Báo cáo Y học: Shifted positioning of the anticodon nucleotide residues of amber suppressor tRNA species by Escherichia coli arginyl-tRNA synthetase pdf
Shiftedpositioningoftheanticodonnucleotideresiduesof amber
suppressor tRNAspecies by
Escherichia coli
arginyl-tRNA synthetase
Daisuke Kiga
1,2
, Kensaku Sakamoto
2
, Saori Sato
1
, Ichiro Hirao
1
and Shigeyuki Yokoyama
1,2
1
Yokoyama CytoLogic Project, ERATO, JST c/o RIKEN, Hirosawa, Wako-shi, Saitama, Japan;
2
Department of Biophysics and Biochemistry,
Graduate School of Science, University of Tokyo, Bunkyo-ku, Japan
Cytidine in theanticodon second position (position 35) and
G or U in position 36 of tRNA
Arg
are required for amino-
acylation byarginyl-tRNAsynthetase (ArgRS) from Escheri-
chia coli. Nevertheless, an arginine-accepting amber
suppressor tRNA with a CUA anticodon (FTOR1D26)
exhibits suppression activity in vivo [McClain, W.H. &
Foss, K. (1988) Science, 241, 1804–1807]. By an in vitro
kinetic study with mutagenized tRNAs, we showed that the
arginylation of FTOR1D26 involves C34 and U35, and that
U35 can be replaced by G without affecting the activity.
Thus, thepositioningofthe essential nucleotides for the
arginylation is shifted to the 5
0
side, by one residue, in the
suppressor tRNA
Arg
. We found that theshifted positioning
does not depend on thetRNA sequence outside the anti-
codon. Furthermore, by a genetic method, we isolated a
mutant ArgRS that aminoacylates FTOR1D26 more
efficiently than the wild-type ArgRS. The isolated mutant
has mutations at two nonsurface amino-acid residues that
interact with each other near the anticodon-binding site.
Keywords: tRNA identity; anticodon; aminoacyl-tRNA
synthetase; genetic screen; kinetic analysis.
The accurate recognition of a tRNAby its aminoacyl-tRNA
synthetase (aaRS) is a vital step in ensuring the fidelity of
translation. An aaRS distinguishes its cognate tRNAs from
the tRNAspecies existing in the same cell bythe recognition
of the particular nucleotideresidues (identity determinants)
that are found only in the cognate tRNAs as one set. In most
tRNA species, these nucleotides are located in the anticodon
moiety and at the discriminator position (position 73) [1].
The identity of tRNA
Arg
involves two anticodon positions:
a cytidine residue in position 35 (C35) and a G or U in
position 36 both contribute to the arginine-accepting activity,
and the effect of base substitutions is much larger for position
35 than for position 36 [2 – 4]. In contrast, position 34 is not
thought to contribute to the reaction, because various base
substitutions are allowed in this position; the naturally
occurring tRNA
Arg
species have inosine, cytidine, and a
modified uridine in this position [5], and tRNA
Arg
tran-
scripts with A34 and C34 exhibit arginylation activity
comparable to that ofthe fully modified tRNA
Arg
[4].
The irrelevance of position 34 and the ambiguous recog-
nition at position 36 are necessary, because there are several
tRNA species with different anticodon sequences for
reading the six arginine codons, which only have G as the
second letter in common. Leucine and serine are also each
encoded with six codons read by several tRNA species, but
their tRNAs have identity determinants located mainly
outside theanticodon [1]. In addition to the anticodon
residues, A20 in the D loop also contributes to the tRNA
Arg
identity in Escherichiacoli and probably in most organisms
other than yeast, but its contribution to the activity is only
comparable to that of G/U36 [2,4,6].
McClain & Foss [6] used ambersuppressortRNA species
to analyze the identity determinants of E. coli tRNA
Arg
.
