letters
© 2002 Nature Publishing Group http://structbio.nature.com
Structural basis of
translational control by
Escherichia coli threonyl
tRNA synthetase
Alfredo Torres-Larios1, Anne-Catherine Dock-Bregeon1,
Pascale Romby2, Bernard Rees1,
Rajan Sankaranarayanan1, Joel Caillet3,
Mathias Springer3, Chantal Ehresmann2,
Bernard Ehresmann2 and Dino Moras1
1
Laboratoire de Biologie et Génomique Structurales, IGBMC, BP163, 67404
Illkirch Cedex, France. 2UPR 9002 du CNRS, IBMC, 15 rue René Descartes,
67084 Strasbourg Cedex, France. 3UPR 9073 du CNRS, IBPC, 13 rue Pierre
et Marie Curie, 75005 Paris, France.
Published online: 15 April 2002, DOI: 10.1038/nsb789
Escherichia coli threonyl-tRNA synthetase (ThrRS) represses
the translation of its own messenger RNA by binding to an
operator located upstream of the initiation codon. The crystal structure of the complex between the core of ThrRS and
the essential domain of the operator shows that the mRNA
uses the recognition mode of the tRNA anticodon loop to initiate binding. The final positioning of the operator, upon
which the control mechanism is based, relies on a characteristic RNA motif adapted to the enzyme surface. The finding of
other thrS operators that have this conserved motif leads to a
generalization of this regulatory mechanism to a subset of
Gram-negative bacteria.
In both prokaryotes and eukaryotes, fast adaptation of a protein intracellular concentration to environmental variations or
cellular state can be achieved at the translational level. Many
RNA-binding proteins in prokaryotic cells are regulated by a
feedback mechanism. In contrast to transcriptional repressors,
the involvement of translational repressors in cellular functions
other than translational regulation is more a rule than an excep-
a
tion1–3. This is the case of threonyl-tRNA synthetase (ThrRS),
which was shown to repress the translation of its own mRNA
in vitro 4 and in vivo5. ThrRS binds to a site called the operator,
which is located in the leader region of thrS mRNA upstream of
the Shine-Dalgarno sequence, and inhibits translational initiation by competing with ribosome binding6. tRNAThr acts as an
antirepressor, and the synthesis of ThrRS in the cell is modulated
by the relative affinities of ThrRS for the operator and for the
cognate tRNAs7.
The thrS operator is composed of four domains (Fig. 1a).
Domain 1 contains the Shine-Dalgarno sequence and the initiation codon and, together with the single-stranded domain 3,
forms the ribosome-binding site8. Domains 2 and 4 are both
specifically recognized by homodimeric ThrRS. The two hairpin
loops in domains 2 and 4 contain base triplets (CGU or UGU)
that behave as tRNAThr anticodons9–11 (Fig. 1b). However, the
two domains are not strictly equivalent. Domain 2 binds ThrRS
with high affinity and is essential for control, whereas domain 4
binds ThrRS with lower affinity and confers optimal control9,12.
Because one whole operator molecule binds to ThrRS, the two
anticodon-like sequences of the operator were proposed to interact with ThrRS by mimicking the binding of the anticodons of
two tRNAs. However, replacing the operator domain 2 loop and
the three adjacent base pairs by the tRNAThr sequence results in a
30-fold decrease of the control efficiency in vivo9, indicating that
the exact nature of the mimicry remains to be defined.
We have previously solved the structure of the class II homodimeric ThrRS in complexed with two molecules of tRNAThr2
isoacceptor13. In that complex, the C-terminal domain recognizes the anticodon loop of the tRNA, and the acceptor arm is
sandwiched between the catalytic and the N-terminal domains.
