Báo cáo Y học: tRNA-dependent amino acid discrimination by yeast seryl-tRNA synthetase pdf

tRNA-dependent amino acid discrimination by yeast seryl-tRNA
synthetase
Ita Gruic-Sovulj
1,2
, Irena Landeka
1,2
, Dieter So¨ll
3
and Ivana Weygand-Durasevic
1,2
1
Department of Chemistry, Faculty of Science, University of Zagreb, Croatia;
2
Rudjer Boskovic Institute, Zagreb, Croatia;
3
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA
The ability of aminoacyl-tRNA synthetases to distinguish
betweensimilaraminoacidsiscrucialforaccuratetrans-
lation of the genetic code. Saccharomyces cerevisiae
seryl-tRNA synthetase (SerRS) employs tRNA-dependent
recognition of its cognate amino acid serine [Lenhard, B.,
Filipic, S., Landeka, I., Skrtic, I., So
¨
ll, D. & Weygand-
Durasevic, I. (1997) J. Biol. Chem. 272, 1136–1141]. Here we
show that dimeric SerRS enzyme complexed with one
molecule of tRNA
Ser
is more specific and more efficient in
catalyzing seryl-adenylate formation than the apoenzyme
alone. Sequence-specific tRNA–protein interactions

enhance discrimination of the amino acid substrate by yeast
SerRS and diminish the misactivation of the structurally
similar noncognate threonine. This may proceed via a
tRNA-induced conformational change in the enzyme’s act-
ive site. The 3¢-terminal adenosine of tRNA
Ser
is not
important in effecting the rearrangement of the serine
binding site. Our results do not provide an indication for a
readjustment of ATP binding in a tRNA-assisted manner.
The stoichiometric analyses of the complexes between the
enzyme and tRNA
Ser
revealed that two cognate tRNA
molecules can be bound to dimeric SerRS, however, with
very different affinities.
Keywords:tRNA
Ser
ÆSerRS complexes; tRNA-dependent
amino acid recognition; amino acid selection; tRNA bind-
ing; covalent cross-linking.
Accurate translation of genetic information is dependent on
the high fidelity of several molecular processes. Different
quality control mechanisms are adapted to prevent or
correct naturally occurring mistakes [1]. Measurements of
the selectivity of aminoacyl-tRNA synthetases in vitro
indicate that the upper limit for error in the selection of
correct amino acid for protein synthesis is in the range of
10
)4

)10
)5
. The frequency of errors involving noncognate
tRNA aminoacylation is, in most cases, 10
)6
or lower [2].
While the selection of the correct tRNA is assumed to occur
as a result of preferential reaction kinetics for the formation
of cognate proteinÆRNA complexes, the differences of the
side chains of amino acids are often sufficient to allow their
specific binding [3]. The basic challenge in achieving high
specificity for the amino acid substrates lies in the rejection
of smaller substrates with similar side chain chemistry or
isosteric amino acids [2]. A number of different pathways
have been proposed to correct misactivation of amino acids
in vivo. The noncognate aminoacyl-adenylate can be
hydrolyzed by several class I synthetases by tRNA-
dependent or tRNA-independent Ôpre-transferÕ editing,
whereas in the Ôpost-transferÕ pathway a mischarged tRNA
is rapidly deacylated in a synthetase-dependent manner
[4–12]. The editing reactions of class II synthetases have
been studied much less than those of class I. Class II
phenylalanyl-tRNA synthetase (PheRS) specifically deacy-
lates Ile–tRNA
Phe
[13], and alanyl-tRNA synthetase
(AlaRS) has been shown to hydrolyze misactivated serine
and glycine [14]. Escherichia coli lysyl-tRNA synthetase
(LysRS) hydrolyzes misactivated homocysteine, homoser-
ine, cysteine, threonine and alanine [15]. Recent reports on

threonyl-(ThrRS) [16,17] and prolyl-tRNA synthetase
(ProRS) [18–20] show clearly that these enzymes misactivate
smaller noncognate amino acids, serine and alanine,
respectively, and therefore require proofreading activity.
However, during the editing process, some of the correct
products are also destroyed [21]. This makes corrective
pathways energetically costly, and they are disfavored in the
cell. Alternatively, the quality of aminoacyl-tRNA synthesis
in the cell can be improved by the tRNA-mediated
mechanisms that enhance the accuracy of amino acid
discrimination. These are based on conformational changes
in the enzymes, induced by the formation of macromolec-
ular complex [22–24] or possibly by the interaction with a
nonsynthetase protein [25,26].
The active site of class II aaRSs contains the motif 2 loop
which is involved in binding of ATP, an amino acid, and the
acceptor end of tRNA. Our earlier investigation revealed
that accurate seryl-tRNA synthesis in Saccharomyces cere-
visiae is accomplished via tRNA-assisted optimization of
amino acid binding to the enzyme active site [27]. To
understand the mechanism by which this occurs, we have
generated a number of SerRS mutants with altered motif 2
Correspondence to I. Weygand-Durasevic, Department of Chemistry,
Faculty of Science, University of Zagreb, Strossmayerov trg 14, 10000
Zagreb, Croatia. Fax: + 385 1 4561177, Tel.: +385 1 4561197,
E-mail:
Abbreviations: aaRS, aminoacyl-tRNA synthetase with amino acid
representingAla,Cys,Gln,Lys,Phe,Pro,SerandThr,thusfor
alanyl- (EC 6.1.1.7), cysteinyl- (EC 6.1.1.16), glutaminyl- (EC
6.1.1.18), lysyl- (EC 6.1.1.6), phenylalanyl- (EC 6.1.1.20), prolyl- (EC

