www.sciencemag.org/cgi/content/full/313/5782/1950/DC1
Supporting Online Material for
Structural Basis of RNA-Dependent Recruitment of Glutamine to the
Genetic Code
Hiroyuki Oshikane, Kelly Sheppard, Shuya Fukai, Yuko Nakamura, Ryuichiro Ishitani,
Tomoyuki Numata, R. Lynn Sherrer, Liang Feng, Emmanuelle Schmitt, Michel Panvert,
Sylvain Blanquet, Yves Mechulam, Dieter Söll,* Osamu Nureki*
*To whom correspondence should be addressed. E-mail: onureki@bio.titech.ac.jp (O.N.);
dieter.soll@yale.edu (D.S.)
Published 30 June 2006, Science 312, 1950 (2006)
DOI: 10.1126/science.1128470
This PDF file includes:
Materials and Methods
SOM Text
Figs. S1 to S5
Table S1
References
Supporting online material
Structural Basis of RNA-Dependent Recruitment of Glutamine to the Genetic
Code
Hiroyuki Oshikane, Kelly Sheppard, Shuya Fukai, Yuko Nakamura, Ryuichiro Ishitani,
Tomoyuki Numata, Lynn R. Sherrer, Liang Feng, Emmanuelle Schmitt, Michel Panvert,
Sylvain Blanquet, Yves Mechulam, Dieter Söll and Osamu Nureki
MATERIALS AND METHODS
Protein purification
Methanobacter thermautotrophicus GatDE protein was overexpressed in Escherichia
coli, as described previously (S1). E. coli strain BL21(DE3) transformed with the
expression vector was cultured, and the harvested cells were resuspended in the buffer,
containing 50 mM Tris-HCl (pH8.0), 0.2 mM EDTA, 10 mM KCl, and 5 mM
2-mercaptoethanol, and then were disrupted by sonication. The supernatant after
centrifugation was heat-treated at 67 ˚C for 30 min., and then was recentrifuged to
exclude the denatured E. coli proteins. The supernatant was loaded onto a Q Sepharose
FF column (Amersham Bioscience), and the proteins were eluted with an NaCl gradient
from 10 mM to 1 M. The eluted sample was dialyzed against 50 mM sodium phosphate
buffer (pH 7.5) with 5 mM 2-mercaptoethanol. Then, 4 M ammonium sulfate was added
to a final concentration of 0.8 M, and the sample was loaded onto a Resource Phe
column (Amersham Bioscience). The eluted sample was dialyzed against 10 mM
Tris-HCl buffer (pH 7.0) containing 10 mM MgCl2 and 5 mM 2-mercaptoethanol, and
was concentrated for crystallization.
tRNA Purification
The gene encoding tRNAGln1 with the preceding sequences of the T7 promoter and th
ribozyme, which is essential to cleave the 5’ end starting with adenosine, was inserted
into the pUC18 plasmid, and the E. coli DH5α strain was transformed with this vector.
The transformant was cultured in 500 ml of LB medium for 16 hours, and the cells were
harvested. The plasmid was purified using a MegaPrep plasmid purification kit
(Qiagen). The purified plasmid was digested by BstNΙ for run-off transcription. In vitro
transcription using T7 RNA polymerase was performed at 37˚C for 15 hours, and the
transcript was phenol-extracted and precipitated with isopropyl alcohol. The pelleted
RNA was dissolved in 20 mM Tris-HCl buffer (pH 7.6) containing 8 mM MgCl2 and
350 mM NaCl, and was annealed by denaturing at 70˚C for 10 min followed by gradual
cooling. The sample was then loaded onto an HPLC equipped with a Resource Q
column (Amersham Bioscience), which was eluted with a gradient from 350 mM to 1
M NaCl. The tRNAGln1 eluted around 600 mM NaCl. The tRNA was ethanol
precipitated and dissolved in 10 mM Tris-HCl buffer (pH 7.0) containing 10 mM MgCl2
and 5 mM 2-mercaptoethanol.
Crystallization
Prior to complex formation, the tRNA was reannealed as described, and then the GatDE
protein and tRNAGln1 were mixed in a molecular ratio of 1:1.2. The complex sample
was incubated at 50 ˚C for 15 min, and then cooled at room temperature. Crystallization
was carried out by the hanging drop vapor diffusion method. After a few days, crystals
appeared in the reservoir solution containing 50 mM Hepes-Na (pH 8.0), 0.2 M NaCl,
10% PEG6000, and 1 mM spermine.
Data collection and structure determination
Crystals were harvested in 1.2-fold concentrated reservoir solution. The crystals were
quite sensitive to light and temperature, and so they were manipulated without light.