Here, we describe the crystal structure of the complex between
domain 2 of the operator (d2RNA) and a dimeric truncated
ThrRS (∆NThrRS), which lacks the N-terminal domains but
shows the same footprint on the operator as the complete
synthetase. Although differences were observed in the case
of the tRNA recognition (Km[ThrRS] = 0.03 µM versus
Km[∆NThrRS] = 0.43 µM), the truncated molecule interacts
with the full operator with the same binding affinity
(Kd[ThrRS] = Kd[∆NThrRS] = 0.01 µM), as measured by
b
Acceptor arm
60
UGUUAAUAAACAA
A
C -70
C U
A
U
A
C C U A U U A U C G G C A C C A 3'
A
U
G
G
5´ G
U
C
G G A U G A U A G C C G p 5'
A-U
U
Y T
C
U-A
U
U-A
U
SD
-50 C
G-C
m 7G
C-G
U-A UAAGGAUAUAAAAUG
G-C
GD
G
G-C
3´
U-A
G-C
G
D
-10
-1 +1
A - U -80
U-A
C-G
20 D
G15
U
G-C
A A
-110 A - U
Lys 246/Lys 249
U-G
D stem
G-C
C-G
A-U
A-U
C-G
C
G-C
U
A
Gly 344/Leu 345
A
C-G
C
AU
A-U
8
-42
G-C
A - A 44
U-A
U-A
Asn
502
G-A
U-A
G-C
Asn
502
C
G
U-A
Ser 347-Arg 349 G - C
-U
Ser 347-Arg 349 A
A-U
U-G
C - G 40
Anticodon-stem
G - C -90
C
G
A-U
-100 C - G
U-G
Asn 575 U
A
A
G
Asn 575
U
U
Arg 583
mt6A
U
Asp 549 U
U
Arg 583
CGU
Asp 549 UC UG
U
-30
G
U GU
Asn 575
Domain 3
Domain 1
Anticodon
arm
Domain 4
Domain 2
Asn 575
Arg 609
Glu 600
Arg 609
Glu 600
Fig. 1 Sequence and secondary RNA structure. a, The thrS mRNA operator. The pseudo anticodon loop is highlighted in blue, the internal loop in yellow, and the Shine-Dalgarno sequence (SD) and the start codon (AUG) in green. The strictly conserved nucleotides are red, and purine or pyrimidine
conservations are purple. Thin lines indicate tertiary interactions. Interactions with synthetase residues are represented by arrows (stars for crosssubunit interactions). The box at the top of domain 2 shows sequence changes that were made to improve the production of d2RNA. By consensus,
the numbering starts with +1 at the start codon; thus, the numbering in the operator region is negative. b, tRNAThr2. The anticodon loop is highlighted in blue and the identity elements noted in red. Tertiary interactions and interactions with the synthetase are represented as in (a).
nature structural biology • volume 9 number 5 • may 2002
343
letters
Internal loop
Domain 2
© 2002 Nature Publishing Group http://structbio.nature.com
-43
E. coli
P.syringae
P.aeruginosa
P.putida
S.putrefaciens
H.influenzae
P.multocida
A.actinomycetes
E.chrysanthemi
Y.pestis
S.typhi
K.pneumoniae
V.cholerae
H.ducreyi
Domain 4
UUUGUAUGUGAUCU
--CCCACGUGACCU
--CCCACGUGGCCU
--CCCACGUGACCU
---CCAUGUGGCCC
---ACACGUCACCU
---ACAAGUAACCU
---ACAAGUCACCU