6.1.1.15), seryl- (EC 6.1.1.11) and threonyl-tRNA synthetase (EC
6.1.1.3); aaRSÆtRNA and aaRS–tRNA, noncovalent and covalent
complexes between synthetase and tRNA, respectively.
(Received 27 May 2002, revised 9 August 2002,
accepted 9 September 2002)
Eur. J. Biochem. 269, 5271–5279 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03241.x
loop residues and showed that these mutations affect
tRNA-dependent amino acid recognition, possibly by
interfering with the flexibility of the motif 2 loop [27]. The
same mode of conformational adjustment has been recently
proposed for maize organellar SerRS [28]. To get better
insight into contribution of tRNA in the amino acid
selection, we have generated nonchargeable, 3¢-truncated
tRNA analogs, which retain the ability of complex forma-
tion with SerRS. These tRNAs significantly influence
enzyme affinity for serine, as revealed by steady-state kinetic
studies in the reaction of pyrophosphate exchange and
decrease the misactivation of threonine by yeast seryl-tRNA
synthetase. The stoichiometric analyses of the complexes
between the enzyme and tRNA
Ser
revealed that two cognate
tRNA molecules can be bound to dimeric SerRS, however,
with very different affinities. The binding of the first
tRNA
Ser
is sufficient for complete readjustment of serine
binding site(s).
EXPERIMENTAL PROCEDURES
The overexpression and purification of S. cerevisiae wild-

type and mutant (E281D, G291A) SerRS has been
described [27]. [
14
C]Serine (166.1 mCiÆmmol
)1
)andtetra-
sodium [
32
P]pyrophosphate (2.44 CiÆmmol
)1
)were
purchased from DuPont NEN. The amino acids were from
Sigma. The purity of threonine was checked by ESI-Ion
Trap mass spectrometry. The signal at m/z value that
corresponds to serine was not recorded.
tRNAs
Yeast tRNA
Ser
and tRNA
Tyr
, purified from total brewer’s
yeast tRNA as described previously [29], accepted 1.2 nmol
of serine and 1.4 nmol of tyrosine per A
260
unit of tRNA,
respectively. tRNA integrity was checked by MALDI-TOF
mass spectrometry. A mass resolution m/Dm ¼ 220 (for
tRNA
Tyr
)and170(fortRNA

Ser
) was achieved in the linear
mode of operation [30]. Nonchargeable substrate analogs
were prepared by stepwise modifications of yeast tRNA
Ser
at the 3¢-end. Intact tRNA
Ser
CCA
was first denatured to enable
better exposition of the 3¢-adenosine diol groups and then
oxidized by periodate treatment essentially as described [31],
except that the concentration of periodate was increased
five-fold. As the oxidized product is known as synthetase
inhibitor, tRNA
Ser
ox
was extensively dialyzed prior to
removal of 3¢-terminal nucleoside by b-elimination with
lysine (pH 8.0), followed by alkaline phosphatase treatment
[32]. The acceptor activity of truncated tRNA
Ser
CC
was not
detectable in the standard aminoacylation assay [27]. All
tRNA substrates were carefully renatured prior the use in
the kinetic assays and complex formation experiments by
heating to 80 °C and the temperature was then allowed to
decrease to 50 °C. Then, MgCl
2
was added, to a final

concentration of 10 m
M
, and the tRNA sample was cooled
to 30 °C. E. coli tRNA
Ser
1
was purchased from Subriden
RNA (Rolling Bay, WA, USA).
Synthesis of seryl-adenylate
The extent of adenylate formation was measured by ATP-
PP
i
exchange at 30 °C [27]. Standard reaction mixtures
contained 100 m
M
Hepes/KOH pH 7.2, 10 m
M
MgCl
2
,
0.5 m
M
[
32
P]PP
i
(4–7 c.p.m.Æpmol
)1
), 10 m
M

KF. For the
K
m
determination of serine the concentration varied from
50 l
M
to 900 l
M
, while ATP–MgCl
2
waskeptconstantat
2m
M
. The concentrations of wild-type and mutant enzymes
were between 50 and 100 n
M
, and nonchargeable tRNAs
were in the range of 100–500 n
M
.TheK
m
for ATP was
determined in the standard reaction mixture with ATP–
MgCl
2
varied from 0.4 l
M
to 50 l
M
, and serine was kept

constant at 900 l
M
.
Misactivation
To study the misactivation of threonine by wild-type and
mutated SerRS (141 n
M
), 0.5 m
M
)50 m
M
threonine was
substituted for serine in the standard reaction mixture
for the pyrophosphate exchange. In the experiment designed
to determine the ability of tRNA
Ser
CC
to suppress threonine
misactivation, SerRS and tRNA
Ser
CC
were 100 n
M
.
Inactivation of SerRS by oxidized tRNA
Ser
The inactivation of the SerRS pyrophosphate exchange
activity by tRNA
Ser
ox

