the complex formation between escherichia coli aminoacyl-trna, elongation factor tu and gtp : the...

Eur. J. Biochem. 108, 213-221 (1980) 0 by FEBS 1980

The Complex Formation between Escherichia coli Aminoacyl-tRNA, Elongation Factor Tu and GTP The Effect of the Side-Chain of the Amino Acid Linked to tRNA

Thomas WAGNER and Mathias SPRINZL

Max-Planck-Institut fur experimentelle Medizin, Abteilung Chemie, Gottingen, and Laboratorium fur Biochemie der Universitat Bayreuth

(Received March 11, 1980)

The interaction between Escherichiu coli aminoacyl-tRNAs and elongation factor Tu (EF-Tu) . GTP was examined. Ternary complex formation with Phe-tRNAPhe and Lys-tRNALYs was compared to that with the respective misaminoacylated Tyr-tRNAPhe and Phe-tRNALys. There was no pronounced difference in the efficiency of aminoacyl-tRNA . EF-Tu . GTP complex formation between Phe-tRNAPh" and Tyr-tRNAphe. However, Phe-tRNALY' was bound preferentially to EF- Tu . GTP as compared to Lys-tRNALY5. This was shown by the ability of EF-Tu . GTP to prevent the hydrolysis of the aminoacyl ester linkage of the aminoacyl-tRNA species. Furthermore, gel filtration of ternary complexes revealed that the complex formed with the misaminoacylated tRNALyS was also more stable than the one formed with the correctly aminoacylated tRNALYs.

Both misaminoacylated aminoacyl-tRNA species could participate in the ribosomal peptide elongation reaction. Poly(U)-directed synthesis of poly(Tyr) using Tyr-tRNAPhe occurred to a comparable extent as the synthesis of poly(Phe) with Phe-tRNAphe. In the translation of poly(A) using native Lys-tRNALYs, poly(Lys) reached a lower level than poly(Phe) when Phe-tRNA',YS was used.

It is concluded that the side-chain of the amino acid linked to a tRNA affects the efficiency of the aminoacyl-tRNA . EF-Tu . GTP ternary complex formation.

The formation of a complex between an aminoacyl-tRNA, elongation factor Tu, and GTP is an obligatory step during peptide chain elongation in bacterial protein biosynthesis [l]. Owing to a high concentration of elongation factor Tu in the Esche- riclziu coli cell [2] it can be assumed that all aminoacyl-tRNA is present in the form of the aminoacyl- tRNA . EF-Tu . GTP ternary complex [3]. Despite the available information on the tertiary structure of tRNA [4] little is known about the molecular details of the interaction between aminoacyl-tRNA and elongation factor Tu. It has been generally expected that elongation factor Tu, a protein which during its functional cycle must recognize all aminoacyl-tRNAs regardless of their specificity, does not interact specifi- cally with the side-chain of the aminoacyl residue attached to the tRNA [5].

We have shown recently that elongation factor Tu binds preferentially to the 2'-aminoacylated isomer

Abbreviation. EF-Tu, elongation factor Tu. Enzymes. Phenylalanyl-tRNA synthetase (EC 6.1.1.20); lysyl-

tRNA synthetase (EC 6.1.1.6); tyrosyl-tRNA synthetase (EC 6.1.1.1); ribonuclease (EC 3.1.27.5); pyruvate kinase (EC 2.7.1.40).

of some tRNAs [6]. This selection is not uniform for all aminoacyl-tRNA species [7], which may indicate that the structure of the amino acid, including its side- chain, influences the mode and the strength of the interaction between aminoacyl-tRNA and EF-Tu . GTP interaction. Misaminoacylated tRNA species are a suitable tool to investigate this hypothesis, since the only variable feature in a pair of correctly and uncorrectly aminoacylated tRNAs is the side-chain of the amino acid. Using misaminoacylated tRNA species we determined the efficiency of aminoacyl- tRNA . EF-Tu . GTP complex formation and the capability of such ternary complexes to participate in the translation of synthetic polynucleotides in vitro.