First, a tRNA
Arg
2
variant, whose anticodon was replaced by
CUA, inserted more Lys than Arg in response to the amber
codon, suggesting that theambersuppressor tRNA
Arg
has
structural features similar to those of tRNA
Lys
. Therefore,
they tried a different approach: an ambersuppressor derived
from tRNA
Phe
was engineered bythe transplantation of A20
together with the three surrounding nucleotides. Actually,
the resultant suppressor (‘F to R’ or FTOR1) inserted Arg
about 10 times more frequently than Lys or Tyr in response
to theamber codon. Furthermore, only Arg was detected
with FTOR1D26, which was made bythe deletion of A26
from FTOR1. As these Arg-inserting amber suppressors
lack both oftheanticodon identity determinants, C35 and
G/U36, of tRNA
Arg
, C34 may be recognized bythe C35-
recognition site of ArgRS. (This mode of recognition is
referred to hereafter as ‘shifted positioning’.) The deletion
of A26 may facilitate the shift of C34 toward the location of
C35, which might enhance the Arg-accepting activity, but
not the Lys- and Tyr-accepting activities [6]. Alternatively,
the A26 deletion may depress the Lys- and Tyr-accepting
activities, but not the Arg-accepting activity, ofthe tRNA
[6].
Recently, the crystal structure ofthe ArgRS
:
tRNA com-
plex from yeast was determined, which revealed a structural
basis for theanticodon recognition by ArgRS [7]. An
interesting finding was that C35 does not make any specific
interactions with amino-acid side chains, but only with
certain backbone atoms in the C-terminal domain. Even
with this structural information, two questions remained.
Correspondence to S. Yokoyama, Department of Biophysics and
Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Fax: 1 81 35841 8057,
Tel.: 1 81 35841 4413, E-mail: yokoyama@biochem.s.u-tokyo.ac.jp
Enzymes: arginyl-tRNAsynthetase from Escherichia coli
(SWISS-PROT entry name ¼ SYR_ECOLI; EC 6.1.1.19).
(Received 2 July 2001, revised 2 October 2001, accepted
3 October 2001)
Abbreviations: aaRS, aminoacyl-tRNA synthetase; ArgRS,
arginyl-tRNA synthetase.
Eur. J. Biochem. 268, 6207–6213 (2001) q FEBS 2001
First, the possibility ofthe ‘shifted positioning’ of C34
needed to be examined. Secondly, the types of amino-acid
replacements that change theanticodon specificity of ArgRS
needed to be identified; because ofthe unique manner of
C35 recognition by ArgRS, it is difficult to rationally design
amino-acid replacements to change theanticodon specificity
on a structural basis.
In the present study, we analyzed FTOR1D26 and its
variants, and found that position 34, but not position 35, is
crucial for the arginylation in E. coli. In addition, this shift
of the crucial position does not depend on the sequence
outside the anticodon. Finally, by genetic selection, we
isolated a mutant ArgRS that arginylates the amber
suppressor tRNAs better than the wild-type enzyme.
MATERIALS AND METHODS
DNA manipulation, sequencing, and PCR amplification
Standard techniques were used for restriction endonuclease
digestion, ligation, and gel electrophoresis [8]. The nucleo-
tide sequence was determined using a Sequencing Pro
Autosequencer core kit for labeled primers (Toyobo, Tokyo,
Japan). PCR was performed using the Gene Amp PCR
system 9700 (Applied Biosystems).
Preparation of ArgRS
The wild-type and mutant ArgRS species were purified by a
histidine-tag system from overproducing cells, to exclude
any contamination bythe endogenous wild-type enzyme.
Thus, each ArgRS preparation has 10 tandem histidine
residues at the N-terminus (the C-terminal tag inactivates
the enzyme). The tagged wild-type enzyme exhibited an
aminoacylation activity similar to the reported values [4,9].
To tag the enzymes, the argS coding sequences were
amplified by PCR with the primers, 5
0
-GGGAATTCCATA
TGAATATTCAGGCTCTTC-3
0
and 5
0
-GAGCGGATCCA
AGCTTCCATTTTCAGAATACATTTAGATGGC-3
0
,and
were then ligated between the Nde I–Bam HI sites of
pET26b (Novagen). The E. coli BLR (DE3) cells (Novagen)
were transformed with this plasmid expressing the tagged
ArgRS, and were then grown at 25 8C in Luria– Bertani
media supplemented with 6% glucose. At the late log phase
of cell growth, isopropryl thio-b-
D-galactoside was added
to the media to a 0.5 m
M concentration, and after 1 h of
induction, the cells were harvested. The enzyme was then
purified by chromatography on Ni-nitrilotriacetic acid
agarose (Qiagen), followed by FPLC using a Resource-Q
column (Amersham Pharmacia Biotech).