After soaking the crystals in harvesting buffer with 10% ethylene glycol for 4 hours for
dehydration, the crystals were soaked into the concentrated reservoir solution with 20%
MPD as a cryo-protectant. Diffraction data were collected at 30 K, with a helium stream,
from the BL41XU beamline at SPring-8, Harima, Japan. The data were processed by
HKL2000 (S2), and analyzed by the CCP4 suite program (S3). Molecular replacement
was performed with the Molrep program (S3), using the M. thermautotrophicus GatDE
structure, generated by homology modeling with the program Modeller (S4) from the
structure of the tRNA-free Pyrococcus horikoshii GatDE (S5), as a search model. The
atomic structure was constructed by the O program (S6), and refined by the CNS
program (S7) to a final free R-factor of 28.9% (Rwork 22.3%) at 3.15 Å resolution (see
Supporting information, Table S1). The asymmetric unit contains a 2:2 complex of
GatDE (GatD, molecules A and B; GatE, molecules C and D) and tRNAGln (molecules
E and F) (Fig. S1). The current model includes all of the aa residues except for residues
A73-A84 (GatD subunit) in one dimer, B75-B84 (GatD) and residues D555-D561
(GatE) in the other dimer, and the terminal adenosine 76 of the two tRNAs.
Structure-based sequence alignment of M. thermautotrophicus GatE and S. aureus GatB
was calculated by the program CE (S13). The complex structure of GatB·ADP·AlF3 (I.
Tanaka, personal communication) was superimposed on the GatE using this alignment,
and the ATP docking model shown in Fig 3A was built. The model was further refined
by energy minimization using the program CNS (S7). The molecular tunnel in Fig. 3B
was calculated by the program CAVER (S12).
Construction of protein and tRNA mutants
GatDE and tRNAGln1 mutants were constructed on the pET20b and pUC18 plasmids
encoding the respective genes, by the use of a Quickchange mutagenesis kit
(Stratagene). Mutations were confirmed by DNA sequencing. The mutant purification
procedures are the same as described above. Purified GatDE enzyme was stored at 4 ˚C
in 20 mM Hepes•Na (pH 7.5), 5 mM MgCl2, 5 mM 2-mercaptoethanol and 50%
glycerol.
Preparation of labeled tRNA
Annealed tRNAGln1 transcript was 32P-labeled on the 3’ terminus by using the E. coli
CCA-adding enzyme and [α-32P] labeled ATP, as previously described with some
modification (S8, S9). Briefly, 16 µM transcript in 50 mM Tris-HCl (pH 8.0), 20 mM
MgCl2, 5 mM DTT and 50 µM NaPPi was incubated for 35 min. at room temperature
with the CCA-adding enzyme and 1.6 µCi/µL of [α-32P] labeled ATP (Amersham
Bioscience). The samples were phenol/cholorform extracted and then passed over a
Bio-spin 30 column (Bio-Rad) to remove excess ATP (S10).
Preparation of aminoacylated tRNA
Transcript was aminoacylated in 50 mM Hepes•KOH buffer (pH 7.2) containing 25
mM KCl, 10 mM MgCl2, 4 mM ATP, and 5 mM DTT with 24 µg/µL of
pyrophosphatase (Roche), 3 µM M. thermautotrophicus GluRS, 1 mM L-Glu, 10 µM
unlabeled transcript and 40 nM 32P-labeled transcript. The reaction was incubated at
37 °C for 90 min. The samples were phenol/cholorform extracted, ethanol precipitated,
resuspended in highly purified water and passed over two Bio-spin 30 columns
(Bio-Rad) to remove excess ATP (S11). Samples without labeled transcript were done
in parallel. To check levels of aminoacylation, 2 µL aliquots at the start and end of the
reactions with labeled tRNA were taken and quenched on ice with 3 µL of 100 mM
sodium citrate (pH 4.74) and 0.66 mg/mL of nuclease P1 (Sigma), and incubated at
room temperature for 35 min. (S8, S10). To separate glutamyl-AMP (Glu-AMP) from
AMP, 1.5 µL of quenched, digested samples were spotted onto a polyethyleneimine
(PEI) cellulose 20 cm x 20 cm thin layer chromatography (TLC) plates (EMD, 5725-7)
and developed for 90 to 120 minutes in 100 mM ammonium acetate and 5% acetic acid
as previously described (S10). The plates were exposed on an imaging plate (FujiFilms)
for 12 hours, scanned using a Molecular Dynamics Storm 860 and quantified using
ImagQuant.