MATERIALS AND METHODS

Unfractionated tRNA from Escherichiu coli MRE 600 cells was a commercial product from Boehringer (Mannheim, Federal Republic of Germany). Ribo- nuclease A from bovine pancreas, pyruvate kinase from rabbit muscle (10 mg/ml, 200 Ujmg), phosphoenolpyruvate, GTP, ATP, poly(A), poly(U) and ben-

214 Aminoacyl-tRNA EF-Tu . GTP Complex

zoylated DEAE-cellulose were also obtained from Boehringer (Mannheim, Federal Republic of Ger- many). Sephadex A-25 and Sepharose 4B from Phar- macia (Uppsala, Sweden) were used. Reversed-phase chromatography (RPC-5) adsorbent was purchased from Miles Laboratories Ltd (Stoke Court, Great Britain). Biogel P-2 was a product of BioRad (Rich- mond, U.S.A.). Ultrogel AcA 44 was supplied by LKB (Bromma, Sweden). Cellulose nitrate filters, type HA (0.45 pm), were from Millipore S.A. (Molsheim, France), cellulose filter discs type 3MM from What- man (Maidstone, Great Britain). Thin-layer electrophoresis was performed on Cel 300-10 plates from Macherey and Nagel (Diiren, Federal Republic of Germany). For the preparation of scintillation cock- tail Scintimix I11 from Merck (Darmstadt, Federal Republic of Germany) was used. 14C-labelled amino acids, specific activity 50 Ci/mol, Stanstar grade, were from Schwarz Radiochemicals (Orangeburg, U.S.A.). These amino acids were used for the analytical assays in the course of purification of individual tRNA species. For the preparative aminoacylation of tRNAs ['4C]phenylalanine (specific activity 5 13 Ci/mol), ['"CI- lysine (specific activity 341 Ci/mol), ['4C]tyrosine (specific activity 518 Ci/mol) and [3H]lysine (specific activity 15 Ci/mmol) all from Amersham-Buchler (Braunschweig, Federal Republic of Germany), were used. The radioactivity retained on the filters was measured after the addition of 3 ml of a solution of 5.5 g Scintimix I11 in 11 toluene to the dry filter. Elution patterns of gel filtrations were monitored upon the addition of 2 ml Aquasol from New England Nuclear (Boston, U.S.A.) to the corresponding radioactive fractions. For liquid scintillation counting a Berthold betaszint BF 8000 spectrophotometer was employed.

Tyrosyl-tRNA synthetase from yeast with a specific activity of 1000 units/mg protein [8] and phenylalanyl- tRNA synthetase from yeast with a specific activity of 1800 units/mg protein [9] were obtained from Drs Faulhammer and von der Haar (Gottingen, Federal Republic of Germany). Partially purified lysyl-tRNA synthetase from E. coli, with a specific activity of 280 units/mg protein, was prepared as described [lo]. Phenylalanyl-tRNA synthetase from E. coli with a specific activity of 800 units/mg protein was a gift from Dr E. Holler (Regensburg, Federal Republic of Germany). The phenylalanyl-tRNA synthetase from yeast used for phenylalanylation of tRNALys from E. coli was incubated for 15 min at 37 "C in a buffer containing 10 mM potassium phosphate pH 7.0 and 10 mM EDTA before its use. This treatment is necessary in order to reduce the rate of the ATPase activity associated with phenylalanyl-tRNA synthetase from yeast [l I].

Homogeneous E. coli elongation factors Tu . GDP and G were isolated from a 100000 x g supernatant, by

the method of Arai et al. [12]. EF-Tu was kept frozen at - 20 "C in a buffer containing 10 mM Tris-HC1 pH 7.5, 10mM MgC12, 100mM KCl and 10pM GDP. EF-G was stored frozen at - 20 "C in 10 mM Tris-HC1 pH 7.5 containing 10 mM MgCl2.

70-S ribosomes from E. coli, washed three times in 1 M NH4C1, were prepared according to Gavrilova and Spirin [13] and were stored as a precipitate in 72% saturated ammonium sulfate at 4°C. Before experiments, aliquots were diluted to a concentration of about 100 A260 units/ml with a buffer containing 60 mM Tris-HC1 pH 7.5, 10 mM MgC12, 30 mM NH4C1, 30 mM KCl and 1 mM dithiothreitol and dialysed against the same buffer at 4 "C.