was studied with 100 n
M
enzyme and
tRNA
Ser
ox
varied from 0 to 1500 n
M
. In order to preform the
macromolecular complex before binding of small substrates,
SerRS and tRNA
Ser
were preincubated under conditions
favorable for noncovalent complex formation (5 min,
30 °C) and the activation reaction was started by addition
of serine and ATP in the standard reaction mixture for
pyrophosphate exchange.
Gel mobility shift assay
SerRSÆtRNA complexes were prepared by incubation of the
enzyme with variable amounts of freshly renatured tRNA,
for 5 min at 30 °C, in buffer containing 30 m
M
Hepes/KOH
pH 7.0 (or Mes/KOH pH 6.0), 10 m
M
MgCl
2
and 1.6%
glycerol, followed by cooling on ice. Glycerol was added to
a final concentration of 7.5% and the preformed complexes

were subjected to electrophoresis on 6% acrylamide/
bisacrylamide (40 : 1) gel containing 5% glycerol in elec-
trophoresis buffer (25 m
M
Tris, 25 m
M
acetic acid, 10 m
M
magnesium acetate; pH 7.2, or 25 m
M
Mes/KOH, 10 m
M
magnesium acetate, pH 6.0). Electrophoresis was carried
out at 4 °C for 3–4 h at 100 V, and the gels ware stained by
standard silver staining procedure.
Covalent SerRS–tRNA
Ser
complexes
Cross-linking reactions were carried out essentially accord-
ing to [33]. The reaction mixture contained 20 m
M
Hepes
pH8.5, 10m
M
MgCl
2
, 1.7% (v/v) glycerol. SerRS was
0.65 l
M
,andtRNA

Ser
ox
/SerRS ratio was varied between 0.5
and 3.0. SerRS and freshly renatured tRNA
Ser
ox
were
incubated for 5 min at 30 °C to allow noncovalent complex
formation and the cross-linking reaction was started with
the addition of 1 lLNaCNBH
3
(0.5 m
M
). The addition of
NaCNBH
3
was repeated after 20 and 40 min. The final
amount of NaCNBH
3
added was 1.5 nmol. The reaction
was stopped after 60 min by the addition of 0.2 volumes of
4.8 m
M
NaBH
4
in 10 m
M
NaOH. After 10 min glycerol
was added to a final concentration of 8%. The whole
reaction mixture was loaded on the 6% nondenaturing

5272 I. Gruic-Sovulj et al. (Eur. J. Biochem. 269) Ó FEBS 2002
acrylamide/bisacrylamide gel (40 : 1) and the electrophor-
esis took place at 4 °C(3h,100 V)with50m
M
Tris, 25 m
M
boric acid and 10 m
M
magnesium acetate, pH 8.0, as
running buffer.
Stoichiometric analysis
Ferguson plot analysis on disc-PAGE [34] was used for
mass determination of covalent SerRS–tRNA
Ser
ox
complexes.
Preformed covalent complexes between SerRS and
tRNA
Ser
ox
were separated on a series of gels containing 5,
6, 7, 8, 9 and 10% acrylamide/bisacrylamide (29 : 1) in
375 m
M
Tris, 5 m
M
MgCl
2
; pH 8.0 alongside 1–10 lgof
native protein molecular weight standards (Sigma). Stack-

ing gel was 4% acryamide/bisacrylamide (40 : 1) in 62 m
M
Tris, 5 m
M
MgCl
2
pH 6.3. Electrophoresis was performed
at 4 °C, 100 V with 50 m
M
Tris, 5 m
M
MgCl
2
pH8.0as
anode buffer and 10 m
M
Tris, 90 m
M
glutamine pH 8.0 as
cathode buffer. For each species, 100 [log(100l)] was
determined and plotted against gel concentration, where l
is equal to the mobility of the species relative to that of the
bromophenol blue tracking dye. Logarithm of negative
slope or retardation coefficient (K
r
) was then plotted as a
function of log molecular mass (kDa) for each protein
standard, and from the evaluated retardation coefficients of
covalent SerRS–tRNA
Ser

ox
complexes their molecular mass
was determined.
RESULTS
Complex formation with modified cognate
and nonmodified heterologous tRNAs
The integrity of the truncated tRNA
Ser
CC
was first checked by
high-resolution polyacrylamide gel electrophoresis (Fig. 1).
Modified tRNA
Ser
CC
appeared intact and its migration, with
respect to mature tRNA
Ser
CCA
, was in satisfying agreement
with the removal of one nucleotide. Additional bands of
lower intensity, visible in both lanes, are probably due to the
presence of other tRNA
Ser
isoacceptors of different length.
In order to determine the ability of modified tRNAs to
participate in complex formation with the cognate synthe-
tase, preformed complexes of SerRS with either tRNA
Ser
CCA
,

tRNA
Ser
ox
or tRNA
Ser
CC
were analyzed by the gel mobility shift
assay (Fig. 2). Under the conditions described in the
Experimental procedures, native, oxidized and truncated
tRNA
Ser
species form complexes of equal mobility (lanes
1–3, respectively). Although the input ratio of enzyme to
tRNA was the same (1 : 2) in all three cases, the shifted
band that corresponds to the enzymeÆtRNA complex is
somewhat weaker in lane 3. This may suggest that the
removal of 3¢-terminal nucleotide slightly influences the
complex stability. The oxidation of 3¢-terminal adenosine in
tRNA
Ser
does not interfere with complex formation and its
stability, in agreement with previously shown results for the
tyrosyl–tRNA synthetase system [31].
When SerRS was mixed with an excess amount of E. coli
tRNA
Ser
and electrophoresis performed as above (Fig. 2,
lane 4), the gel mobility shift analysis revealed a lack of low
mobility bands. This indicates that heterologous complex
was either not formed or its low stability did not allow

