Báo cáo khoa học: Charging of tRNA with non-natural amino acids at high pressure potx

Charging of tRNA with non-natural amino acids
at high pressure
Malgorzata Giel-Pietraszuk and Jan Barciszewski
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
Site-specific incorporation of non-natural amino acids
into proteins is an increasingly emerging field
because of the application of non-natural amino
acids as biophysical probes in structure–function
studies. Moreover, modified peptides may be key
pharmaceuticals for the treatment of a variety of dis-
eases [1]. Among these compounds are protease
inhibitors, a classic example of which are the HIV
protease inhibitors [2,3]. Replacement of methionine
with selenomethionine has been used extensively for
phase determination in protein crystallography, and
the exchange of 4-fluorotryptophan for tryptophan
has been used in NMR analysis. Studies on the
function and properties of proteins require mutants
containing amino acid analogues, for example thiopr-
oline, at multiple sites that do not influence protein
function, including immunogenicity, but may serve as
promising vehicles for targeted drug delivery [4].
Replacement of leucine residues with 5,5,5-trifluoro-
leucine at d-positions of the leucine GCN4-zipper
peptide increases the thermal stability of the coiled-
coil structure [5].
Several strategies have been used to introduce non-
natural amino acids into proteins [1,6]. One of the first
was the derivatization of amino acids at reactive side
chains, for example, conversion of Lys to N
e

-acetyl
lysine. Chemical preparation provides a straightfor-
ward method for the incorporation of non-natural
amino acids using solid-phase peptide synthesis, but
for technical reasons, it remains restricted to small
peptides [7–9]. Development of enzymatic and native
chemical ligations allows us to obtain larger proteins
[10]. General in vitro methods of site-specific incorpor-
ation of the desired amino acid into a protein are
based on chemically charged suppressor tRNA, used
in a translation system [11]. Over 100 non-natural
amino acids have been introduced into proteins of
varying size [12]. The utility of mischarged tRNAs has
Keywords
high pressure; non-natural amino acids;
tRNA charging
Correspondence
Jan Barciszewski, Institute of Bioorganic
Chemistry, Polish Academy of Sciences,
Noskowskiego 12 ⁄ 14, 61-704 Poznan,
Poland
Fax: +49 61 852 05 32
Tel: +48 61 852 85 03 (ext. 132)
E-mail:
(Received 3 October 2005, revised 25 April
2006, accepted 9 May 2006)
doi:10.1111/j.1742-4658.2006.05312.x
We show a simple and reliable method of tRNA aminoacylation with
natural, as well as non-natural, amino acids at high pressure. Such specific
and noncognate tRNAs can be used as valuable substrates for protein

engineering. Aminoacylation yield at high pressure depends on the chem-
ical nature of the amino acid used and it is up to 10%. Using CoA, which
carries two potentially reactive groups -SH and -OH, as a model com-
pound we showed that at high pressure amino acid is bound preferentially
to the hydroxyl group of the terminal ribose ring.
Abbreviations
AARS, aminoacyl–tRNA synthetase; aa-tRNA, aminoacyl-tRNA; Cl-Phe, p-chloro-phenylalanine; Cl-Tyr, 3-chloro-tyrosine; DOPA,
3,4-dihydroxyphenylalanine;
D-Orn, D-ornithine; L-Orn, L-ornithine; Orn-Ado, adenosyl-ornithine; p-Cl-Phe-Ado, adenosyl-p-chlorophenylalanine;
Phe-Ado, adenosyl-phenylalanine; PPO, 2,5-diphenyloxazone.
3014 FEBS Journal 273 (2006) 3014–3023 ª 2006 The Authors Journal compilation ª 2006 FEBS
been expanded by developing chemical acylation of the
unprotected dinucleotide pCpA, followed by enzymatic
ligation to the 3¢-terminus of truncated tRNA using
T4 RNA ligase. This approach has a low acylation
yield of 3–4% [13,14]. An improved version of the
method, based on the acylation of fully protected
5¢pCpCpA resulted in 26% charging [15]. Other meth-
ods of tRNA aminoacylation with non-natural amino
acids take advantage of ribozymes or appropriately
mutated aminoacyl-tRNA synthetases (AARS) charg-
ing tRNA, having specific codons consisting of four or
five bases [16–20]. These methods, in contrast to enzy-
matic aminoacylation, which is generally limited to
natural amino acids or their analogues, enables the
acylation of tRNAs with any non-native amino acid
[16–21]. We have previously shown that yeast tRNA
Phe
can be charged with phenylalanine at high pressure
without a specific AARS and the product, Phe–