In vitro
aminoacylation analyses
Transfer RNA variants were prepared using T7 RNA
polymerase [10]. According to the standard procedure [10],
the tRNA aminoacylation assays were performed at 37 8Cin
40 mL of a buffer [100 m
M Tris/HCl, pH 7.5, 15 mM MgCl
2
,
4m
M ATP, 60 mM [
14
C]arginine (332.1 pCi
:
pmol
21
,New
England Nuclear)] containing various concentrations of the
enzyme and the tRNA. Before the reaction, thetRNA was
renatured at 65 8C for 5 min in the reaction buffer. The
kinetic parameters were obtained from Lineweaver–Burk
plots. The obtained data are the averages of at least two
independent assays. As E. coli ArgRS has a K
m
value of
12 m
M for arginine [9], its concentration in our assay is
nearly saturating.
Bacterial strains and plasmids for the genetic study
The E. coli arginine auxotroph, which we designated as
NR101, is an argE(Am) strain provided in an Interchange kit
(Promega), and confers the tetracycline resistance.
The arginine-inserting ambersuppressor tRNA,
FTOR1D26, reported by McClain & Foss [6], had been
expressed from the gene cloned in pUC18 (which we desig-
nated as pUCsupR) under the control ofthe lpp promoter and
the rrnC terminator. This gene was cloned in the low-copy
vector, pAp102, which has a ColIbP9 replication origin, and
a copy number of 1.7 per cell [11], to generate pApsupR.
The pAp102 vector was a gift from K. Mizobuchi
(Department of Applied Physics and Chemistry, University
of Electro-Communications, Tokyo, Japan).
The E. coli argS gene, with its native promoter and SD
sequence, was inserted between the Bam HI–HindIII sites of
the pACYC184 plasmid to generate pACargS.
Construction of a mutant
argS
library and genetic
selection
In order to prepare a mutant ArgRS library, part ofthe argS
sequence (0.5 kb), from the Eco RV site (amino-acid residue
318) to the C-terminal end, was amplified by error-prone
PCR with the primers, 5
0
-CACCACTGATATCGCCTG
TGCG-3
0
and 5
0
-GAGCAAGCTTCCATTTTCAGAATAC
ATTTAGATGGC-3
0
, where the HindIII site is underlined
[12]. After digestion with these restriction enzymes, the
PCR product was cloned in pACargS, in place of its
counterpart in the wild-type argS sequence, to generate the
pACargS
mu
plasmids.
In order to isolate mutant ArgRS species that amino-
acylate FTOR1D26, strain NR101 was transformed with
pApsupR together with pACargS
mu
, and was then grown for
36 h at 37 8C on an M9 plate supplemented with proline,
methionine, and thiamine (1 m
M each), but not with argi-
nine. The colonies that formed on the plate were isolated,
and after the pACargS
mu
plasmids were extracted, they were
tested again for the potential to suppress the arginine
auxotroph. The Eco RV – HindIII fragments ofthe recovered
argS genes from the plasmids thus selected were cloned
again in pACargS, in place ofthe counterpart in the wild-
type argS, and were then examined again for their
suppressing potential. Finally, the Eco RV – HindIII frag-
ments thus selected were subjected to sequence
determination.
RESULTS AND DISCUSSION
ArgRS recognition of tRNA
Arg
species with a CUA
anticodon
In E. coli and yeast, C35 and G or U in position 36 are
crucial for the arginylation [2,4,13]. In contrast, position
34 is not thought to contribute to the reaction [4,5]. With
this in mind, we investigated the manner by which
ArgRS recognizes FTOR1D26. A wild-type tRNA
Arg
,
tRNA
Arg
2
[14], and FTOR1D26 variants with various
6208 D. Kiga et al. (Eur. J. Biochem. 268) q FEBS 2001
anticodon sequences were prepared by in vitro transcription
with T7 RNA polymerase, and were subjected to in vitro
kinetic analyses. The wild-type tRNA
Arg
2
exhibits an
arginine-accepting activity similar to those reported so far
[4,9], while FTOR1D26 exhibits a 6 Â 10
5
-fold lower
activity than the wild-type (Table 1). The replacement of
C34 by either A or U in FTOR1D26 further reduced the
arginine-accepting activity. This result is in sharp contrast
to the wild-type tRNA
Arg
with C35, in which position 34
is not relevant, but position 35 is strictly required to be C
for the arginine-accepting activity [4,5]. For position 35 of
FTOR1D26, the U35A substitution also reduces the activity,
but U35G does not affect it, which is reminiscent of the
effects ofthe base substitutions in position 36 ofthe wild-
type tRNA
Arg
species. As the specificities of ArgRS for
positions 34 and 35 of FTOR1D26 are exactly the same as
those for positions 35 and 36, respectively, ofthe wild-type
tRNA
Arg
, this indicates that C34 and U35 of FTOR1D26 are
recognized bythe binding pockets for C35 and G/U36,
respectively, ofthe enzyme, as previously proposed [6].