detection under the conditions used. This is an interesting
finding, as E. coli tRNA
Ser
contains a conserved long extra
arm which is considered to be the main recognition element
for all cytosolic SerRS enzymes [35]. Furthermore, this
tRNA is recognized by yeast SerRS in vivo and in vitro [36],
showing that some recognition elements in tRNA are
shared between bacteria and yeast.
Nonchargeable yeast tRNA
Ser
affects serine activation
by SerRS
Kinetic parameters for serine were determined in pyrophos-
phate exchange reaction, catalyzed by yeast SerRS
(Table 1). Under standard conditions in the absence of
tRNA, kinetic parameters are in a very good agreement
with previously reported data [27]. The addition of modified
Fig. 1. Preparation of 3¢-truncated tRNA
Ser
. The size difference
between intact tRNA
Ser
CCA
and truncated tRNA
Ser
CC
was confirmed by
gel electrophoresis (left). The reaction scheme is shown on the right.
Fig. 2. Gel mobility shift assay of SerRS complexes with different

tRNAs. SerRS (9 pmol) was incubated with 18 pmol of tRNA
Ser
CCA
,
tRNA
Ser
ox
,tRNA
Ser
CC
,andE. coli tRNA
Ser
CCA
and subjected to poly-
acrylamide gel under native conditions (lanes 1, 2, 3, and 4, respect-
ively). Noncomplexed tRNA
Ser
(5 pmol) was loaded onto the gel as
electrophoretic mobility marker (lane 5). Full line arrow, noncom-
plexed tRNA; dashed line arrow, SerRSÆtRNA complexes. The bands
migrating between noncomplexed tRNAs and SerRSÆtRNA com-
plexes could be assigned to tRNA oligomers and they also appear in
tRNA lane when a higher amount is loaded (see Fig. 6A, lane 1). This
additional tRNA band appeared when complex formation and elec-
trophoresis were performed at pH 7.0 and above.
Ó FEBS 2002 tRNA-dependent amino acid discrimination (Eur. J. Biochem. 269) 5273
yeast tRNA
Ser
species, which cannot be charged, influences
the kinetics with respect to serine. tRNA

Ser
ox
decreases the K
m
for serine twofold. The reduction of k
cat
may reflect the
interference between the tRNA’s modified 3¢-terminal
adenosine with other substrates in the active site. In the
presence of tRNA
Ser
CC
, which lacks the 3¢-end nucleotide, the
binding of serine is significantly enhanced, as revealed by a
K
m
value decreased by almost an order of magnitude.
Moreover, the K
m
value of 6.0 · 10
)5
M
is very close to that
obtained in the aminoacylation reaction with wild-type
SerRS (6.3 · 10
)5
M
) [27]. An order of magnitude increased
affinity toward serine in the presence of truncated tRNA
Ser

CC
suggests that the 3¢-terminal adenosine of tRNA
Ser
is not
important in effecting the rearrangement of the serine
binding site. It is worth noting that an equimolar concen-
tration of tRNA per dimeric enzyme is capable of inducing
a complete rearrangement of amino acid binding sites, as
judged by full range change of the K
m
value for serine. This
suggests that a
2
Æ(tRNA
Ser
)
1
is a functional complex in seryl-
tRNA formation. The effect of 3¢-truncated tRNA
Ser
on the
activation of serine by the mutated yeast SerRS, bearing two
amino acid changes in the motif 2 loop of the active site
(E281D; G291A), is much less pronounced.
The effect of heterologous tRNA
Ser
on serine binding
Despite the observation that E. coli tRNA
Ser
1

neither is
charged well by yeast enzyme [36], nor forms a stable
noncovalent complex with yeast SerRS (Fig. 2, lane 4), it
affects serine binding properties of yeast synthetase. In the
presence of E. coli tRNA
Ser
1
,theK
m
for serine is decreased
two-fold and the velocity of seryl-adenylate synthesis is
elevated 2.5-fold (Table 1). Thus, interaction of yeast SerRS
with heterologous tRNA
Ser
, that contains the long extra
arm identity element, improves the overall catalytic prop-
erties of the RNAÆsynthetase complex in the pyrophosphate
exchange reaction. Accordingly, even though the binding of
tRNA
Ser
analogs slightly decreased the k
cat
value, which
may be due to the interference of 3¢-CCA
ox
and 3¢-CC
ends with the accessibility of the active site, an observed
increase in k
cat
in the presence of E. coli tRNA suggests that

the tRNAÆSerRS noncovalent complex is catalytically more
efficient in seryl-tRNA formation than noncomplexed
SerRS. On the other hand, the increase in k
cat
may be
consistent with reduced stability of the heterologous
tRNAÆSerRS complex and consequently facilitates substrate
turnover, as previously observed in other systems [23,37]. It
is also important to note that bacterial tRNA
Ser
does not
fully optimize the serine binding site of yeast SerRS, as the
K
m
for serine never reaches the value of 6.5 · 10
)5
M
,
characteristic for aminoacylation of yeast tRNA
Ser
. Never-
theless, heterologous tRNA
Ser
ÆSerRS interactions are suffi-
cient to promote conformational change of SerRS, leading
to increased k
cat
/K
m
value. Thus, tRNA optimized amino

