP
analogue can be regarded as mimicking the nucleotide substrate in its
ground state complex. Therefore, the endogenous GTPase activity of
EF-1
is not involved in the stimulation of ValRS activity.
subunit of EF-1H (20). The finding that neither the native enzyme of 140 kDa nor the truncated
125-kDa ValRS species are susceptible to EF-1
mediated stimulation
also demonstrates that the NH2-terminal extension of ValRS
is not per se engaged in a functional interaction with EF-1
. Noteworthy, the NH2-terminal polypeptide extension
of the multifunctional glutamyl-prolyl-tRNA synthetase, and the p18
auxiliary component of the multisynthetase complex display sequence
similarities with this domain (28). This observation led to the
suggestion that these polypeptide domains could be involved in the
transient anchoring of EF-1
to the complex. Though we were unable to
detect any effect of EF-1
·GTP on the activity of three components
of the multisynthetase complex, namely lysyl-, aspartyl-, and
methionyl-tRNA synthetases, we cannot rule out the possibility that a
specialized adaptor molecule could be involved to provide a functional
interaction between EF-1
and those synthetases, a role played by the
EF-1
subunits in the case of the ValRS·EF-1H complex.
-induced activation of tRNAVal aminoacylation by
ValRS requires the whole ValRS·EF-1H complex to occur. Neither
bacterial EF-Tu can substitute for mammalian EF-1
, nor free
dissociated ValRS can mimic the ValRS component of the ValRS·EF-1H
complex. Furthermore, the isolated guanine-nucleotide
exchange factors, EF-1
or EF-1
, cannot individually sustain
the activation of ValRS by EF-1
. All these results illustrate the
direct connection between regulation of ValRS activity and the adequate
association of ValRS and EF-1
within a macromolecular assemblage of
defined composition and structure. The ValRS·EF-1H complex should
provide a structural support for the functional interaction of ValRS
with EF-1
. A synoptic model is outlined in Fig.
6. ValRS is tightly associated to the
,
, and
subunits of EF-1H. EF-1
is loosely bound to this
complex: (i) it is easily dissociated from the other components
following chromatography on a Mono Q column (21) or following
incubation with aminoacyl-tRNA and GTP (17); (ii) catalytic amount of
the ValRS·EF-1H complex can efficiently exchange GTP for GDP from a
100-fold molar excess of EF-1
·[3H]GDP (Fig. 3).
Therefore, EF-1
·GDP can be recycled into EF-1
·GTP by the
exchange factors bound to ValRS even in the absence of synthetase
activity, at least in vitro (Fig. 6, top right).
In parallel, the ValRS component of the complex is able to catalyze tRNAVal aminoacylation even in the absence of GTP or of an
excess of free EF-1
, that is when the elongation factors are not
functioning (Fig. 6, top left). However, the concomitant
functioning of ValRS and of the EF-1 subunits is accompanied by a
2-fold increase in the valylation rate. Since the
kcat parameter is primarily affected, the
EF-1
·GTP facilitated ValRS activity is most likely the result of a
protein-protein interaction that induces a conformational change in
ValRS, and promotes the release of Val-tRNAVal from the
enzyme, suggesting that product release is the rate-limiting step.
Coupling of these two parallel reactions would favor the direct
transfer of the aminoacylated tRNA to the elongation factor, with
formation of the ternary complex
EF-1
·GTP·Val-tRNAVal (Fig. 6, bottom).
In vivo, this coupling could be responsible for the
channeling of tRNA reported by Deutscher and co-workers (8, 10). Using
a permeabilized cell system, they observed that exogenously added tRNA
or aminoacyl-tRNA are not effective precursors for protein synthesis,
suggesting that there is a channeled tRNA cycle in mammalian cells. In
the case of the ValRS system, our results suggest that the release of
the aminoacylated tRNA from the synthetase could be controlled by the
availability of free EF-1
, therefore providing a rational
explanation for channeling. In that connection, it should be stressed
that a great deal of data reports that members of the EF-1H and of the
ValRS·EF-1H complexes are the targets of phosphorylation events (21,
29-32). Hyperphosphorylation of elongation factor 1
is also
observed in virus infected cells (33). The functional significance of these modifications is poorly understood but could alter translational efficiency. Whether these postranslational modifications have a role on
the EF-1
-induced stimulation of ValRS, and therefore on tRNA
channeling in vivo remains to be shown, but could be a means
to regulate the efficiency of tRNA delivery for protein synthesis.
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Fig. 6.
Channeling of tRNAVal in the
valyl-tRNA synthetase·elongation factor 1H complex. The
ValRS·EF-1H complex is a dimer of an elementary core made of
equimolar amounts of each subunit. For clarity, only one monomeric core
containing one copy of ValRS and of the ,
,
, and
subunits
of EF-1 is shown. Two parallel reactions take place on this complex:
specific recognition of tRNAVal by the synthetase followed
by its aminoacylation by valine and association of EF-1
·GDP to the
and/or
subunits of EF-1H to exchange its nucleotide with GTP.
The EF-1
·GTP-dependent stimulation of
tRNAVal aminoacylation suggests that these two reactions
are concerted mechanisms. Formation of the ternary complex
EF-1
·GTP·Val-tRNAVal on the ValRS·EF-1H complex
would be responsible for the vectorial transfer of tRNA from the
synthetase to the ribosome.
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ACKNOWLEDGEMENTS |
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The valuable comments from an anonymous reviewer are gratefully acknowledged.
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FOOTNOTES |
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* This work was supported in part by grants from the CNRS and the Association pour la Recherche sur le Cancer (France), by Grant 5. 4/73 from Ministry for Science and Technologies of Ukraine, and by Grant 96-1594 from International Association for the Promotion of Cooperation with Scientists from the New Independent States of the Former Soviet Union.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 East European Fellowship from EMBO.
Recipient of a short term fellowship from FEBS.
** To whom correspondence should be addressed: Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. Tel.: 33-1-69-82-35-05; Fax: 33-1-69-82-31-29; E-mail: marc.mirande{at}lebs.cnrs-gif.fr; . .
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ABBREVIATIONS |
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The abbreviations used are:
ValRS, valyl-tRNA
synthetase;
DTE, 1,4-dithioerythritol;
GMP-PNP, ;
-imidoguanosine
5'-triphosphate.
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REFERENCES |
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