-UUGCACGUGAGCU
-UUGCAUGUGAUCU
UUUGUAUGUGAUCU
UUUGUAUGUGCUCU
---ACAAGUGUUGC
--UACAAGUAACCU
-32
Domain 1
-20
UUCGUGU
UUGGUAG
UUGGUAG
UUGGUAG
UACGUGU
UUCGUAU
CUCGUAU
UGCGUAU
UUCGUGU
GUCGUGA
UGCGUAU
UUCGUGC
UUGGUGU
CUCGUAU
UAAGGAAUUAGA-AUG
UAAGGAUAUAAC-AUG
UAAGGACAUAAA-AUG
--AGGAUAAUAC-AUG
AAGGAUACAAC-AUG
AC-loop
AUUUGUUG
-ACUGUGA-ACUGUGA-ACUGUGGUCCUGUACU
AAUGGUAG-AGAGUAGGCUUGUAAAAUUGUGGAAAUGUGGAAUGGUUGAGAGGUAA-
+1
UAAGGAUAUAAA-AUG
AGAGGAGGCGCC-AUG
AGAGGAGGCGCC-AUG
AGAGGAGGCGCC-AUG
CAAGGAUAUUUC-AUG
UAAGGAAAAAACAAUG
AAAGGAAAACUU-AUG
GAAGGAUUUAUU-AUG
AC-loop
AUUGCGAACCAAUUUAGC
E. coli
CCUGCC
P.syringae
GCCGCAAGGCGCCUGCC
P.aeruginosa
AAAGGCUUCUGCC
P.putida
CGCAAGGCGGCUAAGAC
S.putrefaciens
GCCUAAUUUCGCUCGUUC
H.influenzae
CCGCACUUUUGCCUUAUUC
P.multocida
CCGCACUUGGUUUGUCC
A.actinomycetes
GGCGGAACUGAUUUAAC
E.chrysanthemi
GUUGAGUACCUAUUCGAC
Y.pestis
GUUGUGCGCCAAUUUAGC
S.typhi
GGUCGUGCUCCUAUUUUCGC
K.pneumoniae
V.cholerae
H.ducreyi
GCUAGAUGGUUUCGCAAU
GGCAGG
GGCAGGCCGGCCCGGC
GGCAGGCUUCCUUU
GUCUUAACCUCGCGAG
GAAUGAAGCCAAGUAGGGC
GAAUGAGUCUAAAGUGCGG
GGAUGAUAAAAGUGCGG
GUUAAAUCCUUUUCCACC
GUUGAGUUGAGUACUUAAC
GCUAGAUGGAUACACAAC
GCGGGAACUGCGCCUUAACC
competition with tRNA aminoacylation. The isolated domain 2
behaves as a competitive inhibitor of tRNA aminoacylation, with
a Kd of 0.5 µM. This is the first structure of an aminoacyl-tRNA
synthetase in complex with an RNA other than tRNA. The comparison of the two complexes shows how the two RNAs use their
characteristic structural properties to serve distinct functions,
while the protein partner does not undergo any significant conformational change. An analysis of the available nucleotide
sequences of the leader regions of prokaryotic thrS genes has led
to the identification of 14 operator-like regions in a subset of the
Gram-negative bacteria (Fig. 2).
Overall structure
The crystal asymmetric unit contains eight RNA molecules and
eight synthetase monomers, forming three noncrystallographic
and two crystallographic dimers. After molecular replacement
using the coordinates of ∆NThrRS14 and solvent flattening, the
initial eight-fold averaged density map clearly showed the RNA
molecules (Fig. 3a). The high redundancy due to noncrystallographic symmetry enhanced the quality of the maps, which were
readily interpretable at the atomic level despite the moderate resolution of the data (3.5 Å). The d2RNA forms a 2:1 complex with
the homodimeric enzyme. The structure of the protein does not
show any significant difference from the reported structures of
free ∆NThrRS or the ThrRS core in the tRNA complex.