Amidotransferase assays
Transamidation assays were carried out in 1x AdT buffer (100 mM Hepes•KOH, pH 7.2,
30 mM KCl, 12 mM MgCl2, and 5 mM DTT) with 2.6 mM glutamine (Gln), 4 mM
ATP, 500 nM 32P-labeled Glu-tRNAGln, and 50 nM enzyme. Reactions were carried out
at 37 °C over 5 min at which point 2 µL aliquots were quenched on ice with 3 µL of 100
mM sodium citrate, pH 4.74 and 0.66 mg/mL of nuclease P1 (Sigma) and incubated at
room temperature for 35 minutes (S10). To separate the glutaminyl-AMP (Gln-AMP)
from Glu-AMP and AMP, 1.5 µL of the digested samples were spotted onto PEI
cellulose 20 cm x 20 cm TLC plates (EMD, 5725-7) and developed for 90 to 120
minutes in 100 mM ammonium acetate and 5% acetic acid (S10). The plates were
exposed on an imaging plate, scanned and quantified as described above.
Glutaminase assays
They were carried out at 37°C for one hour in 1x AdT buffer, 4 mM ATP, 15 µM
unlabeled Glu-tRNAGln, 25 µM L-[14C]Gln and 50 nM enzyme. Reactions were
quenched with 0.3 M sodium acetate, pH 5.0, ethanol precipitated and the supernatant
was dried as previously described (S11). The pellet was dissolved in 2.1 µL of water.
Cellulose 20 cm x 20 cm TLC plates (Sigma) were spotted with 1.0 µL of the reactions
and developed for 7 hours under acidic conditions (isopropanol:formic acid:water,
20:1:5) to separate L-[14C]Gln from L-[14C]Glu (S11). The plates were exposed on an
imaging plate, scanned and quantified as described above.
ATPase assays
ATPase assays were carried out in 1x AdT buffer with 2.4 mM L-Gln, 20 µM unlabeled
ATP, 0.025 µCi/µL of [α-32P] ATP, 15 µM unlabeled Glu-tRNAGln and 50 nM enzyme
for 12 minutes at 37°C. 1.5 µL of the reactions were spotted onto PEI cellulose 20 cm x
20 cm TLC plates and developed in 0.75 M KH2PO4 for 105 minutes to separate ADP
from ATP. The plates were exposed on an imaging plate, scanned and quantified as
described above.
Gel retardation assay
The annealed tRNA was mixed with the proteins at a ratio of 1:1. The mixture was
incubated at 50 ˚C for 15 min, and fractionated through a 5% acrylamide gel (100 V), in
a buffer solution containing 10 mM Tris-HCl (pH 8.0) and 10 mM MgCl2, for 1 hour at
room temperature. The gel was stained with toluidine blue and CBB to detect both
tRNA and proteins.
Amidotransferase assays of tRNA mutant
For the tRNA mutants to improve levels of charging, aminoacylation and
transamidation reactions were carried out together. The reactions were carried at 37 °C
in 50 mM Hepes͌KOH buffer (pH 7.2) containing 25 mM KCl, 10 mM MgCl2, 4 mM
ATP, and 5 mM DTT with 24 µg/mL of pyrophosphatase, 50 nM M.
thermautotrophicus GluRS, 50 nM, M. thermautotrophicus GatDE, 1 mM L-Glu, 4 mM
L-Gln, 1 µM unlabeled transcript and 5 nM 32P-labeled transcript for 90 minutes at
which point 2 µL aliquots were quenched on ice with 3 µL of 100 mM sodium citrate,
pH 4.74 and 0.66 mg/mL of nuclease P1 (Sigma). The quenched reactions were
incubated at room temperature for 35 minutes. 1.5 µL of the digested samples were
spotted onto PEI cellulose 20 cm x 20 cm TLC plates (EMD, 5725-7) and developed for
90 to 120 minutes in 100 mM ammonium acetate and 5% acetic acid (S9). The plates
were exposed on an imaging plate, scanned and quantified as described above.
Supporting Online Text
Glu-tRNAGln-dependent activation of the GatD glutaminase
Our previous biochemical analysis demonstrated Thr101, Thr177, Asn178, and Lys254
in the GatD AnsA-like domain 1 are catalytically important for the liberation of
ammonia from glutamine (S14). Like the P. abyssi apoenzyme structure, Thr101, the
putative catalytic nucleophile, is 8.4 Å away from the active site of GatD (Fig. S5). As
compared with the crystal structure of the type I L-asparaginase from P. horikoshii
(S15), in both GatDE structures the β-hairpin carrying the Thr is shifted away from the
catalytic center. It has been proposed that conformational changes upon Glu-tRNAGln
binding allow the β-hairpin to shift the Thr into a different position, as the GatDE
glutaminase was active only in the presence of Glu-tRNAGln and not tRNAGln (S14). A
similar activation is seen in GatCAB (S16). Our present structure differs from the
apoenzyme structure in that the helical region of the cradle domain (residues 53-72 in M.
thermautotrophicus) is rotated by 60° (Fig. S5). In the P. abyssi tRNA-free structure the
region overhangs and anchors the β-hairpin. In our tRNA complex structure, the
rotation would allow for release of the β-hairpin (Fig. S5). Therefore, the present
GatDE•tRNAGln structure might represent a transient state preceding the fully active
conformation of GatD in which Thr101 would enter the active site for action as the
nucleophile.