We then examined the effect ofthe deletion of A26. The
arginine-accepting activity of FTOR1 is higher than that of
FTOR1D26 in vitro (Table 1). However, FTOR1 inserts Lys
and Tyr, in addition to Arg, but FTOR1D26 predominantly
inserts Arg, in response to theamber codon [6]. As previously
proposed [6], the two possible reasons why the deletion of
A26 makes thetRNA much more specific to arginine are (a)
the arginine-accepting activity, but not the lysine- and
tyrosine-accepting activities, ofthetRNA was increased,
and (b) the recognitions ofthetRNAby LysRS and TyrRS
were more impaired than that by ArgRS. The present results
clearly indicate that the latter reason is the case for the
A26 deletion of FTOR1. The C34A and C34U substitutions
of FTOR1 both reduce the arginine-accepting activity, as
in the case of FTOR1D26 (Table 1). Therefore, the shifted
positioning occurred in both the presence and absence of
A26 in FTOR1.
We next assayed theambersuppressortRNA derived
from tRNA
Arg
2
, whose nucleotide sequence is significantly
different from that of FTOR1D26. This amber suppressor
tRNA, tRNA
Arg
2
(CUA), shows a higher arginine-accepting
activity in vitro than those of FTOR1D26 and FTOR1
(Table 1). In contrast, tRNA
Arg
2
(CUA) inserts more lysine
than arginine in vivo in response to theamber codon, while
the other two tRNAs mainly insert arginine [6]. The nucleo-
tide sequence of tRNA
Lys
is more similar to that of
tRNA
Arg
2
(CUA) than to those of FTOR1D26 and FTOR1,
which are derived from tRNA
Phe
. Again, the substitution of
C34 in tRNA
Arg
2
(CUA) decreased the arginine acceptance
(Table 1), indicating that neither the irregular conformation
of FTOR1D26 nor thetRNA sequence outside the anticodon
is important for theshifted positioning. In summary, the
change in thetRNA framework from tRNA
Arg
2
(CUA) to
FTOR1, as well as the A26 deletion of FTOR1, increased
the arginine specificity by decreasing the recognition by
LysRS and TyrRS much more than that by ArgRS.
Genetic selection of mutant ArgRS molecules
To explore the accommodation ofthesuppressortRNA by
ArgRS, mutant enzymes facilitating theamber suppression
by thetRNA were isolated by genetic methods. In the
complex structure of ArgRS
:
tRNA, C35 is recognized only
by the main-chain atoms ofthe enzyme [7], and this finding
raised the question of what types of amino-acid replace-
ments change theanticodon specificity ofthe enzyme.
For the mutant isolation, we constructed a genetic system
to detect the increased activity of a mutant enzyme for
FTOR1D26. When expressed from a high-copy plasmid
(pUCsupR), FTOR1D26 by itself exhibits a suppression
activity. When expressed from a single copy plasmid
(pApsupR), FTOR1D26 does not complement the poor
growth ofthe E. coli argE(Am) mutant on minimal medium
(data not shown): this cannot be rescued bythe over-
production of ArgRS from the plasmid pACargS (a multi-
copy plasmid carrying argS ), which allowed us to isolate
mutant argS genes that promote theamber suppression by
FTOR1D26.