The structure of d2RNA consists of a loop and a continuous
but kinked helix (Fig. 4a). d2RNA occupies the same binding site
as the anticodon arm of the tRNA (Fig. 3b). d2RNA mimics not
only the anticodon stem and loop but also the short D-stem,
which is stacked on the anticodon stem in the tRNA
(Figs 1b, 3b). The same residues recognize analogous nucleotides
in both anticodon stem loops (Figs 1, 4b,d), and mutations of
these residues affect similarly both aminoacylation and control
(data not shown). The similarity of recognition includes the
cross-subunit interactions that involve two loops of the catalytic
domain and the RNA backbone at positions –38 and –39 of
344
SD
GGGUCACCACUGCAAA
GGGUCACCACUAGG-GGGUCACCACUAGG-GGGUCACCACUAGG-GGGUCACCACUGG--GGGUGACCACUGU--GGGUUACCACUGU--GGGUGACCACUGU--GGCUCACCACUGCAGGGGUCACCACUGCAGGGGUCACCACUGCAAA
GGGGCACCACUGCAAA
GCAACACCACUGU--GGGUUACCACUGUAC-
Fig. 2 Sequence alignments of the leader
sequences of thrS. BLAST (GCG package)
searches found eight homologs of d2RNA
in the microbial genomes of the NCBI server. The other sequences were obtained
after preliminary alignment with respect
to the nucleotide sequence of E. coli thrS,
followed by investigation of the 200
upstream nucleotides for stable hairpin
formation (mFOLD, GCG package). The 14
aligned sequences are represented with
the strictly conserved nucleotides highlighted in red, and purine- or pyrimidineconserved nucleotides in purple (the
variable domain 3 is not shown). Red lines
indicate the pseudo anticodon and internal loop regions. The Shine-Dalgarno
sequence and AUG start codon are highlighted in green. The thin black line indicates base-paired regions.
d2RNA. The pseudo anticodon loop of d2RNA adopts the characteristic conformation observed for the anticodon loop in the
ThrRS–tRNA complex13, with six out of seven nucleotides
splayed out (Fig. 4a). Thus, both loops bind ThrRS by the same
induced-fit mechanism. The two identity nucleotides G(-32)
and U(-31), which are essential for both aminoacylation and
control, make a lateral base pair lying on the hydrophobic platform of the anticodon-binding domain (Fig. 4b). The conserved
purine G(-30), 3′ adjacent to the pseudo anticodon, lies on the
surface of the protein. Its equivalent in the tRNA is the hypermodified nucleotide at position 37. Here, ordered solvent molecules fill the gap due to the absence of hypermodification
(Fig. 3a).
The mRNA stem
Despite these similarities, the stem of d2RNA does not superimpose perfectly on the anticodon arm of the tRNA but shows a
small but consistent divergence from the tRNA backbone,
starting from the U•G pair at the bottom of the stem (Fig. 3b).
Sequence comparison of a collection of the leader regions of thrS
genes shows that this U•G base pair and the three 3′ stacked guanines are conserved (Figs 2, 4a). Such a sequence has not been
seen in any tRNAThr isoacceptor. Because these residues make no
contact with the protein, their conservation is probably a consequence of an important structural role. They induce an overwinding of the helix, which brings the backbone of d2RNA
closer to the protein surface at the level of the cross-subunit
interactions. This difference from tRNA accounts for the observation that the anticodon loop and the three adjacent base pairs of
tRNAThr do not efficiently substitute for the analogous part of
domain 2, as far as the control function is concerned9.
A remarkable characteristic of domain 2 is the presence of an
asymmetrical internal loop, involving a single uridine, U(-43),
on the 5′ strand facing a CAC triplet (bases from –19 to –21) on
the 3′ strand (Fig. 1a). This loop is, with the anticodon-like
motif, a pivotal element of the specific operator function. Its
nature structural biology • volume 9 number 5 • may 2002
letters
© 2002 Nature Publishing Group http://structbio.nature.com
a
Fig. 3 Electron density and overall view of the complex.
a, Stereo view of the simulated annealed omit-map, contoured
at 1 σ level, showing one d2RNA molecule (not included in the
calculation). b, View of the complex of ∆NThrRS with two
d2RNA molecules (red). The positions of the tRNA molecules in
the ThrRS–tRNA complex13, obtained after superposition of
the protein core, are yellow. As in the following figures, the
two monomers of the synthetase are gray and green. In the
crystal, RNA molecules from two different dimeric complexes
stack end to end, producing an almost continuous A-RNA
helix. c, Similar view in which ∆NThrRS has been replaced by
the full wild type ThrRS, showing the position of the N-terminal domains. The locations of the adjacent ribosome-binding
sites (domain 1 containing the Shine-Dalgarno sequence and
domain 3) are indicated. In vivo, domain 2 of the second
monomer is replaced by domain 4 (ref. 9).