acid activation can occur irrespectively of aminoacylation,
as shown previously for GlnRS [37]. This finding can be
explained by the fact that discrimination between
cognate and noncognate tRNA occurs predominantly
during the transfer reaction [24].
Binding of ATP to yeast SerRS is not mediated
by tRNA
The analysis of kinetic parameters for ATP in seryl-
adenylate formation did not reveal considerable differences
between the K
m
values for ATP in the presence and absence
of tRNAs
Ser
(Table 1). Furthermore, the nature of non-
chargeable or heterologous tRNA
Ser
did not affect the ATP
binding properties of yeast SerRS. Thus, contrary to the
observation based on crystallographic studies of Thermus
thermophilus SerRS complexes [38], our biochemical experi-
ments do not provide indications for tRNA-assisted rear-
rangement of ATP binding site. We have previously
observed a 6.3-fold decrease in the apparent affinity of
ATP for the yeast enzyme in the aminoacylation reaction
compared to the PP
i
exchange [27]. This could be explained
by possible interference of correctly positioned Ser–tRNA
Ser

with ATP binding. If so, the transfer of acyl moiety to
tRNA decreases the affinity of SerRS toward ATP.
Misactivation of threonine by wild-type and mutant
SerRS
Slight misactivation of threonine by wild-type yeast SerRS
(about 0.4%, based on the comparison of turnover numbers
for cognate and noncognate amino acid) has been observed
in the pyrophosphate exchange reaction, in which 0.5–
50 m
M
threonine was substituted for serine (Fig. 3).
Table 1. The influence of nonchargeable tRNAs
Ser
and heterologous tRNAs on seryl-adenylate synthesis by yeast SerRS. Kinetic parameters were
determined in the PP
i
exchange reaction. Numbers in parenthesis denote the concentration of tRNA. The enzymes were at a concentration of
100 n
M
.
K
m
for ATP
(l
M
)
K
m
for Ser
(l

M
)
k
cat
(s
)1
)
k
cat
/K
m, Ser
(sÆl
M
)
)1
SerRS
wt
Without tRNA 14 500 3.9 8 · 10
)3
With tRNA
Ser
ox
(500 n
M
) 13 250 0.94 4 · 10
)3
With tRNA
Ser
CC
(100 n

M
) 16 70 4.9 70 · 10
)3
With tRNA
Ser
CC
20 60 1.8 30 · 10
)3
With tRNA
Ser
CCA
E. coli (100 n
M
) ND 260 10 39 · 10
)3
With tRNA
Ser
CCA
E. coli (500 n
M
) 9 300 10 33 · 10
)3
SerRS (E281D; G291A)
Without tRNA 40 850 0.056 6.59 · 10
)5
With tRNA
Ser
CC
(100 n
M

) ND 490 0.030 6.12 · 10
)5
With tRNA
Ser
CC
(500 n
M
) ND 350 0.028 8.00 · 10
)5
5274 I. Gruic-Sovulj et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Although threonine concentration used in vitro corresponds
to approximately 1–100 · K
m
for serine, these data may be
relevant in vivo, as the average amino acid concentrations in
yeast cells are in the millimolar range [39]. Glycine and
alanine showed no detectable activation in PP
i
exchange
assay. Mutated SerRS, bearing the alterations at two amino
acid positions in the motif 2 loop of the active site (E281D;
G291A) exhibits more pronounced misactivation with
threonine (Fig. 3, inset). This was expected, as the kinetic
analysis of the aminoacylation and amino acid activation
reactions revealed that substitution of the class II invariant
glycine has dramatic effect on seryl-adenylate formation,
resulting in severe reduction of catalytic efficiency [27]. The
other altered amino acid, E281, is conserved in the active site
of seryl-tRNA synthetases and also has an important
functional role in yeast SerRS [27]. Based on our experi-

mental data, amino acid substitutions in the active site of
mutant SerRS (E281D; G291A) decrease the ability of the
synthetase to discriminate against noncognate substrates.
The addition of tRNA
Ser
CC
decreases the misactivation
by yeast SerRS
Figure 3 compares the extent of threonyl-adenylate synthe-
sis by yeast SerRS with and without tRNA addition. The
decreased level of misactivation observed in the presence of
cognate, but nonchargeable tRNA, could be due to
hydrolytic editing as described for several other aaRSs
[16,18,40,41] or the consequence of tRNA-optimized amino
acid discrimination. For the reasons discussed below, we
favor the latter hypothesis. Nevertheless, our results point
out the importance of tRNAÆsynthetase complex formation
in the accuracy of amino acid selection, which may
contribute to fidelity of translation in vivo.
Analysis of the complexes between SerRS and tRNA
Ser
Our kinetic experiments show that equimolar concentration
of tRNA per dimeric enzyme induces a complete rearrange-
ment of amino acid binding sites, as judged by full range
change of the K
m
value for serine (Table 1). This is an
indication that a
2
Æ(tRNA