Isolation of tRNAP"' and tRNALys from E. coli

tRNALyS and tRNAPhe from E. coli were purified by chromatography of bulk tRNA from E. coli on a column of benzoylated DEAE-cellulose [14]. 2 g tRNAb"lk were dissolved in 50 ml equilibration buffer. The column (5 x 30 cm) was equilibrated with 0.2 M NaCl, 0.01 M MgS04, 0.02 M sodium acetate pH 5.2, and 2 % dimethylformamide, and elution was performed with a 2 x 2-1 gradient from 0.4 M to 1.1 M NaCl. tRNALyS was eluted with 0.69 M NaCl, tRNAPhe with 0.90 M NaCl. The acceptor activities of the pooled tRNALy' and tRNAPhe fractions after benzoylated DEAE-cellulose chromatography were 200 pmol l y ~ i n e / A ~ ~ ~ unit and 800 pmol phenylalanine/A~~~ unit of tRNA respectively. The crude tRNAs were collected by ethanol precipitation, low-speed centrifugation, followed by desalting on a Biogel P-2 column and evaporation in vucuo [15]. tRNALyS was further purified by chromatography on Sepharose 4B [16]. 12000 A260 units of the crude tRNALyS in 25 ml 2.5 M ammonium sulfate, 0.01 M MgS04 and 0.02 M sodium acetate, pH 4.5, were applied onto a column of Se- pharose 4B (60 x 3 cm) equilibrated with 2 M ammonium sulfate, 0.01 M MgS04 and 0.02 M sodium acetate pH 4.5. The column was washed with a linear gradient from 2 M to 1 M ammonium sulfate (2 x 2 1). The lysine-accepting activity emerged at an ammonium sulfate concentration of 1.51 M. The corresponding pooled fractions had an acceptor activity of 500 pmol l y ~ i n e / A ~ ~ ~ unit tRNA. The solution was dialysed extensively against H20 at 4 "C, then concentrated and desalted as described above. In the last purification step 2000 A260 units of the tRNALys preparation in 5 ml buffer containing 0.35 M NaCl, 0.01 M MgS04 and 0.02 M sodium acetate, pH 5.2, were chromato- graphed on a reversed-phase chromatography 5 column [17] (120x 1 cm) equilibrated with the same buffer. The tRNAs were eluted from the column using a linear gradient from 0.35 M to 0.8 M NaCl(2 x 1 1). With the help of a Milton Roy instrument minipump the flow rate was kept constant at 0.4ml/min. The

T. Wagner and M. Sprinzl 215

pressure on the column head was 5 atmospheres (0.5 MPa). The material appearing with 0.42 M NaCl could be aminoacylated to a maximum extent with lysine. After collecting the appropriate fractions, the tRNALys, which had an acceptor activity of 1450 pmol lysinelA260 unit of tRNA, was precipitated, desalted and concentrated according to the standard procedure. The final yield was 250 A260 units. An aqueous solution was stored frozen at - 20 "C.

After benzoylated DEAE-cellulose chromatography, tRNAPhe could be obtained in a sufficiently pure form upon rechromatography on a reversed- phase chromatography 5 column. The procedure was the same as that described for tRNALYs. tRNAPhe was eluted with 0.63 M NaCl. From a total of 1000 A260

units of applied crude tRNAPhe 300 A260 units of pure tRNAPhe, with an acceptor activity of 1500 pmol phenylalanine/A260 unit of tRNA, were recovered.