sequence is highly conserved among Gram-negative bacteria
(Fig. 2), and its importance in regulation was demonstrated in
Escherichia coli by mutating C(-19) or C(-21) into uridines. This
resulted in a 15- and 40-fold decrease in the affinity of C(-19)
and C(-21), respectively, for ThrRS, and a 15-fold reduction of
the control in vivo15. The structure reveals a compact stack of
three base triples. Each of the three 3′ strand residues (C(-19),
A(-20) and C(-21)) interacts with an adjacent base pair, allowing
U(-43) to bulge out (Fig. 4c). The base of A(-20) lies in the
minor groove of the RNA, available for contacts with the
enzyme, and forms a distorted triple with the base pair
A(-23)-U(-41) at the bottom of the stack. In the upper base
triple, U(-18)-A(-44)•C(-21), the ribose-phosphate chain of the
3′ strand makes a sharp turn as it folds backwards. Notably, this
turn is positioned at the same place as the sharp P10 turn in the
tRNA, which is located at the hinge between the acceptor stem
and the D-stem (Fig. 3b). Thus, the internal loop motif adds new
elements of molecular mimicry, including key tertiary structure
features, beyond the anticodon stem. Three similar motifs were
observed in the crystal structure of the 23S and 16S ribosomal
RNAs16,17. They are built upon two or three base triples and a
bulged-out residue on the 5′ strand. In all cases, the adenine
equivalent to A(-20) lies fully accessible in the minor groove, as
in the structure of d2RNA, and the ribose phosphate backbone
at the bulged-out residue is distorted and protruding. In d2RNA,
this enables a novel interaction to take place between the ribose
of U(-43) and the base of U(-29) of the pseudo anticodon loop.
This ‘axial interaction’, parallel to the helix axis, is made possible
by the splayed out conformation of the anticodon-like loop promoted by ThrRS binding and the intrinsic curvature of the stem.
No strong sequence constraint is observed at position (-29),
although uridines and guanines are preferred (Fig. 2). Because
the 2′ hydroxyl group from U(-43) may hydrogen bond with any
nature structural biology • volume 9 number 5 • may 2002
b
c
of the four bases, a strict conservation at this position is not necessary. The importance of the axial interaction in the control is
confirmed by the observation that mutation of the highly conserved Arg 583, which plays a role in the correct orientation of
U(-29) (Fig. 4b), affects the in vivo control more severely than
tRNA aminoacylation18.
The overwinding of the d2RNA helix and the internal loop
structure allow a better fit with the enzyme than tRNA. The contact area at the level of the cross-subunit interactions (820 Å2) is
larger than in the tRNA complex (720 Å2). Additional contacts
are made. The base A(-20) rests on a shoulder of the catalytic
domain made by the loops involved in the cross-subunit interactions (Fig. 3b). The base ring stacks on Leu 345, and the ribose
O2′ hydrogen bonds to the main chain amino group of Gly 344
(Fig. 4d). Right after the internal loop and a few nucleotides
from the Shine-Dalgarno sequence, the mRNA backbone at
U(-18) and G(-17) make contacts with Lys 246 and Lys 249. The
two Lys residues are widely conserved in most ThrRSs, whereas
the sequence Gly 344/Lys 345 is strongly correlated with the
presence of a putative operator.