tRNA
Phe
, was the correct substrate for protein biosyn-
thesis [22,23].
Here, we show that aminoacylation of tRNA at high
pressure may be used to prepare aminoacyl-tRNA
(aa-tRNA) using any natural or non-natural amino
acid. Using CoA, we also show that amino acid binds
specifically to the ribose ring at high pressure. Applica-
tion of MS provides evidence that charging occurs at
the hydroxyl group of the 3¢-end ribose.
Results
TRNA
Phe
aminoacylation with non-natural amino
acids at high pressure
Aminoacylation of tRNA
Phe
using natural and non-
cognate amino acids was carried out at 6 kbar as
described in Experimental procedures. Charging of
tRNA
Phe
with 3-chloro-tyrosine (Cl-Tyr), l-ornithine
(l-Orn), d-ornithine (d-Orn), 3,4-dihydroxyphenylala-
nine (DOPA) and p-chloro-phenylalanine (Cl-Phe) was
analysed using PAGE (Fig. 1A,B). The amounts of
amino acid incorporated into 1600 pmol of yeast
tRNA
Phe

, estimated on the basis of imagequant, were
40, 80, 96, 72 and 144 pmol, respectively (Table 1).
The amounts of Val–tRNA
Val
and Leu–tRNA
Val
cal-
culated from the scintillation measurement of gel
slices, obtained after fluorography (Fig. 1C), were
123 and 60 pmol per 1600 pmol of Escherichia coli
tRNA
Val
(Table 1). The yield of the yeast tRNA
Phe
charging with natural amino acids is shown in
Table 1. Time-dependent aminoacylation of tRNA
with tryptophan showed that the best result, 96 pmol
of Tyr per 1600 pmol of tRNA
Phe
, was obtained after
30 min of pressure at 6 kbar (Fig. 2). The yield of
charging of crude tRNA from wheatgerm with lysine
at high pressure was 333 pmol Lys per 40 lg
tRNA, and in the enzymatic reaction was 133 pmol
(Fig. 3).
1234
123
12 3
4
56

aa-tRNA
Phe
tRNA
Phe
aa-tRNA
Phe
aa-tRNA
Val
tRNA
Phe
A
B
C
Fig. 1. Detection of aa-tRNA using acidic ⁄ urea gel electrophoresis.
Reactions were performed as described in the Experimental proce-
dures. Aminoacylation of tRNA with different amino acids carried at
6 kbar for 6 h. (A) Aminoacylation of yeast tRNA
Phe
. 1, Control
[5¢-
32
P]tRNA
Phe
at 6 kbar for 6 h in a reaction buffer without amino
acid; 2, control [5¢-
32
P]tRNA
Phe
incubated at normal pressure for
6 h with Cl-Tyr; 3, [5¢-

32
P]tRNA
Phe
with Cl-Tyr; 4, [5¢-
32
P]tRNA
Phe
with L-Orn, [5¢-
32
P]tRNA
Phe
with D-Orn, [5¢-
32
P]tRNA
Phe
with DOPA.
(B) Aminoacylation of yeast tRNA
Phe
.1,[5¢-
32
P]tRNA
Phe
not treated
at high pressure; 2, [5¢-
32
P]tRNA
Phe
at 6 kbar for 5 h in reaction
buffer without amino acid; and 3, [5¢-
32