Ser
)
1
is a functional complex in seryl-
tRNA formation. In order to determine whether SerRS can
bind more than one cognate tRNA molecule under various
conditions, several experimental approaches were employed.
(a) Inactivation of SerRS by oxidized tRNA
Ser
.The
dependence of the velocity of the pyrophosphate exchange
reaction on the concentration of the tRNA
Ser
ox
is shown in
Fig. 4. The concentration of oxidized tRNA
Ser
was from 0 to
1500 n
M
, while the other substrates were held at constant
concentration (2 m
M
ATP and 1 m
M
serine), in the standard
pyrophosphate exchange reaction. The velocity decreased to
17% when the concentration of the tRNA
Ser
ox

reached the
value of about 1200 n
M
. As the active site titration performed
without tRNA [42] showed that dimeric enzyme possesses
two active sites at which the activation of serine occurs,
almost total inactivation of the a
2
-dimeric SerRS with a
nonchargeable tRNA
Ser
suggests that two tRNAs could be
simultaneously bound per dimeric protein. (b) Gel mobility
shift assay. SerRS forms only one type of complex which was
sufficiently stable to be detected by the gel mobility shift
assay, as shown in Fig. 5. The complex of the same
electrophoretic mobility was detected with SerRS/tRNA
Ser
ratios 2 : 1, 1 : 1, 1 : 2, 1 : 3, 1 : 4 and 1 : 5 (Fig. 5, lanes 2–7,
respectively). Uncomplexed tRNA
Ser
was subjected on the
same gel as electrophoretic mobility marker (Fig. 5, lane 1).
SerRS in complex with two bound tRNA
Ser
was not detected
by the mobility shift assay despite the broad variation in pH
and ionic strength. To test the specificity of the method,
Fig. 3. tRNA
Ser

CC
decreases the misactivation of
threonine by yeast SerRS. Threonyl-adenylate
formation was compared with and without
tRNA
Ser
CC
. Noncognate aminoacyl-adenylate
formation (pmol amino acid-AMP per pmol
SerRS per minute) was measured at different
amino acid concentrations (0.5–50 m
M
,
which corresponds approximately to
1–100 · K
m
for serine) in a standard pyro-
phosphate exchange reaction. Glycine and
alanine showed no detectable activation. The
inset shows misactivation of threonine by
wild-type and mutant SerRS (E281D;
G291A).
Fig. 4. Dependence of seryl-adenylate formation velocity on the con-
centration of tRNA
Ser
ox
. To facilitate formation of noncovalent complex,
SerRS and tRNA
Ser
ox

were incubated for 5 min at 30 °Cpriorto
addition to the reaction mixtures for pyrophosphate exchange.
Ó FEBS 2002 tRNA-dependent amino acid discrimination (Eur. J. Biochem. 269) 5275
SerRS was incubated with the noncognate yeast tRNA
Tyr
present either in the equimolar amount or in high molar
excess (Fig. 5, lanes 8 and 9, respectively), followed by the
electrophoresis on the native polyacrylamide gel. No band of
retarded electrophoretic mobility relative to noncomplexed
tRNA
Tyr
was detected. (c) Covalent cross-linking. Oxidized
tRNA possesses 3¢-terminal dialdehyde groups able to form
Schiff bases with lysine side chain amino groups. Considering
the nucleophilicity of the lysine side chain amino groups,
cross-linking experiments were performed in the pH range
8.0–9.0. In all cases, two complexes, presumably SerRS–
tRNA
Ser
ox
and SerRS–(tRNA
Ser
ox
)
2
were detected (data not
shown). The largest amount of both complexes was obtained
at pH 8.5. SerRS and tRNA
Ser
ox

wereincubatedinratios2 : 1,
1 : 1, 1 : 2 and 1 : 3 (Fig. 6A, lanes 2, 3, 4 and 5, respectively)
under conditions described in the Experimental procedures.
A higher mobility band, appearing in lanes with SerRS/
tRNA
Ser
ox
ratios 1 : 1–1 : 3 (Fig. 6A, lanes 3–5) could be
assigned to SerRS–(tRNA
Ser
ox
)
2
. Uncomplexed SerRS and
tRNA
Ser
ox
were used as mobility markers (Fig. 6A, lanes 6 and
1, respectively). Our results indicate that Schiff bases formed
between yeast SerRS and tRNA
Ser
ox
were stable even without
reduction, as approximately the same amount of both cross-
linked products were formed both with and without reducing
reagents (NaCNBH
3
and NaBH
4
) (data not shown). On the

contrary, Madore et al. [33] could not detect the GlnRS–
(tRNA
Gln
)
2
complex without treatment with NaCNBH
3
.
Stoichiometric analysis
Ferguson plot analysis [34] was performed in order to
confirm predicted stoichiometry of detected covalent com-
plexes between SerRS and tRNA
Ser
ox
. Relative mobilities of
the protein standards and two covalent complexes were
determined on a series of nondenaturing discontinuous gels
of increasing polyacrylamide concentration having constant
acrylamide/bisacrylamide ratio and their logarithm was
plotted as a function of the gel concentration (Fig. 6B). The
negative slope of each line [representing the retardation
coefficient (K
r
) for species] was defined and log K
r
was
plotted against log molecular mass (kDa) (Fig. 6C). Deter-
mined K
r
values for covalent complexes were 9.2 (lower