Functional implications
In summary, the anchoring of domain 2 is primarily governed by
base-specific interactions between the anticodon-like loop and
the anticodon-binding domain, in a mode mimicking the tRNA
anticodon binding. The sequence-dependent specific structure of
the stem, locked by the axial interaction, properly positions the
internal loop. This in turn imposes the exact pathway of d2RNA
at the surface of ThrRS, which differs slightly from that of the
tRNA anticodon arm, enabling formation of additional mRNAspecific interactions. The structure of the d2RNA complex suggests that, in the complete ThrRS–operator complex, domains 1
and 3, which build the ribosome binding site, emerge from the
345
© 2002 Nature Publishing Group http://structbio.nature.com
letters
a
b
c
d
Fig. 4 Structure and interactions of d2RNA a, Structure of d2RNA. The nucleotides in red are conserved in the putative operator sequences (pink indicates purine or pyrimidine conservation). b, Stereo view showing the interactions between ∆NThrRS and the pseudo anticodon, materialized by
dotted lines (U34(O2)…Asp 549(Oδ) is not shown)). The interactions are essentially the same as in the tRNA–ThrRS complex. Ala 607, which faces the
lateral base pair U(-31)•G(-32), is conserved in all ThrRSs whose gene contains a putative operator in the leader region. c, The motif of three base
triples at the internal loop. From bottom to top, A(-20) (amino group) interacts with U(-41) (O2) paired to A(-23) (red base triple); C(-19) (amino group)
interacts with G(-42) (N3) paired with C(-22) (magenta); and C(-21) makes a reverse-Hoogsteen interaction with A(-44) paired to U(-18) (blue). The drop
of control observed upon mutation of C(-19) and C(-21) into uridines is consistent with the involvement of the amino groups in the base triples.
d, Stereo view showing the protein interactions with the stem. The strictly conserved residues Ser 347, Tyr 348 and Arg 349 (dark green loop) bind the
ribose-phosphate backbone at the level of A(-39) and U(-38). A(-20) stacks upon Leu 345, with its ribose O2′ binding to the main chain at Gly 344.
Another loop of the catalytic domain (light green) brings Asn 502 into contact with N2 of G(-40), the only contact with a base, and with O4′ of A(-23).
The gray ribbon represents the part of the catalytic domain that brings Lys 246 and Lys 249 into contact with the phosphate oxygens at (-17) and (-18).
The latter interactions involve the subunit in contact with the pseudo anticodon, the other are cross-subunit contacts
synthetase core at the level of its junction with the
N-terminal domain (Fig. 3c). In the thrS operator, the ShineDalgarno region is not directly recognized by ThrRS. Because
there is no evidence for a ThrRS-induced conformational change
in this region, the ribosome entry is probably impeded by a simple steric hindrance effect. The bulky N-terminal domain, which
is expected to be close to the Shine-Dalgarno sequence and the 3′
part of domain 3, probably participates in this effect. Such a
mechanism does not impose stringent constraints on the molecular structure of the enzyme, as shown by changing the pseudo
anticodon to the Met-specific anticodon, which switched the
specificity of control from ThrRS to MetRS11. The relative position and size of the catalytic domain of the class I MetRS probably
explains the repression effect in this case.
The conservation of domain 2, which contains the major
identity elements (G(-32) and U(-31)) and the internal loop,
and its position relative to the Shine-Dalgarno sequence in the
leader regions of thrS genes from Gram-negative bacteria suggest that these organisms share a common regulatory mechanism with E. coli (Fig. 2). Most of these putative operators
contain a second hairpin homologous to domain 4, which possesses the anticodon determinants but is much less conserved
than domain 2. This bipartite organization reflects an adapta346
tion of the RNA to the dimeric nature of ThrRS to enhance
competition with tRNAs.
Many other RNA binding proteins are strongly suspected to
regulate their own expression through a competition mechanism involving mimicry between their natural substrate and
their mRNA regulatory site — for example, the spliceosomal
protein U1A19 or ribosomal proteins L30 (ref. 20) and S15
(ref. 21). The structure of ThrRS with its two challenging ligands
provides the first opportunity to precisely describe both the
extent and the limit of mimicry at the molecular level. The
prominent role of the anticodon in triggering tRNA recognition,
shared by many synthetases22–24, has been diverted by the mRNA
operator to ensure the first step of recognition. In addition,
d2RNA has evolved a motif that precisely locks the helical
domain on the surface of the protein in a position preventing
ribosome binding. This contrasts with tRNA, whose final positioning results from the recognition of other determinants
(particularly in the accepting stem). The sequence characteristics of d2RNA and tRNAThr indicate that they are unlikely to have
a common evolutionary origin. The similarities between d2RNA
and the tRNA molecule are probably explained by constraints
arising from the binding requirements of ThrRS and the
competition that is the basis for the regulatory mechanism.