P]tRNA
Phe
with Cl-Phe; 4,
[5¢-
32
P]tRNA
Phe
with Phe. (C) Aminoacylation of E. coli tRNA
Val
.1,
Control [
14
C]Val-tRNA
Val
from T. thermophilus aminoacylated enzy-
matically, 2, [
14
C]Leu-tRNA
Val
;3,[
14
C]Val-tRNA
Val
acylated under
high pressure. Bands were visualized by fluorography.
M. Giel-Pietraszuk and J. Barciszewski Charging of tRNA with non-natural amino acids
FEBS Journal 273 (2006) 3014–3023 ª 2006 The Authors Journal compilation ª 2006 FEBS 3015
HPLC-MS analysis
Charging of tRNA with non-natural amino acids was
also confirmed using HPLC-MS analysis. The HPLC

chromatogram of aa-tRNA, partially hydrolysed with
RNaseA, showed a peak at a retention time of 9.99 min;
this was identified using ESI-MS as adenosyl-phenyl-
alanine (Phe-Ado) (Fig. 4A). Two major signals at
m ⁄ z ¼ 415 and 437 corresponded to [M + 1]
+
and
[M + Na]
+
ions, respectively. The ESI-MS spectrum of
tRNA aminoacylated with Chl-Phe showed signals at
m ⁄ z ¼ 415 and 457, corresponding to [M + 1]
+
and
[M + Na]
+
of Phe-Ado, respectively, and m ⁄ z ¼ 450
corresponding to [M + 1]
+
of adenosyl-p-chloroph-
enylalanine (p-Cl-Phe-Ado) (Fig. 4B). The strongest sig-
nal on the ESI-MS spectrum, m ⁄ z ¼ 419, recorded for
aminoacylation of tRNA with ornithine, originated
from adenosyl-ornithine (Orn-Ado), whereas the signals
at m ⁄ z ¼ 331 and 389 derived from its decomposition
products. The first corresponded to fragmentation of a
five-membered ring sugar by releasing 29 mass units,
and the second by breaking the C–C bond between
ribose and the methyl group (Fig. 4C) [24,25].
Activity of high pressure-charged tRNA

in protein biosynthesis
Activity of [
14
C]Phe–tRNA
Phe
aminoacylated under
high pressure has been checked previously in an in vitro
translation assay using poly-(U)-programmed ribo-
somes [23]. Here we also show that [
14
C]Val–tRNA
Val
prepared at high pressure was active in an in vitro
transcription ⁄ translation assay. This means that high
pressure-charged tRNA is a good substrate in protein
synthesis (Fig. 5).
Aminoacylation of CoA at high pressure
The data clearly show that high pressure induces acyla-
tion of the ribose OH group. In order to check
Table 1. Yield of aminoacylation of tRNA with different amino acids.
Amino acid
pmole amino acid per 1600 pmole tRNA
tRNA
Phe
(yeast)
crude tRNA
(wheatgerm)
tRNA
Val
(E. coli)

3-Chlorotyrosine 40 (2.5%)
a
Chlorophenylanine 144 (9%)
a
L-Ornithine 80 (5%)
a
D-Ornithine 96 (6%)
a
DOPA 72 (4.5%)
a
Phenylalanine [160 (10%)
a
] 116
b
247
b
–
Tyrosine 114
b
(7.1%) 163
b
–
Tryptophan 109
b
(6.8%) 173
b
–
Arginine 96
b
(6.0%) 170

b
–
Lysine 95
b
(5.9%) 204
b
–
Histidine 82
b
(5.1%) – –
Valine 74
b
(4.6%) 134
b
123
c
Leucine 71
b
(4.4%) 124
b
60
c
Glycine 59
b
(3.6%) –
Glutamic acid 58
b
(3.6%) –
Methionine 54
b

(3.3%) –
Alanine 52
b
(3.5%) –
a
IMAGEQUANT measurement of [5¢-
32
P]tRNA separated on
acidic ⁄ urea PAGE.
b
Filter binding assay of[
3
H] or[
14
C]-amino acids.
c
Scintillation counting of gel slabs containing [
3
H] or [
14
C]-amino
acids.
A
B
Fig. 2. Aminoacylation of yeast tRNA
Phe
with [
3
H]Trp at 6 kbar pres-
sure as a function of (A) tRNA concentration and (B) time.