Aminoacylation Using Cognate Systems

The aminoacylation assay contained 150 mM Tris- HCI pH 7.65, 50rnM KCl, 10mM MgS04, 5 m M dithiothreitol, 2 mM ATP, 0.02 mM radioactively labelled amino acid, 3 pM tRNA and 20 units/ml of aminoacyl-tRNA synthetase. For an analytical assay usually a total volume of 100 p1 was incubated at 37 "C and at appropriate time intervals 10-p1 aliquots were spotted on Whatman 3 MM paper discs, which were then washed twice with 5% aqueous trichloroacetic acid followed by washings in ethanol and ether prior to the determination of residual radioactivity. For preparative aminoacylation, up to 1 ml of the reaction mixture was incubated at 37°C for 20 min, then chilled on ice and extracted with the same volume of ice-cold phenol saturated with 10 mM sodium acetate pH 4.5. The aminoacyl-tRNA in the aqueous phase was precipitated with 2.5 volumes of ethanol at - 20°C for 2 h and pelleted by centrifugation. The pellet was washed with 1 ml 70% aqueous ethanol, twice with ethanol, dried under vacuum and dissolved in 5 mM sodium acetate pH 4.5. The final concentration of an aminoacylated tRNA was 5 - 10 pM. The extent of aminoacylation was determined by trichloroacetic acid precipitation and was generally better than 1300 pmol amino a~id /A26~ unit of tRNA.

Misaminoacylation of E. coli tRNAs

Phenylalanylation of tRNALyS from E. coli was achieved by phenylalanyl-tRNA synthetase from yeast in a reaction mixture containing 10 mM Tris-HC1 pH 9.0, 10 mM MgS04, 0.5 mM ATP, 0.02mM [I4C]- phenylalanine, 3 pM tRNALys and 500 units/ml phenylalanyl-tRNA synthetase from yeast. The aminoacylation reaction and the isolation of the aminoacylated

tRNA was carried out as described above. The extent of misaminoacylation was of 1530 pmol ['"Clphenyl- alanine/A2bU unit of tRNALyS.

Tyrosylation of tRNAPhe from E. coli was performed using tyrosyl-tRNA synthetase from yeast and by adopting the reaction conditions described by Giege et al. [18]. The reaction mixture contained 15 mM Tris-HC1 pH 8.5, 15 mM MgC12, 20% (v/v) dimethylsulfoxide, 1 mM ATP, 0.02 mM [14C]tyrosine, 4.5 pM tRNAPhe from E. coli and 500 units/ml tyrosyl- tRNA synthetase from yeast. For preparative aminoacylation a total volume of 0.5 ml was incubated at 37°C for 20 min and then chilled on ice. After the addition of 50 p1 1 M sodium acetate, pH 4.5, the reaction mixture was diluted with 1 ml water and applied onto a column of Sephadex A-25 (1 x 2 cm) equilibrated with 20 mM sodium acetate, pH 5.2, containing 100mM NaCl and 10mM MgCI2. The column was first washed with the same buffer to remove the protein and free [14C]tyrosine, then with the same buffer containing 500 mM NaCl to remove the adenosine nucleotides. [14C]Tyr-tRNAPhe was eluted with equilibration buffer contaning 1 M NaCI,, then desalted on Biogel P2, evaporated to dryness, dissolved in 5 mM sodium acetate pH 4.5 and stored at - 20 "C as described above. The recovery of the aminoacyl-tRNA was about 80 %, the extent of aminoacylation was 1360 pmol [14C]tyrosine/A260 unit of tRNAPhe.

All aminoacyl-tRNA species were analysed prior to their use by electrophoresis on cellulose thin-layer plates. The ['4C]amin~acyl-tRNA (about 20 000 counts/min) in a solution of 10 p1 containing 50 mM potassium phosphate pH 7.0 was incubated with 2 pl pancreatic ribonuclease (2 mg/ml) at 30 "C for 5 min. The mixture was spotted onto a cellulose thin-layer plate thereafter. In parallel, the untreated ['"Clamino- acyl-tRNA and the corresponding '"C-labelled amino acid were applied to the plate as controls. The electrophoresis was run at 25 V/cm for 3 h using 20% aqueous acetic acid adjusted to pH 3.5 with ammonia. The dry plates were scanned for 14C radioactivity on a Berthold thin-layer scanner. Using appropriate standards the extent of aminoacylation could be estimated. The electrophoretic analysis of the aminoacyl-tRNAs was used especially to exclude the possibility of non-covalent binding of the 14C-labelled amino acid to the synthetase or to the tRNA. When dimethylsulfoxide is present in the reaction mixture during misaminoacylation an extentive binding of this nature was observed. The trichloroacetic acid precipitation assay does not allow one to differentiate between a covalent or a non-covalent binding of an amino acid to tRNA or to proteins. However, by purification of ['4C]Tyr-tRNATY' by Sephadex A-25 chromatography (see above) the non-covalently bound [14C]tyrosine could be quantitatively removed.