Table 1. The kinetic parameters for the aminoacylation ofthe tRNA
Arg
variants bythe wild-type ArgRS and the argS1 mutant enzyme. ND,
not determined because ofthe very low activation ofthetRNAbythe enzyme variant. “Framework” indicates a moiety oftRNA outside the anticodon.
tRNA
Wild-type ArgRS argS1
Framework Anticodon
k
cat
(s
21
)
K
m
(mM)
k
cat
/K
m
(M
21
:
s
21
)
k
cat
(s
21
)
K
m
(mM)
k
cat
/K
m
(M
21
:
s
21
)
tRNA
Arg
2
(wild-type) ACG 17 1.0 1.7 Â 10
7
2.8 0.44 6.4 Â 10
6
FTOR1D26 CUA 9.3 Â 10
24
34 27 3.1 Â 10
23
32 97
FTOR1D26 UUA ND ND ,10 ND ND ,10
FTOR1D26 AUA ND ND ,10 ND ND ,10
FTOR1D26 CGA 1.1 Â 10
23
38 29 3.7 Â 10
23
32 1.2 Â 10
2
FTOR1D26 CAA ND ND ,10 ND ND ,10
FTOR1 CUA 2.8 Â 10
23
14 2.0 Â 10
2
6.4 Â 10
23
12 5.3 Â 10
2
FTOR1 UUA ND ND ,20 1.1 Â 10
23
19 58
FTOR1 AUA ND ND ,20 8.6 Â 10
24
11 78
tRNA
Arg
2
CUA 3.1 Â 10
22
4.5 6.9 Â 10
3
8.4 Â 10
22
5.6 1.5 Â 10
4
tRNA
Arg
2
UUA ND ND ,700 ND ND ,700
tRNA
Arg
2
AUA ND ND ,700 ND ND ,700
q FEBS 2001 Unusual manner ofanticodon recognition (Eur. J. Biochem. 268) 6209
Fig. 1. Amino-acid replacements found in the ArgRS mutants. The amino-acid sequences from residue 318 to the C-terminus (residue 577) of the
wild-type E. coli ArgRS and its mutants are shown. The secondary structures are assigned according to the crystal structure of yeast ArgRS
determined Cavarelli et al. [20].
6210 D. Kiga et al. (Eur. J. Biochem. 268) q FEBS 2001
On average, a few base substitutions were introduced in
random positions in the C-terminal region of ArgRS by
error-prone PCR; this region ranges from residue 318, a
position between the HIGH and KMSKS sequences, to the
C-terminus. Ofthe 10
7
argE(am) cells with pApsupR that
were transformed by these randomly mutagenized argS
genes, 10
2
cells formed colonies on the minimal plates
without arginine after an incubation at 37 8C for 36 h. After
excluding the revertant cells, 26 different mutant argS genes
were isolated with the amino-acid replacements listed in
Fig. 1, which will be discussed later.
Recognition ofthesuppressortRNAspeciesby a
mutant ArgRS
To characterize the mutant ArgRSs, we tried to purify the
mutant enzymes. The histidine tag was added to the
N-terminus ofthe enzyme to exclude the contamination by
the endogenous wild-type ArgRS. As all but one of the
mutant enzymes with the histidine tag were found in the
insoluble fraction ofthe cell lysates, we hereafter focused on
the only soluble mutant enzyme, argS1, with two amino-
acid replacements, Met to Val in position 460 and Tyr to Asp
in position 524. In order to investigate the effects of these
replacements individually, the argS mutants with either
replacement were constructed by site-directed mutagenesis,
and were subjected to the same test used for the mutant
selection; neither ofthe single mutants could form colonies
on the plate for the selection. Both replacements were thus
shown to be necessary for the ability of argS1 to suppress
argE(Am) when FTOR1D26 is expressed simultaneously
(data not shown).
AsshowninTable1,theargS1 mutant enzyme
arginylates FTOR1D26 3.6-fold more efficiently, and the
wild-type tRNA
Arg
2.7-fold less efficiently, than the wild-
type ArgRS. Thus, thetRNA specificity of argS1 changes by
9.7-fold, compared with that ofthe wild-type ArgRS. The
activities of argS1 for the other two tRNAs with the CUA
anticodon, FTOR1 and tRNA
Arg
2
(CUA), were also higher
than that ofthe wild-type enzyme.
The effects oftheanticodon base substitutions in
FTOR1D26 were examined for argS1, and the shifted
positioning oftheanticodonresidues was also observed for
this mutant enzyme (Table 1); the substitutions of C34U,
C34A, and U35A all reduce the arginine-accepting activity,
whereas U35G has no effect on the activity. In addition, the
substitutions of C34, in both FTOR1 and tRNA
Arg
2
(CUA),
all reduce the arginine-accepting activity bythe mutant
enzyme.