U G
17 G
18 G
U
A
A
U
10
A
C
C
A
U
C
A 70
G
G
G
60
C
C
A
U
U CGC C
G
AGCG G
50
U
C
C
U
A
GC
G
G
C
C 40
C
A
A
G
U
C
C
C
G
G
U G GG
A U C CU G
C
U
G
30 G
G
C
U A
17 G
18 G
C
U
U
G
G
C C A
A
10
U G UG
U
G
C
U
C
C
G
G
A
A U C CU G
A
U
G
30 G
A
C
U
C
G
30 G
C
C
G
U
U
C
M. thermautotrophicus tRNAGlu
U G
M. thermautotrophicus tRNAGln
2
A
C
C
A
C
G
A 70
G
G
C
60
A
C
A
U
G GCC C
U
G
U
C
U C GG G
50
U
C
A
AA
G
G
C
C 40
G
A
A U C CU U
U
G
G
M. thermautotrophicus tRNAGln
1
C U G
10
U G GG
G
U
C
A
A
A
G
U
C
C
C
G
A
C
C
A
U
C
A 70
G
G
G
60
C
C
A
U
U CGC C
G
AGCG G
50
U
C
C
U
A
UC
G
A
C
C 40
U
A
U
A
C
U G
A
G
G
U
A
10
CUCG
U
A
A G A GC G
U
U
C
30 G
G
C
G
C
C
G
C
C
G
A
C
C
G
C
G
G 70
C
G
G
60
G
C
A
C
U GUC C
G
A C AG G
50
U
C
C
U
U
UU
G G
A
G
C 40
C
A
U
A
G U U
M. thermautotrophicus tRNAAsn
Fig. S3. Secondary structures of tRNAGln, tRNAGln and tRNAAsn from M. thermautotrophicus. In tRNAGlns, nucleosides of which the bases are specifically recognized by
GatE are colored red. The twisted G18:C56 base pair in the complex are colored green.
The two guanines conserved in the tRNA D-loop are enclosed with black boxes. The
anti-determinants in tRNAGlu and tRNAAsn are enclosed with blue circles.
α helix
C75
T101
T177
K254
C74
β hairpin
N178
F ig. S 5. P utative activation of the G atD glutaminas e s ite by the binding of tR NAG ln to
the G atE s ubunit, via the s ubunit interface interactions . G atD and G atE are colored cyan
and violet, res pectively. T he helical regions in the G atE cradle domain (res idues 53-72 in
M. thermautotrophicus G atE ) with conformational change between the complex form
(dark blue) and the apo form (green), are repres ented. T he indicated β-hairpin of G atD
s ubunit carries T hr101, the catalytic nucleophile, and is s ugges ted to s hift upon the
enzyme activation to relocate T hr101 to the catalytic triad (T hr177, As n178, and Lys 254)
of the glutaminas e. T he bound C C A-terminus of tR NA is indicated.
Supporting Online Table
Table S1 Summary of data collection and refinement statistics
GatDE•tRNAGln complex
Data collection
Wavelength (Å)
1.0
Resolution (Å)
50-3.15 (3.26-3.15)
Unique reflections
53,897
Redundancy
5.3 (3.8)
Completeness (%)
98.9 (97.5)
I/σ(I)
10.5 (4.9)
Rsyma
0.104 (0.351)
Refinement statistics
Resolution (Å)
50-3.15
c
d
Rwork (%) / Rfree (%)
22.3 / 28.9
Number of atoms
Protein
16,180
RNA
3,140
metal
4
Water
209
2
Average B-factor (Å )
69.0
Average rms B-factor (Å2)
main-chain
1.383
side-chain
1.590
Cross-validated sigma-A coordinate error (Å)
0.54
rms deviation
Bond lengths (Å)
0.008
Bond angles (˚)
1.32
Dihedral angles (˚)
22.2
Improper angles (˚)
1.02
Ramachandran plot
Most favorable (%)
76.5
Additionally allowed (%)
20.2
Generously allowed (%)
2.4
The numbers in parentheses are for the last shell.
a
Rsym = Σ|Iavg – Ii|/ΣIi.
b
Rcullis = Σ||FPH + FP| - FH(calc)|/ Σ|FPH|.
c
Rwork = Σ|Fo – Fc|/ΣFo for reflections of working set.
d
Rfree = Σ|Fo – Fc|/ΣFo for reflections of test set (7.5% of total reflections).
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