216 Aminoacyl-tRNA . EF-Tu . GTP Complex

Spontuneous Hydrolysis of the Amino Acid threitol, with 10 pg poly(A), 28 pg EF-Tu . GTP ,from Aminoucyl-tRNA in the Absence (prepared as described in the preceeding section), und in the Presence of EF-Tu . GTP 30 pg EF-G and 75 pmol aminoacyl-tRNA in a total

The rate of hydrolysis of the amino acid from aminoacyl-tRNA was measured by determining the remaining amount of aminoacyl-tRNA in the reaction mixture. The aminoacyl-tRNA was allowed to hydro- lyze in a buffer containing 15 mM MgC12, 75 mM Tris-HC1 pH 7.5, 75 mM NH4CI, 7 mM dithiothreitol, 10 mM phosphoenolpyruvate pH 7.0, 0.5 mM GTP and 150 pg/ml pyruvate kinase. EF-Tu . GDP was added in different amounts to give the indicated final concentrations. In order to convert EF-Tu . GDP to EF-Tu . GTP preincubation for 10 rnin at 37 "C was necessary. Varying amounts of the aminoacyl-tRNA were then added with the concentration at the beginning of each experiment as indicated. Reaction mixtures had a final volume of 150 p1 and were incubated at 37 "C. Aliquots of 15 pl were withdrawn at appropriate time intervals and spotted onto What- man 3MM filter discs. The radioactivity insoluble in 5 aqueous trichloroacetic acid was determined by

volume of 75 pl at 37°C. In controlexperiments either EF-Tu . GTP or EF-G was omitted. The kinetics of polypeptide synthesis with Lys-tRNALYs or Phe- tRNA'-Y' were determined by removing 10-p1 aliquots at appropriate time intervals. The dependence of the rate of the polymerisation reaction on the concentration of EF-Tu . GTP was measured using 0.43 A260

unit of 70-S ribosomes and 40 pmol aminoacyl-tRNA in a reaction mixture of the same volume.

For the determination of poly(Lys) the samples were chilled on ice and hydrolyzed after the addition of 10 pl 1 M NaOH at 37 "C for 10 min. The samples were neutralised with 10 pl 1 M acetic acid at 0°C and 2 ml of an ice-cold solution of 0.25 sodium tungstate in 5 % aqueous trichloroacetic acid was added [19]. The solutions were kept on ice for 10 rnin and then filtered through Millipore nitrocellulose membranes.

The formation of poly(Phe) was assayed as described in the following section.

the- standard procedure.

Gel Filtration ofAminoacvl-tRNA . EF-Tu . GTP Ternary Complexes

Poly ( U ) -Dependent Polypeptide Synthesis

0.57 A260 unit of 70-S ribosomes was incubated for 10 rnin at 30 "C in 100 p1 of a reaction mixture con-

EF-Tu. GTP was prepared in a solution containing 15 niM MgCI2, 10 mM Tris-HC1 pH 7.5, 100 mM KCl, 7.7 mM phosphoenolpyruvate pH 7.0, 1.8 mM GTP, 800 pg/ml of pyruvate kinase and 28 pM EF-Tu . GDP by incubation for 10 rnin at 37 "C. Ternary complexes were formed with a mixture of 45.0 pmol [3H]Lys-tRNALYS ( [3H]lysine was diluted to a final specific activity of 2.5 Ci/mmol with non- radioactive lysine prior to the aminoacylation of the tRNALY') and 45.9 pmol [l4C]Phe-tRNALyS (['"CI- phenylalanine had a specific activity of 513 mCi/mmol) upon the addition of 15 pl of an ice-cold EF-Tu . GTP solution. The mixture, which had a final volume of 30 pl, was kept at 0 "C for 2 min and then applied to a column of Ultrogel AcA 44 (0.8 x 28 cm) equilibrated with 10 mM MgC12, 10 mM Tris-HC1 pH 7.5 and 100 mM KCI. The gel filtration procedure was essentially the same as that described by Sprinzl et al. [6]. Fractions of about 0.5 ml were collected and the radioactivity was measured as described above. In the control experiment EF-TU . GTP was omitted and the corresponding amount of the respective buffer was added to the mixture of the aminoacyl-tRNAs before gel filtration.