Fig. 3. High-pressure aminoacylation of tRNA crude from wheat-
germ with [
14
C] Lys at 6 kbar (r) in a control experiment, enzymat-
ic charging with crude aa-tRNA synthetase was carried out at
ambient pressure (n).
Charging of tRNA with non-natural amino acids M. Giel-Pietraszuk and J. Barciszewski
3016 FEBS Journal 273 (2006) 3014–3023 ª 2006 The Authors Journal compilation ª 2006 FEBS
whether the hydroxyl group of ribose is preferentially
acylated, we used CoA as a model. CoA and trypto-
phan were subjected to a pressure of 10 kbar overnight
followed by TLC. Bands of free substrates and amino-
acyl–CoA were visualized using UV light and then
stained with ninhydrin. The yield of this reaction was
 8% (Fig. 6). Analysis of dephosphorylated CoA
(CoA[OH]), aminoacylated with different amino acids
carried out using TLC (Figs 7 and 8) showed the fol-
lowing reaction yields: 50, 90, 32, 23 and 28% for Ala,
Gly, Val, Phe and Lys, respectively. Tryptophan
bound to CoA[OH] and acetyl-CoA[OH] with yields of
17 and 29%, respectively. The aminoacylation of
CoA[OH] with tryptophan was essentially completed
in 3 h and, after that, a slow decrease in product
concentration was observed (Fig. 9A). The pressure
dependence of CoA aminoacylation was linear
(Fig. 9B).
Discussion
The preparation of tRNA charged with non-natural
amino acid is a critical step in the synthesis of modified
protein. All methods of preparing aa-tRNA charged

with non-native amino acids are complicated and time-
consuming [1–19]. In this study, we developed a general
method of tRNA aminoacylation using any amino acid.
0
10
20
20 40 60
[
14
C]Val incorporated [pmole]
time [min]
Fig. 5. In vitro transcription ⁄ translation assay. Analysis of
14
C-labelled Val incorporation into protein was carried out by scintil-
lation counting of trichloroacetic acid-insoluble material.
A
B
C
D
Fig. 4. HPLC-MS analysis of an aminoacylation reaction of yeast
tRNA
Phe
with different amino acids carried out at 6 kbar for 5 h. Sig-
nals at (A) m ⁄ z ¼ 415 and 437 correspond to [M + H]
+
and [M + Na]
+
of the Phe-Ade, respectively; (B) m ⁄ z ¼ 415 and 437 correspond to
[M + H]
+

and [M + Na]
+
of the Phe-Ade, respectively, m ⁄ z ¼ 450
to [M + H]
+
of the p-Chl-Phe-Ade; (C) m ⁄ z ¼ 419 to [M + H]
+
of
the Orn-Ade; (D) other signals correspond to the disintegration
products Orn-Ade formula showing disintegration products [24,25].
M. Giel-Pietraszuk and J. Barciszewski Charging of tRNA with non-natural amino acids
FEBS Journal 273 (2006) 3014–3023 ª 2006 The Authors Journal compilation ª 2006 FEBS 3017
For that purpose, we used a high-pressure technique.
High pressure is currently used in many areas of bio-
technology. Its mechanism of action includes the deci-
sive role of water structure [26,27]. High pressure allows
preparation of aa-tRNA in one step, without an enzyme
or additional modification of the tRNA molecule. We
have previously shown that tRNA
Phe
could be charged
with Phe at high pressure without the need for a specific
aa-tRNA synthetase. Aminoacylation occurred only at
the 3¢-end of tRNA [22] and pressure-aminoacylated
Phe–tRNA
Phe
was a normal substrate for peptide syn-
thesis on the ribosome [23].
We tested our method using Cl-Tyr, Cl-Phe, l-Orn,
d-Orn and DOPA. Aminoacyl–tRNA formation

analysed on acidic PAGE showed small shift in
32
P-labelled tRNA (Fig. 1A,B). For comparison of the
results, we used tRNA
Val
charged with [
14
C]Val and
[
14
C]Leu, and analysed them using acidic PAGE visu-
alized with fluorography (Fig. 1C). The results, estima-
Fig. 7. TLC of CoA[OH] aminoacylation carried out at 6 kbar for 6 h
in buffer: 0.1
M imidazole–HCl pH 6.6, 20 mM MgCl
2
,10mM EDTA.
The TLC plate was developed in butanol-1 ⁄ acetic acid ⁄ water (1:1:1
v ⁄ v ⁄ v) and visualized with a 0.1% ethanolic solution of ninhydrin.
Lanes are as follows: (1) CoA[OH], (2) CoA[OH] + Ala, (3) Ala,
(4) CoA[OH] + Gly, (5) Gly, (6) CoA[OH] + Phe, (7) Phe, (8)
CoA[OH] + Val, (9) Val. Position of CoA[OH], in circles, was visual-
ized under UV light. The arrows show the position of the products.
Fig. 6. Aminoacylation of CoA[OH] with [
14
C]Trp at high pressure.
Graphic representation shows the distribution of radioactivity on a
TLC plate: (d)CoA+[
14
C]Trp, (n) CoA[OH] + [

