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

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.. Results TRNAPheaminoacylation with

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 5,5,5-trifluoro-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 Ne-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: Jan.Barciszewski@ibch.poznan.pl

(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.

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, havcharg-ing specific codons consistcharg-ing 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 tRNAPhe

can be charged with phenylalanine at high pressure

without a specific AARS and the product, Phe–

tRNAPhe, 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

TRNAPheaminoacylation with non-natural amino

acids at high pressure

Aminoacylation of tRNAPhe using natural and

non-cognate amino acids was carried out at 6 kbar as

described in Experimental procedures Charging of

tRNAPhe with 3-chloro-tyrosine (Cl-Tyr), l-ornithine

(l-Orn), d-ornithine (d-Orn),

3,4-dihydroxyphenylala-nine (DOPA) and p-chloro-phenylala3,4-dihydroxyphenylala-nine (Cl-Phe) was

analysed using PAGE (Fig 1A,B) The amounts of

amino acid incorporated into 1600 pmol of yeast

tRNAPhe, estimated on the basis of imagequant, were

40, 80, 96, 72 and 144 pmol, respectively (Table 1)

The amounts of Val–tRNAVal and Leu–tRNAVal

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

tRNAVal (Table 1) The yield of the yeast tRNAPhe

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 tRNAPhe, 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)

4

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 tRNAPhe 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]tRNAPhe with Cl-Tyr; 4, [5¢- 32

P]tRNAPhe 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]tRNAPhe 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, [14C]Leu-tRNAVal; 3, [14C]Val-tRNAVal acylated under high pressure Bands were visualized by fluorography.

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 [14C]Phe–tRNAPhe 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 [14C]Val–tRNAVal 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 tRNAPhe

(yeast)

crude tRNA (wheatgerm)

tRNAVal (E coli) 3-Chlorotyrosine 40 (2.5%) a

Chlorophenylanine 144 (9%)a

L -Ornithine 80 (5%) a

D -Ornithine 96 (6%) a

Phenylalanine [160 (10%) a ] 116 b 247 b –

a

IMAGEQUANT measurement of [5¢- 32 P]tRNA separated on

acidic ⁄ urea PAGE b

Filter binding assay of[3H] or[14C]-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).

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

14 C

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].

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 tRNAPhecould 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–tRNAPhe 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

32P-labelled tRNA (Fig 1A,B) For comparison of the results, we used tRNAVal charged with [14C]Val and [14C]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 m M MgCl 2 , 10 m M 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 [14C]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 + [14C] Trp, (r) acetyl-CoA[OH] + [14C]Trp The signal at

posi-tion 7 corresponds to Trp-CoA, at posiposi-tion 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 5000 6000

0 1500 3000 4500 6000 7500 9000

[3’OH]CoA Phe-[3’OH]CoA

Fig 8 Aminoacylation of CoA[OH] with (A) [14C]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.

ted using the imagequant calculation, as well as by

scintillation measurements of bands corresponding to

[14C-aa]tRNAVal, 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 tRNAPhe and tRNAVal 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–tRNAPhe at high pressure,

measured as a function of tRNAPhe 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¢- or

3¢-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].

without any additional substrate modification In

addi-tion, we showed that the terminal OH group is

acylat-ed preferentially

Experimental procedures

Transfer RNA

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

Pharmacia (Little Chalfont, UK)

Aminoacylation of tRNA at high pressure

Aminoacylation of tRNAs was carried out at 6 kbar for

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 MgCl2, and 1 mm M EDTA The solutions were pressured in 35 lL or 1 mL 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%

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]

with an intensifying screen Distribution of aa-tRNAs

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

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

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

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¢- or 3¢-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.

ously [44] Translation was carried out for 20, 40 and 80 min

CH3COOK, 30 mm NH4Cl, 14 mm MgCl2, 0.1 mm EDTA,

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

inhibitor, 26% (v⁄ v) S30, 0.2–0.6 lm mRNA and 5 lm

polymerase, and 0.5–2 nm of a covalently closed plasmid

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

RNaseA in 0.1 m imidazole–HCl buffer pH 6.6 containing

separated by HPLC⁄ ESI ⁄ MS on Waters ⁄ Micromass ZQ

mass spectrometer (Manchester, UK) A sample was injected

eluted with a gradient of solvent A (95% water, 5%

B in (A + B) within 10 min was applied, followed by

iso-cratic elution for 20 min with 100% methanol The source

Dephosphorylation of CoA and acetyl-CoA

CoA or acetyl-CoA (4 mmol) was dephosphorylated for 1 h

ammo-nium acetate buffer, pH 5.3, in a total volume of 10 lL

Dephosphorylated CoA (CoA[OH], CoA,

acetyl-CoA[OH]) was purified on a cellulose F plate (Merck,

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

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

visual-ized under UV, amino acid with ninhydrin staining,

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 reacscintilla-tion 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 [14C]Val-tRNAVal

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