Between the wild-type and mutant enzymes, the differ-
ence in the aminoacylation activity ofthesuppressor tRNA
species is mainly derived from the difference in the k
cat
values, not in the K
m
values. It reminds us that the substi-
tution ofthe identity determinants in theanticodon of
tRNA
Arg
mainly decreases the k
cat
values [2,4]. The muta-
tions therefore seem to facilitate the unusual signal
Fig. 2. Mapping ofthe yeast ArgRS residues that correspond to the
two amino-acid replacements in the argS1 mutant of E. coli ArgRS
on the yeast ArgRS
:
tRNA complex structure. (A) The crystal
structure ofthe yeast ArgRS
:
tRNA complex [7] used as a working
model ofthe E. coli system. In the argS1 mutant of E. coli ArgRS,
Met460 and Tyr524 are replaced by Val and Asp, respectively. The
E. coli Met460 and Tyr524 correspond to Leu489 and Phe555,
respectively, of yeast ArgRS. On the ribbon model of yeast ArgRS, the
positions of Leu489 and Phe555 are indicated with the side chains (ball-
and-stick) in green and magenta, respectively. ThetRNA main chain is
shown by a wire model, mainly colored yellow, while a part of the
anticodon stem (positions 39–41), which interact with the H18 and H22
helices bearing Leu489 and Phe555, respectively, are shown in red. The
three nucleotideresiduesoftheanticodon are indicated by orange
sticks. (B) The detailed structure around Leu489 and Phe555 in the
yeast ArgRS
:
tRNA complex. The H18 helix and the Leu489 side chain
are shown in green, and the H22 helix and the Phe555 side chain are
shown in magenta. The side chains of Tyr488 on the H18 helix and
those of His559, Ser562, and Ser563 on the H22 helix, shown by balls
and sticks, form a protein surface that contacts the anticodon-stem
residues in positions 39–41, shown by sticks. The E. coli ArgRS
residues that correspond to the yeast ArgRS residues are indicated in
parentheses. All the figures were prepared using the programs
MOLSCRIPT [21] and RASTER3D [22].
q FEBS 2001 Unusual manner ofanticodon recognition (Eur. J. Biochem. 268) 6211
transduction from the C35- and G/U36-binding pockets
upon the binding of some nucleotides in the noncognate
CUA anticodon to the catalytic domain ofthe enzyme.
Structural elements for the change in thetRNA specificity
of
argS1
The argS1 mutant enzyme involves two mutations, M460V
and Y524D, as described above. In the sequence alignment
of the ArgRSs from E. coli and yeast, the E. coli Met460
corresponds to the yeast Leu489, which is located on an a
helix, H18 (Fig. 2), in the reported structure ofthe yeast
ArgRS
:
tRNA complex [7]. The other argS1 mutation,
Y524D, ofthe E. coli ArgRS occurs in the position that
corresponds to position 555 in the yeast ArgRS. This
position is located on another a helix, H22 (Fig. 2). These
two helices form part ofthe a helix bundle that constitutes
the anticodon-binding site. In the helix bundle ofthe yeast
ArgRS, these two amino-acid residues are below the protein
surface, and interact directly with each other (Fig. 2). The
mutations in these positions should induce some structural
change on the surface ofthe helices, which contact the
backbones ofthenucleotideresidues in positions 39–41
within theanticodon stem ofthetRNA (Fig. 2).
Upon binding oftheanticodonofthe wild-type tRNA
Arg
to the site on the yeast ArgRS, theanticodon loop undergoes
a large conformational change [7]; upon the putative binding
of C34 and U35 ofthesuppressortRNAspecies to the
C35- and G/U36-binding sites, respectively, ofthe enzyme
(‘shifted positioning’), a somewhat different conformational
change oftheanticodon loop is likely to be required. This
alternative anticodon-loop conformational change of the
suppressor tRNA is probably much less favorable for amino-
acylation than that ofthe wild-type tRNA. In the argS1
mutant enzyme, the unusual structure due to the mutations
of the anticodon-stem-docking site may facilitate the alter-
native anticodon-loop conformational change ofthe sup-
pressor tRNA, but it may hamper the anticodon-loop
conformational change ofthe wild-type tRNA.
