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

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

Keywords high pressure; non-natural amino acids; tRNA charging

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.

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

is

an increasingly

leucine at d-positions of the leucine GCN4-zipper peptide increases the thermal stability of the coiled- coil structure [5].

[1]. Among these

for

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

Site-specific incorporation of non-natural amino acids into proteins emerging field the application of non-natural amino because of acids as biophysical probes in structure–function studies. Moreover, modified peptides may be key pharmaceuticals for the treatment of a variety of dis- eases 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 targeted drug delivery [4]. Replacement of leucine residues with 5,5,5-trifluoro-

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.

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Charging of tRNA with non-natural amino acids

A

aa-tRNAPhe

tRNAPhe

5

6

1

2

3

4

B

aa-tRNAPhe tRNAPhe

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 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].

4

1

2

3

C

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.

aa-tRNAVal

Results

TRNAPhe aminoacylation with non-natural amino acids at high pressure

1

2

3

[14C]Leu-tRNAVal; 3,

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 (A) Aminoacylation of yeast tRNAPhe. 1, Control 6 kbar for 6 h. [5¢-32P]tRNAPhe at 6 kbar for 6 h in a reaction buffer without amino acid; 2, control [5¢-32P]tRNAPhe incubated at normal pressure for 6 h with Cl-Tyr; 3, [5¢-32P]tRNAPhe with Cl-Tyr; 4, [5¢-32P]tRNAPhe with L-Orn, [5¢-32P]tRNAPhe with D-Orn, [5¢-32P]tRNAPhe with DOPA. (B) Aminoacylation of yeast tRNAPhe. 1, [5¢-32P]tRNAPhe not treated at high pressure; 2, [5¢-32P]tRNAPhe at 6 kbar for 5 h in reaction buffer without amino acid; and 3, [5¢-32P]tRNAPhe with Cl-Phe; 4, [5¢-32P]tRNAPhe with Phe. (C) Aminoacylation of E. coli tRNAVal. 1, Control [14C]Val-tRNAVal from T. thermophilus aminoacylated enzy- [14C]Val-tRNAVal acylated under matically, 2, high pressure. Bands were visualized by fluorography.

is

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).

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-phenylalanine (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 shown in charging with natural amino acids 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

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Table 1. Yield of aminoacylation of tRNA with different amino acids.

pmole amino acid per 1600 pmole tRNA

Amino acid

crude tRNA (wheatgerm)

tRNAVal (E. coli)

tRNAPhe (yeast)

40 (2.5%)a 144 (9%)a 80 (5%)a 96 (6%)a 72 (4.5%)a

Fig. 3. High-pressure aminoacylation of tRNA crude from wheat- germ with [14C] Lys at 6 kbar (r) in a control experiment, enzymat- ic charging with crude aa-tRNA synthetase was carried out at ambient pressure (n).

– – – – – – 123c 60c

3-Chlorotyrosine Chlorophenylanine L-Ornithine D-Ornithine DOPA Phenylalanine Tyrosine Tryptophan Arginine Lysine Histidine Valine Leucine Glycine Glutamic acid Methionine Alanine

[160 (10%)a] 116b 114b (7.1%) 109b (6.8%) 96b (6.0%) 95b (5.9%) 82b (5.1%) 74b (4.6%) 71b (4.4%) 59b (3.6%) 58b (3.6%) 54b (3.3%) 52b (3.5%)

247b 163b 173b 170b 204b – 134b 124b – – – –

separated

[5¢-32P]tRNA

a on of IMAGEQUANT measurement acidic ⁄ urea PAGE. bFilter binding assay of[3H] or[14C]-amino acids. cScintillation counting of gel slabs containing [3H] or [14C]-amino acids.

A

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 tRNA with ornithine, originated aminoacylation of 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].

B

Activity of high pressure-charged tRNA in protein biosynthesis

Fig. 2. Aminoacylation of yeast tRNAPhe with [3H]Trp at 6 kbar pres- sure as a function of (A) tRNA concentration and (B) time.

[14C]Phe–tRNAPhe aminoacylated under Activity of 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).

HPLC-MS analysis

Aminoacylation of CoA at high pressure

Charging of tRNA with non-natural amino acids was also confirmed using HPLC-MS analysis. The HPLC

The data clearly show that high pressure induces acyla- the ribose OH group. In order to check tion of

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A

Fig. 4. HPLC-MS analysis of an aminoacylation reaction of yeast tRNAPhe 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].

B

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 (cid:1) 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 respectively. The aminoacylation of 17 and 29%, 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 linear dependence of CoA aminoacylation was (Fig. 9B).

Discussion

C

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.

20

10

] e l o m p [ d e t a r o p r o c n

D

]

i l a V C 4 1 [

0

20

60

40 time [min]

In

vitro

assay. Analysis

transcription ⁄ translation

of Fig. 5. 14C-labelled Val incorporation into protein was carried out by scintil- lation counting of trichloroacetic acid-insoluble material.

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A

Lys

Lys-[3’OH]CoA

[3’OH]CoA

0

1000 2000 3000 4000

5000

6000

[cpm]

B

Phe

Phe-[3’OH]CoA

[3’OH]CoA

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 + [14C]Trp, (n) CoA[OH] + [14C]Trp, (m) acetyl- CoA + [14C] Trp, (r) acetyl-CoA[OH] + [14C]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.