nature structural biology • volume 9 number 5 • may 2002
letters
© 2002 Nature Publishing Group http://structbio.nature.com
Table 1 Data collection and refinements statistics
Data collection
Space group
a (Å)
b (Å)
c (Å)
β (°)
Resolution range (Å)1
Reflections1
Observed
Unique
Rmerge1
Completeness (%)1
<I / σ (I)>1
P2
188.4
101.7
199.3
114.4
29.80–3.46 (3.58–3.46)
330,893 (33,319)
88,408 (8,790)
0.063 (0.346)
97.9 (82.8)
15.7 (4.7)
Refinement
Resolution range (Å)1
29.80–3.50 (3.66–3.50)
Reflections used
I>2σ
78,345 (7,473)
In Rfree
7,775 (793)
R-factor
0.251 (0.328)
Rfree
0.288 (0.365)
Number of molecules in asymmetric unit
Protein
8
RNA
8
Water2
170
R.m.s. deviation between NCS-related molecules (Å)
Protein
0.06
RNA3
0.13
R.m.s. deviations
Bond lengths (Å)
0.012
Bond angles (°)
1.5
Values in parentheses are for the highest resolution shell.
Only water molecules with at least three noncrystallographic equivalents were retained.
3On seven RNA molecules (see Methods).
1
2
Methods
RNA and ThrRS preparations. D2RNA (from nucleotides –12 to
–49) was synthesized by the T7 polymerase standard method and
purified by gel electrophoresis, followed by chromatography on a
MonoQ column (Pharmacia). To enhance the level of transcription,
the sequence of the first three base pairs was changed (Fig. 1).
When introduced into the complete operator, this change did not
affect binding of ThrRS or regulation in vivo8. Furthermore, the
mutated domain 2 competitively inhibits tRNAThr aminoacylation as
efficiently as the wild type domain 2. ∆NThrRS (residues 242–642 of
ThrRS) was prepared as described13, but an additional step of
hydrophobic interaction chromatography was required to eliminate
trace amounts of RNAse.
Crystallization and data collection. Crystals were obtained in
ammonium sulfate (1.9 M) buffered with sodium cacodylate,
pH 6.6, at 4 °C, from a solution containing 88 µM ∆NThrRS, 200 µM
d2RNA, 10 mM MgCl2 and 10 mM ThrAMS (an analog of the
threonyl-adenylate). The crystals were quickly soaked in the mother
liquor solution containing 25% (v/v) glycerol and then flash frozen
in liquid ethane before data collection. A 3.5 Å resolution data set
(Table 1) was collected at the ID14-2 beam line at the ESRF
(Grenoble, France) on a ADSC Q4 CCD detector. All of the data were
nature structural biology • volume 9 number 5 • may 2002
processed with DENZO and SCALEPACK25. The asymmetric unit contains eight RNA molecules and eight synthetase monomers. The solvent content is 68%.
Structure determination and refinement. The structure was
solved by molecular replacement using the coordinates of
∆NThrRS (PDB entry 1EVK)14. The four copies of the ∆NThrRS dimer
localized with AMoRe26 were used for the initial phasing. A fifth
dimer was then seen in the map, as well as the RNA molecules,
which always formed pairs by the stacking of the first base pair of
the stem. The model was built using O27 and refined with CNS28.
Tight noncrystallographic symmetry restraints (weight =
300 kcal mol–1 Å–2) were applied on the coordinates of the eight
protein monomers, with looser restraints (100 kcal mol–1 Å–2) on
seven of the eight RNA molecules. One RNA molecule showed
significant differences with the other ones, due to close contacts
with its equivalent around the crystallographic two-fold axis, and
was not restrained.