It has been reported that mutations in positions other than
the anticodon-interacting amino-acid residues increase the
aminoacylation of a nonsense suppressor tRNA; for E. coli
glutaminyl-tRNA synthetase, the mutations are outside the
anticodon-binding site, and are located near the core region
of tRNA in the enzyme
:
tRNA complex [15]. It was argued
that these mutations affect the process of transmitting the
signal from theanticodon binding domain to the active site,
and make the enzyme bind C35 in the pocket for U35, which
is conserved in all ofthe glutamine tRNAs. On the other
hand, on the surface ofthe putative anticodon-recognition
helix of E. coli methionyl-tRNA synthetase, there are two
acidic residues that reportedly serve as negative discrimi-
nants against noncognate tRNA anticodons through a direct
electrostatic repulsion; the replacement of one of these
residues does not affect the activity ofthe cognate tRNA, but
rather increases the activity ofthe noncognate ones [16].
The mutations in the 26 ArgRS mutants isolated in this
work exhibit a tendency to converge at certain positions
(Fig. 1), although their contributions to the mutant pheno-
type have not yet been confirmed, and the mutant enzymes
have not been characterized. Structural and genetic studies
have suggested that the H16, H18, and H22 helices and
the V loop of yeast ArgRS are probably involved in the
recognition oftheanticodon moiety [7,17]. The conver-
gence ofthe above mutations in these domains suggests that
their effects are associated with theanticodon recognition by
the E. coli enzyme.
Misrecognition of an apparently irrelevant anticodon
sequence has been also reported for yeast ArgRS [13,18,19],
which misarginylates tRNA
Asp
transcripts with an efficiency
similar to to that ofthe arginylation ofthe cognate tRNA
Arg
species. This misrecognition is due to the existence, in the
anticodon region, of an alternative set of nucleotides con-
tributing to the arginylation. In addition, for the yeast ArgRS,
the replacement of Y491H (position 462 in the E. coli
numbering) probably affects its anticodon specificity,
causing a misarginylation ofthe noncognate tRNA
Asp
with
a GUC anticodon sequence [17]. Notably, Tyr491 is also
located on the H18 helix, and is only two residues down-
stream of Leu489, the counterpart of Met460 found in the
E. coli argS1 mutant.
ACKNOWLEDGEMENTS
We thank Dr J. Cavarelli (Biologie et Genomique Structurales, Institut
de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/
ULP) and coworkers, for allowing us to use the published coordinates of
the ArgRS
:
tRNA complex before public release. We also thank Dr
K. Mizobuchi for providing us the pAp102 vector. This work was
supported in part bythe Japan Society for the Promotion of Science
under the Research for the Future program (JSPS-RFTF 96I00101).
REFERENCES
1. Giege, R., Sissler, M. & Florentz, C. (1998) Universal rules and
idiosyncratic features in tRNA identity. Nucleic Acids Res. 26,
5017–5035.
2. Schulman, L.H. & Pelka, H. (1989) Theanticodon contains a major
element ofthe identity of arginine transfer RNAs. Science. 246,
1595–1597.
3. McClain, W.H., Foss, K., Jenkins, R.A. & Schneider, J. (1990)
Nucleotides that determine EscherichiacolitRNA (Arg) and tRNA
(Lys) acceptor identities revealed by analyses of mutant opal and
amber suppressor tRNAs. Proc. Natl Acad. Sci. USA 87,
9260–9264.
4. Tamura, K., Himeno, H., Asahara, H., Hasegawa, T. & Shimizu, M.
(1992) In vitro study of E. colitRNA (Arg) and tRNA (Lys) identity
elements. Nucleic Acids Res. 20, 2335–2339.
5. Sprinzl, M., Horn, C., Brown, M., Ioudovitch, A. & Steinberg, S.
(1998) Compilation oftRNA sequences and sequences of tRNA
genes. Nucleic Acids Res. 26, 148–153.
6. McClain, W.H. & Foss, K. (1988) Changing the acceptor identity of
a transfer RNA by altering nucleotides in a ‘variable pocket’.
Science. 241, 1804–1807.
7. Delagoutte, B., Moras, D. & Cavarelli, J. (2000) tRNA amino-
acylation byarginyl-tRNA synthetase: induced conformations
during substrates binding. EMBO J. 19, 5599 –5610.
8. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular
Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor
University Press, Cold Spring Harbor, New York.
9. Lin, S.X., Shi, J.P., Cheng, X.D. & Wang, Y.L. (1988) Arginyl-
tRNA synthetase from Escherichia coli, purification by affinity
chromatography, properties, and steady-state kinetics. Biochem-
istry. 27, 6343–6348.
10. Nureki, O., Niimi, T., Muramatsu, T., Kanno, H., Kohno, T.,
Florentz, C., Giege, R. & Yokoyama, S. (1994) Molecular recog-
nition ofthe identity-determinant set of isoleucine transfer RNA
from Escherichia coli. J. Mol. Biol. 236, 710–724.
6212 D. Kiga et al. (Eur. J. Biochem. 268) q FEBS 2001
11. Clewell, D.B. & Helinski, D.E. (1970) Existence of the
colicinogenic factor-sex factor ColI-b-P9 as a supercoi led circular
DNA-protein relaxation complex. Biochem. Biophys. Res. Com-
mun. 41, 150–156.
12. Cadwell, R.C. & Joyce, G.F. (1992) Randomization of genes by
PCR mutagenesis. PCR Methods & Applications. 2, 28–33.
13. Sissler, M., Giege, R. & Florentz, C. (1996) Arginine aminoacyla-
tion identity is context-dependent and ensured by alternate recog-
nition sets in theanticodon loop of accepting tRNA transcripts.
EMBO J. 15, 5069–5076.
14. Chakraburtty, K. (1975) Primary structure of tRNA
Arg
II
of E. coli B.
Nucleic Acids Res. 2, 1787–1792.
15. Rogers, M.J., Adachi, T., Inokuchi, H. & Soll, D. (1994) Functional
communication in the recognition oftRNAbyEscherichia coli
glutaminyl-tRNA synthetase. Proc. Natl Acad. Sci. USA 91,
291–295.
16. Schmitt, E., Meinnel, T., Panvert, M., Mechulam, Y. & Blanquet, S.
(1993) Two acidic residuesofEscherichiacoli methionyl-tRNA
synthetase act as negative discriminants towards the binding of
non-cognate tRNA anticodons. J. Mol. Biol. 233, 615 –628.
17. Geslain, R., Martin, F., Delagoutte, B., Cavarelli, J., Gangloff, J. &
Eriani, G. (2000) In vivo selection of lethal mutations reveals two
functional domains in arginyl-tRNA synthetase. RNA 6, 434–448.
18. Sissler, M., Giege, R. & Florentz, C. (1998) The RNA sequence
context defines the mechanistic routes by which yeast arginyl-
tRNA synthetase charges tRNA. RNA 4, 647–657.
19. Sissler, M., Eriani, G., Martin, F., Giege, R. & Florentz, C. (1997)
Mirror image alternative interaction patterns ofthe same tRNAwith
either class I arginyl-tRNAsynthetase or class II aspartyl-tRNA
synthetase. Nucleic Acids Res. 25, 4899–4906.
20. Cavarelli, J., Delagoutte, B., Eriani, G., Gangloff, J. & Moras, D.
(1998)
L-Arginine recognition by yeast arginyl-tRNA synthetase.
EMBO J. 17, 5438–5448.
21. Kraulis, P.J. (1991) MOLSCRIPT: a program to produce both
detailed and schematic plots of protein structures. J. Appl.
Crystallogr. 24, 946–950.
22. Merritt, E.A. & Bacon, D.J. (1997) Raster3D. Photorealistic
Molecular Graphics. Methods Enzymol. 277, 505–524.
q FEBS 2001 Unusual manner ofanticodon recognition (Eur. J. Biochem. 268) 6213
. Shifted positioning of the anticodon nucleotide residues of amber suppressor tRNA species by Escherichia coli arginyl -tRNA synthetase Daisuke Kiga 1,2 , Kensaku. the backbones of the nucleotide residues in positions 39–41 within the anticodon stem of the tRNA (Fig. 2). Upon binding of the anticodon of the wild-type tRNA Arg to the site on the yeast ArgRS, the anticodon. for the aminoacylation of the tRNA Arg variants by the wild-type ArgRS and the argS1 mutant enzyme. ND, not determined because of the very low activation of the tRNA by the enzyme variant. “Framework”