mobility band) and 10.9 (higher mobility band), corres-
ponding to molecular masses of 136 kDa and 183 kDa., i.e.
to one and two tRNA
Ser
bound to SerRS dimer, respectively
(Fig. 6C). Molecular masses of SerRSÆtRNA
Ser
and SerR-
SÆ(tRNA
Ser
)
2
complexes obtained by MALDI-MS analysis
were 136 kDa and 163 kDa, respectively [29], in very good
agreement with those determined by native electrophoresis.
Percentage of error, calculated as (estimated molecular mass
– reported molecular mass/reported molecular mass) · 100,
was in the range of 0 and 12%, i.e. the same as in previously
published experiments performed by this method [43].
DISCUSSION
tRNA-dependent amino acid discrimination as a
possible quality control mechanism in seryl-tRNA
formation
An order of magnitude of increased affinity toward serine in
the presence of nonchargeable 3¢-truncated tRNA
Ser
con-
firms our previous finding that SerRS modulates its affinity
for serine in a tRNA-dependent manner [27]. We have
showninthispaperthatcognatetRNAÆSerRS interactions

increase the stringency of amino acid discrimination. This
may proceed via a tRNA-induced conformational change in
the enzyme’s active site. In agreement with the observation
of Cusack et al. [38] that the alteration of glycines may
influence loop flexibility in T. thermophilus SerRS, our
results revealed that the activation of serine by mutated
yeast SerRS (E281D; G291A) was less significantly affected
by 3¢-truncated tRNA
Ser
. This indicates that the flexibility of
the motif 2 loop is important for structural readjustment of
the amino acid binding site. Based on the structural and
functional resemblance among seryl-tRNA synthetases [27],
wesuggestthatinyeast,likeinT. thermophilus [44], docking
interactions between the long variable arm of tRNA
Ser
and
the N-terminal coiled coil of SerRS govern the positioning
of the acceptor stem in the active site of the enzyme. Either
these docking interactions or the interaction with the
acceptor stem, induce conformational changes in the serine
binding sites, which optimize the enzyme for seryl-adenylate
formation. As a consequence, the misactivation of structur-
ally similar noncognate amino acid is prevented. As recently
shown for the aspartyl-system, hierarchical recognition of
the synthetase and its cognate tRNA is crucial for formation
of the active macromolecular complex [45]. In T. thermo-
philus SerRS, serine specificity is guaranteed by two
hydrogen bond interactions with the side chain hydroxyl
group and by the size of the binding pocket [46]. Model

building studies on this enzyme [46] revealed that binding of
glycine and alanine would be unfavorable because of the
absence of hydrogen bonding capacity, and many other
amino acids would be too large. The hydroxyl group of
structurally similar threonine also makes an important
contribution to specificity of recognition by ThrRS [16,46].
Besides a protein side chain, a zinc ion serves as a specific
recognition cofactor for the hydroxyl group of threonine in
ThrRS. Although serine is weakly misactivated by ThrRS,
the enzyme needs proofreading or editing activity to correct
this misactivation. On the other hand, mechanistically
analogous zinc ion-mediated amino acid discrimination by
cysteinyl-tRNA synthetase (CysRS) fully assures specific
Fig. 5. Gel mobility shift assay with different amounts of tRNA. SerRS
(9 pmol) was incubated with 4.5, 9, 18, 27, 36 and 45 pmol of tRNA
Ser
andwith9and45pmolofyeasttRNA
Tyr
prior to electrophoresis on
polyacrylamide gel under native conditions (lanes 2, 3, 4, 5, 6, 7 and 8,
9, respectively). Noncomplexed tRNA
Ser
was loaded on the gel as
electrophoretic mobility marker (lane 1). Complex formation and
electrophoresis were performed under pH 6.0. Full line arrow, non-
complexed tRNA; dashed line arrow, SerRSÆtRNA complexes.
5276 I. Gruic-Sovulj et al. (Eur. J. Biochem. 269) Ó FEBS 2002
synthesis of cysteinyl-adenylate, without serine or threonine
activation [47]. The discrimination against threonine by
SerRS may rely partly on the fact that its methyl group

would be in an unfavorable hydrophilic environment in the
active site of enzyme [46]. However, this amino acid is still
misactivated by yeast SerRS apoenzyme in vitro, especially
when the enzyme is altered in the active site. The misacti-
vation by wild-type enzyme is diminished in the presence of
cognate tRNA. In the model, we suggested that such
tRNA-optimized amino acid recognition may act as a
quality control mechanism that lowers the extent of
misactivation and thus editing would not be necessary.
Another possibility, although much less likely, is that tRNA
directs erroneous threonyl-adenylate from the active site to
the center for editing. This needs to be further investigated.
Thus far, the only experimental finding regarding the ability
of SerRS to correct errors is related to pretransfer editing,
which seems to be negligible with any amino acid tested,
including threonine [2]. Additionally, SerRS displays
AMP- and pyrophospate-independent deacylation of cog-
nate aminoacyl-tRNA in the presence of thiols, which
mimics editing of homocysteine [15]. Our experiments show
that the 3¢-terminal adenosine of cognate tRNA
Ser
does not
seem to be important in bringing about the rearrangement
of the serine binding site. However, less specific noncovalent
interactions between yeast enzyme and E. coli tRNA
Ser
induce less pronounced conformational change in the active
site, revealing the importance of cognate complex formation
for accurate amino acid recognition.
Anticooperative binding of tRNA