Coordinates. Atomic coordinates have been deposited in the
Protein Data Bank (accession code 1KOG)
Acknowledgments
We thank F. Winter for help with purification of the macromolecules and the
ESRF staff in Grenoble for assistance during data collection. A.T.L. is a recipient of
a scholarship from Consejo Nacional de Ciencia y Tecnología (Mexico). This work
was supported by funds from Aventis, the Institut National de la Santé et de la
Recherche Médicale, the Centre National de la Recherche Scientifique and the
Université Louis Pasteur.
Competing interests statement
The authors declare that they have no competing financial interests.
Correspondence should be addressed to D.M. email: [email protected]
Received 13 December, 2001; accepted 25 February, 2002.
1. Springer, M., Portier, C. & Grunberg-Manago, M. In RNA structure and function
(eds Simons, R.W. & Grunberg-Manago, M.) 377–413 (Laboratory Press, Cold
Spring Harbor; 1998).
2. Draper, D.E. J. Mol. Biol. 293, 255–270 (1999).
3. Hentze, M.W. Trends Biochem. Sci. 19, 101–103 (1994).
4. Lestienne, P., Plumbridge, J.A., Grunberg-Manago, M. & Blanquet, S. J. Biol.
Chem. 259, 5232–5237 (1984).
5. Springer, M. et al. J. Mol. Biol. 185, 93–104 (1985).
6. Moine, H. et al. J. Mol. Biol. 216, 299–310 (1990).
7. Comer, M.M., Dondon, J., Graffe, M., Yarchuk, O. & Springer, M. J. Mol. Biol.
261, 108–124 (1996).
8. Sacerdot, C. et al. Mol. Microbiol. 29, 1077–1090 (1998).
9. Romby, P. et al. EMBO J. 15, 5976–5987 (1996).
10. Springer, M., Graffe, M., Butler, J.S. & Grunberg-Manago, M. Proc. Natl. Acad.
Sci. USA 83, 4384–4388 (1986).
11. Graffe, M. et al. Science 255, 994–996 (1992).
12. Brunel, C. et al. J. Mol. Biol. 227, 621–634 (1992).
13. Sankaranarayanan, R. et al. Cell 97, 371–381 (1999).
14. Sankaranarayanan, R. et al. Nature Struct. Biol. 7, 461–465 (2000).
15. Brunel, C. et al. Biochimie 75, 1167–1179 (1993).
16. Ban, N., Nissen, P., Hansen, J., Moore, P.B. & Steitz, T.A. Science 289, 905–920
(2000).
17. Wimberly, B.T. et al. Nature 407, 327–339 (2000).
18. Springer, M., Graffe, M., Dondon, J. & Grunberg-Manago, M. EMBO J. 8,
2417–2424 (1989).
19. Oubridge, C., Ito, N., Evans, P.R., Teo, C.H. & Nagai, K. Nature 372, 432–438
(1994).
20. Mao, H., White, S.A. & Williamson, J.R. Nature Struct. Biol. 6, 1139–1147 (1999).
21. Nikulin, A. et al. Nature Struct. Biol. 7, 273–277 (2000).
22. Briand, C. et al. J. Mol. Biol. 299, 1051–1060 (2000).
23. Moulinier, L. et al. EMBO J. 20, 5290–5301 (2001).
24. Cusack, S., Yaremchuk, A., Krikliviy, I. & Tukalo, M. Structure 6, 101–108 (1998).
25. Otwinowski, Z. & Minor, W. In Macromolecular crystallography Vol. 276 (eds
Carter, C.W. & Sweet, R.M.) Part A, 307–326 (Academic Press, New York; 1997).
26. Navaza, J. Acta Crystallogr. A 50, 157–163 (1994).
27. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Acta Crystallogr. A 47,
110–119 (1991).
28. Brünger, A.T. et al. Acta Crystallogr. D 54, 905–921 (1998).
347