14
C]Trp, (m) acetyl-
CoA + [
14
C] Trp, (r) acetyl-CoA[OH] + [
14
C]Trp. The signal at posi-
tion 7 corresponds to Trp-CoA, at position 9 free Trp was detected.
The reaction was analysed by TLC on cellulose with fluorescence
indicator F254 and developed in an isobutyric acid solution, the TLC
plate was cut into pieces as shown in the left-hand panel and the
radioactivity was counted in scintillator solvent using Beckmann
Apparatus LS 5000 TA. The position of the substrates was visual-
ized under UV light.
A
B
[cpm]
[cpm]
Lys
[3’OH]CoA
Lys-[3’OH]CoA
Phe
0 1000 2000 3000 4000 60005000
0 1500 3000 4500 6000 90007500
[3’OH]CoA
Phe-[3’OH]CoA
Fig. 8. Aminoacylation of CoA[OH] with (A) [
14
C]Lys and (B)
[

14
C]Phe. Reactions were carried out at 10 kbar pressure for 12 h.
Aminoacyl-CoA[OH] was separated from free amino acids using
TLC cellulose F. Diagrams show the distribution of radioactivity on
the TLC plate measured in scintillator solvent.
Charging of tRNA with non-natural amino acids M. Giel-Pietraszuk and J. Barciszewski
3018 FEBS Journal 273 (2006) 3014–3023 ª 2006 The Authors Journal compilation ª 2006 FEBS
ted using the imagequant calculation, as well as by
scintillation measurements of bands corresponding to
[
14
C-aa]tRNA
Val
, were similar. The yield of tRNA
charging at high pressure with non-natural amino acids
was between 2.5 and 9%, similar to data obtained for
aminoacylation with natural amino acids (Table 1).
Analysis of tRNA
Phe
and tRNA
Val
charging with a
series of natural amino acids showed that the best
yields were obtained for aromatic amino acids, but
aminoacylation using amino acids with an aliphatic
side chain was less efficient (Table 1). This can be
explained by chemical activation of the carbonyl group
by aromatic moiety. This observation is consistent with
the suggestion that the aromatic ring of some amino
acids is stabilized by association with an adenine ring.

A similar effect was observed for Phe-AMP ester
[28,29]. Synthesis of Trp–tRNA
Phe
at high pressure,
measured as a function of tRNA
Phe
concentration,
showed the highest yield after 30 min (Fig. 2A) [30].
Longer incubation decreased the amount of product
(Fig. 2B). Aminoacylation of crude tRNA with Lys
at high pressure was approximately 2.5 times higher
compared with the enzymatic reaction, which was due
to misacylation (Fig. 3, Table 1).
To obtain more data on tRNA charging at the 3¢-
end, we performed MS analysis of a product after lim-
ited hydrolysis of aa-tRNA with RNaseA. MS analysis
showed that the signals corresponded to Phe-Ade, p-Cl-
Phe-Ade and l-Orn-Ade (Fig. 4). In the spectrum for
l-Orn-Ade, in addition to the highest peak, other
signals were observed. One of them, at m ⁄ z ¼ 331,
suggests that the 2¢-OH group becomes esterified
(Fig. 4D). The high-pressure aminoacylation occurred
preferentially at the OH group of the terminal ribose
ring. The 3¢-phosphate-free CoA molecule carried two
potentially reactive sites, a thiol group and a 2¢-or3¢-
OH group of ribose and, owing to this, we found it to
be a very good substrate for high-pressure aminoacyla-
tion (Figs 6–9). It has previously been reported that the
thiol group of CoA can be acylated by AARS [31].
Furthermore, it was shown that AARSs are able to