0

1500 3000 4500 6000

7500

9000

[cpm]

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

(2) CoA[OH] + Ala,

(1) CoA[OH],

(6) CoA[OH] + Phe,

(7) Phe,

(5) Gly,

preparation of aa-tRNA in one step, without an enzyme or additional modification of the tRNA molecule. We have previously shown that tRNAPhe 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–tRNAPhe was a normal substrate for peptide syn- thesis on the ribosome [23].

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 MgCl2, 10 mM 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. (3) Ala, Lanes are as follows: (4) CoA[OH] + Gly, (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.

shift

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

We tested our method using Cl-Tyr, Cl-Phe, l-Orn, d-Orn and DOPA. Aminoacyl–tRNA formation analysed on acidic PAGE showed small 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-

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A

compared with the enzymatic reaction, which was due to misacylation (Fig. 3, Table 1).

that

B

(A) Time-dependent aminoacylation of CoA with Trp at aminoacylation of

(B) Pressure-dependent

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 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.

Fig. 9. 10 kbar pressure. CoA[OH].

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.

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, tRNAPhe concentration, measured as a function of 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

In summary, we have shown that the high pressure method could be used to prepare aa-tRNA in one step,

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Charging of tRNA with non-natural amino acids

Aminoacylation of tRNA at high pressure

Detection of aa-tRNA using acidic/urea gels electrophoresis

Aminoacylation of tRNAs was carried out at 6 kbar for 5 h in a mixture containing 1 lm [5¢-32P]tRNAPhe (1–5 lm tRNAPhe, 0.1 lm tRNAVal 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 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.

Fig. 10. Putative mechanism of tRNA aminoacylation at high pres- In the first step, high pressure induces a lowering of pH sure. 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.

without any additional substrate modification. In addi- tion, we showed that the terminal OH group is acylat- ed preferentially.

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].

Experimental procedures

Transfer RNA

Enzymatic aminoacylation of tRNA

[5¢-32P]-tRNAPhe was identified by autoradiography. Gels were exposed overnight at )70 (cid:1)C to X-ray films in a cassette with an intensifying screen. Distribution of aa-tRNAs labelled with [14C] 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 (cid:1)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].

tRNAPhe, E. coli

pH 7.5, containing 0.01 m MgCl2,

Uniformly radiolabelled amino acids

tRNAVal, CoA and acetyl-CoA Yeast 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).

Coupled in vitro transcription/translation

Five, 10, 15 and 20 lg of crude tRNA from wheatgerm were dissolved in a buffer containing 50 lL of 0.1 m 4 mm Tris ⁄ HCl, b-mercaptoethanol, 2 mm ATP and 2 lm [14C]-lysine. After 20 min of incubation with crude plant AARS at 37 (cid:1)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-tRNAVal from Thermus thermophilus (220 cpmÆpmole)1) used as a control was a gift from M. Sprinzl (Bayreuth University, Germany).

l-[5-3H]Trp [3H]Met

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The in vitro translation reaction was based on an E. coli S30 lysate (strain D10) and was performed as described previ- [14C]Gly (235 mCiÆmmol)1), [14C]Phe (360 mCiÆmmol)1), (250 mCiÆmmol)1), [14C]Arg (210 mCiÆmmol)1), [14C]Tyr [14C]Leu (230 mCiÆmmol)1) (235 mCiÆmmol)1), [14C]Lys and [14C]His (215 mCiÆmmol)1) were from UVVVR (Pra- (28 CiÆmmol)1), gue, Czech Republic); (15 CiÆmmol)1) and G-[3H]Glu (53 CiÆmmol)1), [14C]Val (45 mCiÆmmol)1) were purchased from Amersham Pharmacia (Little Chalfont, UK).

M. Giel-Pietraszuk and J. Barciszewski

Charging of tRNA with non-natural amino acids

TLC of aminoacyl-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.

rifampicin, 0.1 mgÆmL)1

HPLC/ESI/MS analysis

ously [44]. Translation was carried out for 20, 40 and 80 min at 37 (cid:1)C in a 240 mL reaction mixture containing the follow- ing components: 50 mm Hepes-KOH pH 7.6, 70 mm CH3COOK, 30 mm NH4Cl, 14 mm MgCl2, 0.1 mm EDTA, 0.02% NaN3, 40 lg [14C]Val-tRNAVal (18 500 c.p.m. ⁄ A260), 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 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-[14C]Val into the synthesized proteins was determined by liquid scintillation counting of the trichlo- roacetic acid-insoluble material as described previously [44].

(or aminoacyl-acetyl-CoA[OH]) The aminoacyl-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, [14C]-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 [14C]Val-tRNAVal.

References

Dephosphorylation of CoA and acetyl-CoA

1 Hendrickson TL, Crecy-Lagard V & Schimmel P (2004) Incorporation of non-natural amino acids into proteins. Annu Rev Biochem 73, 147–176. 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 (cid:1)C for 15 min with 1 unit of RNaseA in 0.1 m imidazole–HCl buffer pH 6.6 containing 20 mm MgCl2 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 C18 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 (cid:1)C and the desolvation temperature was 300 (cid:1)C. Nitrogen was used as the nebulizing and desol- vation gas at flow rates of 100 and 600 LÆh)1, respectively.

2 Abdel-Rahman HM, Al-Karamany GS, El-Koussi NA, Youssef AF & Kiso Y (2002) HIV protease inhibitors: peptidomimetic drugs and future perspectives. Curr Med Chem 9, 1905–1922.

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CoA or acetyl-CoA (4 mmol) was dephosphorylated for 1 h at 37 (cid:1)C with 1 unit of nuclease P1 (Sigma) in 50 mm 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

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