Ser
The finding that a high molar surplus of yeast-oxidized
tRNA
Ser
in a PP
i
exchange reaction deactivated more
Fig. 6. Covalent complexes between SerRS and tRNA
Ser
ox
. (A) Detection on the gel. SerRS (9 pmol) was incubated with 4.5, 9, 18 and 27 mol of
tRNA
Ser
ox
inthepresenceofNaCNBH
3
and subjected to polyacrylamide gel under native conditions (lanes 2, 3, 4, and 5, respectively). Non-
complexed tRNA
Ser
ox
(5 pmol) and SerRS (9 pmol) was loaded onto the gel as an electrophoretic mobility marker (lanes 1 and 6, respectively).
Covalent cross-linking reactions were stopped by the addition of NaBH
4
. (B) Representative Ferguson analysis. A logarithmic function of relative
mobility for each of the protein standards and two covalent complexes, SerRS–tRNA
Ser
ox
and SerRS–(tRNA
Ser
ox

)
2
, was plotted against the poly-
acrylamide concentration and fitted to the regression. Protein standards are indicated: (a) a-lactalbumin (molecular mass 14.2 kDa), (b and c)
carbonic anhydrase (two charge isomers with molecular mass 29.0 kDa), (d) chicken egg albumin (molecular mass 45.0 kDa), (e and f) bovine
serum albumin monomer (molecular mass 66 kDa) and dimer (molecular mass 132.0 kDa), respectively, (g and h) urease trimer (molecular mass
272.0 kDa) and hexamer (molecular mass 545.0 kDa), respectively. 1 : 1 and 1 : 2 denote SerRS–tRNA
Ser
ox
and SerRS–(tRNA
Ser
ox
)
2
. R
2
values were
in the range 0.9914–0.999 for all protein standards and both covalent complexes. (C) Representative plot of log K
r
vs. log of molecular mass (kDa).
K
r
values for protein standards were derived by a Ferguson analysis and log K
r
was plotted as a function of log molecular mass (kDa). Protein
standards (a–h) are as indicated in (B). Interpolations of log K
r
values derived for the SerRS–tRNA
Ser
ox

and SerRS–(tRNA
Ser
ox
)
2
complexes (dashed
lines) indicate molecular weights of 136 and 183 kDa, respectively. R
2
value was 0.993.
Ó FEBS 2002 tRNA-dependent amino acid discrimination (Eur. J. Biochem. 269) 5277
than 80% of yeast SerRS activity led to the conclusion
that two tRNA
Ser
ox
molecules could be simultaneously
bound to dimeric SerRS. This was confirmed by covalent
cross-linking. However, SerRSÆ(tRNA
Ser
)
2
complex was not
stable enough to be detected by the gel mobility shift assay
under the conditions of relatively broad pH and ionic
strength ranges. This suggests that two tRNA
Ser
binding
sites on yeast SerRS are nonequivalent, which is in
agreement with previously reported studies of others which
revealed that two binding sites for tRNA
Ser

differ by about
two orders of magnitude in their binding constants [48,49].
Both, SerRSÆtRNA
Ser
and SerRSÆ(tRNA
Ser
)
2
, complexes
were also detected by MALDI-MS [29]. The specificity of
the MALDI-MS measurements was confirmed in the
competition assay. The SerRSÆ(tRNA
Ser
)
2
complex was
not detected on the control gel mobility shift assay
conducted under the same conditions as the MALDI-MS
measurements. This could be the consequence of increased
electrostatic forces as the dielectric constant of the environ-
ment is lowered during solvent evaporation and in vacuum.
For prokaryotic systems, crystallographic studies showed
that T. thermophilus SerRS form two types of noncovalent
complexes with cognate tRNA
Ser
;SerRSÆtRNA
Ser
and
SerRSÆ(tRNA
Ser

)
2
[44,50], while Escherichia coli SerRS
forms only the SerRSÆ(tRNA
Ser
)
2
complex [51]. Borel et al.
[52] were able to detect both 1 : 1 and 1 : 2 E. coli SerRSÆ
tRNA
Ser
complexes by zone interference gel electrophoreses
and concluded that binding of the tRNA was positively
cooperative.
It is tempting to speculate as to why yeast SerRS binds
two tRNA
Ser
molecules with nonequivalent affinity. Are
these binding sites apriorinonequivalent, or does binding of
the first tRNA leads to a conformational change which
lessens the affinity towards tRNA at the second site? Results
presented in this paper show undoubtedly that binding of
the equimolar amount of tRNA
Ser
induces complete
conformational optimization of the serine binding sites
which results in efficient and accurate seryl-adenylate
formation. The binding of one tRNA may promote
structural rearrangement of SerRS which decreases the
affinity for the second tRNA.

ACKNOWLEDGMENTS
We thank Sinisa Stipanicic for recording ESI-MS spectrum of the
threonine sample and Tomislav Kamenski and Marko Mocibob for
assistance in complex formation studies and Ferguson plot analysis. We
are indebted to Professor Kucan for critical discussions. This work was
supported by grants from International Centre for Genetic Engineering
and Biotechnology, Trieste, the Ministry of Science and Technology of
the Republic of Croatia, and National Institutes of Health (NIH/
FIRCA).
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Ó FEBS 2002 tRNA-dependent amino acid discrimination (Eur. J. Biochem. 269) 5279