utilize noncognate amino acids in the aminoacylation
of CoA, and in the acylation of mini helix of RNA
[32]. The equilibrium of CoA acylation was shifted
towards an aa-S-CoA formation [33]. In the case of
high-pressure induced aminoacylation of CoA, we
observed that the -OH group was acylated preferen-
tially. The [HS]CoA acylation yield with Trp was 8%,
whereas for [HS]CoA[OH] and [acetyl-S]CoA[OH] it
was 17 and 29%, respectively.
The detailed mechanism for the aminoacylation
reaction of tRNA at high pressure remains unknown.
Recently, we obtained new information about the con-
formation of tRNA at elevated pressure [26]. It is
known that high pressure lowers the pH of water.
Because of this, the carbonyl group of the amino acid
becomes protonated [27], which creates a positively
charged carbon reactive towards nucleophilic attack by
the ribose -OH group. Such acylation does not occur
at normal pressure or at high pressure without imidaz-
ole, which is a commonly occurring group in the active
centres of many enzymes and plays an important role
in electron transfer. Imidazole catalyses the aminoacyl
transfer from adenylate anhydride to the 2¢OH groups
along the RNA backbone [34]. The nitrogen of imidaz-
ole attracts a proton from the hydroxyl group, which
facilitates nucleophilic attack (Fig. 10). The entire pro-
cess is induced by high pressure and does not proceed
without it. We showed that high pressure influences
the conformation of tRNA because of rearrangements
in the structure of water [26]. These changes most

probably create a binding pocket anchoring side chain
in the amino acid, which brings the substrates closer.
In summary, we have shown that the high pressure
method could be used to prepare aa-tRNA in one step,
A
B
Fig. 9. (A) Time-dependent aminoacylation of CoA with Trp at
10 kbar pressure. (B) Pressure-dependent aminoacylation of
CoA[OH].
M. Giel-Pietraszuk and J. Barciszewski Charging of tRNA with non-natural amino acids
FEBS Journal 273 (2006) 3014–3023 ª 2006 The Authors Journal compilation ª 2006 FEBS 3019
without any additional substrate modification. In addi-
tion, we showed that the terminal OH group is acylat-
ed preferentially.
Experimental procedures
Transfer RNA
Yeast tRNA
Phe
, E. coli tRNA
Val
, CoA and acetyl-CoA
were purchased from Sigma (St Louis, MO, USA). Crude
tRNA from wheatgerm and AARS were purified by us
[35,36]. Ala, Gly, Arg, Glu, His, Leu, Lys, Met, Phe, Tyr,
Cl-Phe, Cl-Tyr, l-Orn and d-Orn were purchased from
Sigma, and DOPA was from Behringwerke AG (Marburg,
Germany).
Uniformly radiolabelled amino acids
[
14

C]Phe (360 mCiÆmmol
)1
), [
14
C]Gly (235 mCiÆmmol
)1
),
[
14
C]Arg (210 mCiÆmmol
)1
), [
14
C]Tyr (250 mCiÆmmol
)1
),
[
14
C]Lys (235 mCiÆmmol
)1
), [
14
C]Leu (230 mCiÆmmol
)1
)
and [
14
C]His (215 mCiÆmmol
)1
) were from UVVVR (Pra-

gue, Czech Republic); l-[5-
3
H]Trp (28 CiÆmmol
)1
),
G-[
3
H]Glu (53 CiÆmmol
)1
), [
3
H]Met (15 CiÆmmol
)1
) and
[
14
C]Val (45 mCiÆmmol
)1
) were purchased from Amersham
Pharmacia (Little Chalfont, UK).
Aminoacylation of tRNA at high pressure
Aminoacylation of tRNAs was carried out at 6 kbar for
5 h in a mixture containing 1 lm [5¢-
32
P]tRNA
Phe
(1–5 lm
tRNA
Phe
, 0.1 lm tRNA

Val
or 40–50 lg of crude tRNA),
and 0.1 mm of nonlabelled amino acid (or radioactively
labelled amino acid mixed with nonlabelled tRNA to
obtain the desired specific activity per mmol), 0.1 m imidaz-
ole–HCl buffer pH 6.6, 20 mm MgCl
2
, and 1 mm M
EDTA. The solutions were pressured in 35 lLor1mL
Teflon vessels placed in high-pressure cell (Unipress, War-
saw, Poland). After pressuring, aa-tRNA was precipitated
with ethanol, dried and dissolved in water.
Detection of aa-tRNA using acidic/urea gels
electrophoresis
aa-tRNA was purified from free tRNA on a 6.5% poly-
acrylamide gel (19:1 acrylamide ⁄ bisacrylamide v ⁄ v) with
8 m urea in 0.1 m sodium acetate buffer, pH 5.0. Electro-
phoresis was carried out at 600 V until the Bromophenol
blue reached the bottom of the gel [37].
[5¢-
32
P]-tRNA
Phe
was identified by autoradiography. Gels
were exposed overnight at )70 °C to X-ray films in a cassette
with an intensifying screen. Distribution of aa-tRNAs
labelled with [
14
C] was detected by fluorography. After elec-
trophoresis the gel was treated with dimethylsulfoxide for

20 min in order to remove water, and soaked with 10% 2,5-
diphenyloxazone (PPO) in dimethylsulfoxide for 2 h. Excess
PPO was removed with water, and the gel was dried and
exposed to X-ray film in a cassette with an intensifying screen
at )70 °C for 48 h. Radioactivity was measured by scintilla-
tion counting of individual gel slices [38,39]. Charging the
efficiency of crude tRNA was monitored using the filter
binding method [40].
Enzymatic aminoacylation of tRNA
Five, 10, 15 and 20 lg of crude tRNA from wheatgerm
were dissolved in a buffer containing 50 lL of 0.1 m
Tris ⁄ HCl, pH 7.5, containing 0.01 m MgCl
2
,4mm
b-mercaptoethanol, 2 mm ATP and 2 lm [
14
C]-lysine. After
20 min of incubation with crude plant AARS at 37 °C, the
reaction mixture was spotted onto Whatmann 3 mm filter
paper, washed once in 10% ice-cold trichloroacetic acid,
twice in 5% trichloroacetic acid and, finally, with ethanol
[41,42]. The radioactivity of aa-tRNA was measured by
scintillation counting [43]. Valyl-tRNA
Val
from Thermus
thermophilus (220 cpmÆpmole
)1
) used as a control was a gift
from M. Sprinzl (Bayreuth University, Germany).
Coupled in vitro transcription/translation

The in vitro translation reaction was based on an E. coli S30
lysate (strain D10) and was performed as described previ-
Fig. 10. Putative mechanism of tRNA aminoacylation at high pres-
sure. In the first step, high pressure induces a lowering of pH
and protonation of amino acid. A proton from the 2¢-or3¢-OH
group is transferred to imidazole and a lone oxygen pair attack
activates the carbon of the amino acid. Releasing of the high
pressure causes dehydration of the intermediate product and
aa-tRNA formation.
Charging of tRNA with non-natural amino acids M. Giel-Pietraszuk and J. Barciszewski
3020 FEBS Journal 273 (2006) 3014–3023 ª 2006 The Authors Journal compilation ª 2006 FEBS
ously [44]. Translation was carried out for 20, 40 and 80 min
at 37 °C in a 240 mL reaction mixture containing the follow-
ing components: 50 mm Hepes-KOH pH 7.6, 70 mm
CH
3
COOK, 30 mm NH
4
Cl, 14 mm MgCl
2
, 0.1 mm EDTA,
0.02% NaN
3
,40lg[
14
C]Val-tRNAVal (18 500 c.p.m. ⁄ A
260
),
0.2 mm of each amino acid (Val omitted), 1 mm each of ATP
and GTP, 0.5 mm each of CTP and UTP, 30 mm phospho-

enolpyruvate, 10 mm acetyl phosphate, 4% poly(ethylene
glycol) 2000, 20 lgÆmL
)1
rifampicin, 0.1 mgÆmL
)1
total
E. coli tRNA, 0.1 mm folinic acid, 100 unitsÆmL
)1
RNase
inhibitor, 26% (v ⁄ v) S30, 0.2–0.6 lm mRNA and 5 lm anti-
ssrA oligonucleotide, 1 lgÆmL
)1
leupeptin, 2 lgÆmL
)1
aproti-
nin, 1 lgÆmL
)1
pepstatin, 500 unitsÆmL
)1
T7 phage RNA
polymerase, and 0.5–2 nm of a covalently closed plasmid.
The incorporation of l-[
14
C]Val into the synthesized proteins
was determined by liquid scintillation counting of the trichlo-
roacetic acid-insoluble material as described previously [44].
HPLC/ESI/MS analysis
Ten micrograms of aa-tRNA obtained at high pressure were
purified from free amino acid on a Sephadex G-75 column
and subsequently digested at 37 °C for 15 min with 1 unit of

RNaseA in 0.1 m imidazole–HCl buffer pH 6.6 containing
20 mm MgCl
2
and 1 mm EDTA. The hydrolysed tRNA was
separated by HPLC ⁄ ESI ⁄ MS on Waters ⁄ Micromass ZQ
mass spectrometer (Manchester, UK). A sample was injected
using an autosampler onto a Waters Nova Pak C
18
RP-18
column (150 · 3.9 mm) at a flow rate of 0.5 mLÆmin
)1
and
eluted with a gradient of solvent A (95% water, 5% aceto-
nitrile v ⁄ v) and solvent B (45% water, 50% methanol, 5%
acetonitrile: v ⁄ v ⁄ v). A linear gradient from 100% A to 100%
B in (A + B) within 10 min was applied, followed by iso-
cratic elution for 20 min with 100% methanol. The source
temperature was 120 °C and the desolvation temperature
was 300 °C. Nitrogen was used as the nebulizing and desol-
vation gas at flow rates of 100 and 600 LÆh
)1
, respectively.
Dephosphorylation of CoA and acetyl-CoA
CoA or acetyl-CoA (4 mmol) was dephosphorylated for 1 h
at 37 °C with 1 unit of nuclease P1 (Sigma) in 50 m m ammo-
nium acetate buffer, pH 5.3, in a total volume of 10 lL.
Dephosphorylated CoA (CoA[OH], acetyl-CoA, acetyl-
CoA[OH]) was purified on a cellulose F plate (Merck, Darm-
stadt, Germany) in a solvent containing isobutyric acid ⁄
25% ammonium hydroxide ⁄ water (15:0.25:7.25 v ⁄ v ⁄ v). The

spots corresponding to CoA[OH] and acetyl-CoA[OH] were
visualized at UV light, scraped out and eluted with water.
Aminoacylation of CoA at high pressure
Aminoacylation of CoA was carried out at 10 kbar for
16 h in a mixture containing 1 mm CoA ( acetyl -CoA,
CoA[OH], acetyl-CoA[OH]), 10 mm labelled amino acid in
the buffer used for the tRNA aminoacylation. Trp-CoA
purified on a TLC plate was dissolved in 20 lL of 0.1 m
Tris ⁄ HCl pH 8.2 and left at room temperature for 14 h.
Random deacylation was observed on TLC.
TLC of aminoacyl-CoA
The aminoacyl-CoA[OH] (or aminoacyl-acetyl-CoA[OH])
was analysed by TLC on cellulose F (Merck) in solvent
containing isobutyric acid ⁄ ammonium hydroxide ⁄ water
(15:0.25:7.25 v ⁄ v ⁄ v). The positions of free CoA were visual-
ized under UV, amino acid with ninhydrin staining,
[
14
C]-labelled amino acids were located by measuring of the
samples in scintillation counter (Beckman, Fullerton, CA,
USA). For that purpose, the TLC plate was cut into pieces
and the amount of radioactivity was measured using scintilla-
tion counting [43]. Each reaction was repeated five times and
the per cent yield of aminoacylation and standard deviation
were calculated based on five independent measurements.
Acknowledgements
We thank Ms Sylwia Dolecka and Ms Ewa Powalska
for their assistance in laboratory work. We thank also
Prof Dr Volker A. Erdmann and Dr Torsten Lamla
from the Free University in Berlin for help in carrying

out the transcription ⁄ translation assay and Prof Math-
ias Sprinzl from Bayreuth University for providing us
with [
14
C]Val-tRNA
Val
.
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