Poly(A)-Dependent Polypeptide Synthesis

taining 60 mM Tris-HCI pH 7.65,70 mM KC1, 10 mM MgC12, 1 mM dithiothreitol, 1 mM GTP, 1 mM ATP, 1 mM phosphoenolpyruvate and 50 pg poly(U). Then 27 pg EF-Tu . GTP (prepared as described above), 40 pg EF-G and 40 pmol [14C]Phe-tRNAPh' or ['"CI- Tyr-tRNAPhe were added. The total volume of the redction mixture was 125 p1 with a Mg2+ concentration of 10 mM. The incubation was continued at 37°C and at indicated time intervals 15 pl of the mixture were withdrawn and spotted onto Whatman 3MM filter discs. The formation of poly(Phe) or poly(Tyr) was determined by washing the filters once in 5 % aqueous trichloroacetic acid at 90 "C for 10 rnin followed by two washings in cold 5 2) trichloroacetic acid, one wash in ethanol and one wash in ether [20]. The residual radioactivity on the filters was measured.

RESULTS

Misaminoucylation of tRNAs in Heterologous Systems

tRNALyS from Esrherichia coli can be misaminoacylated with phenylalanine by yeast phenylalanyl- t RNA synthetase. Whereas the aminoacylation of this tRNA with lysine cdtalysed by lysyl-tRNA synthetase was found to be optimal at pH 7.6 and 50 mM KCI, the misaminoacylation of tRNALys with phenylalanine gave the best yield at pH 9.0 without KCI. -

0.72 A260 unit of 70-S ribosomes was incubated in a buffer containing 60 mM Tris-HC1 pH 7.8, 30 mM KCI, 30 mM NH4C1, 10 mM MgC12, 1 mM dithio-

To achieve misaminoacylation it is crucial to treat the yeast phenylalanyl-tRNA synthetase with EDTA. It was suggested recently [ l l ] that this enzyme pos-


Table 1. Aminoucylution and misaminoarylation of E. coli tRNAs The reaction conditions are given under Materials and Methods

tRNA species Aminoacyl-tRNA Source synthetase specific for

Amino acid Extent of aminoacylation

pmol/Az60 unit tRNA

tRNALyS lysine E. coli lysine 1450 tRNALyS phenylalanine yeast phen ylalanine 430 tRNALy' phenylalanine yeast (EDTA-treated) phenylalanine 1530 tRNAPh' phenylalanine E. c d i phenylalanine 1500 tRNAPh' tyrosine yeast tyrosine 1360

sesses a Zn2+-dependent ATPase activity. As a consequence of this side-reaction the relatively slow misaminoacylation of tRNALyS by the Zn2+-saturated enzyme is limited because of a fast consumption of ATP. The extent of misaminoacylation reaches that of the correct aminoacylation only when the Zn2+ ions are complexed by the addition of EDTA to the enzyme (Table 1). The kinetics of the non-cognate aminoacylation of tRNALyF with phenylalanine and of the cognate aminoacylation reaction were measured and compared. Very similar K , values for tRNALy' from E. coli in the homologous and in the heterologous systems were observed to be 6.4 pM with lysyl-tRNA synthetase from E. coli and 5.5 pM with phenylalanyl-tRNA synthetase from yeast. The rate of phenylalanylation of tRNALyS from E. coli by yeast phenylalanyl-tRNA synthetase is, however, about two hundred times slower than the phenylalanylation of the cognate yeast tRNAPhe by the same enzyme

The misaminoacylation of tRNAPhe from E. coli with tyrosine was achieved using yeast tyrosyl-tRNA synthetase. The reaction was performed at low ionic strength and in the presence of 20 % dimethylsulfoxide according to Giege el al. [18]. A large amount of the synthetase had to be used to get quantitative misaminoacylation in this case. In addition of the precipitation of ['4C]Tyr-tRNAPhe a coprecipitation of [14C]tyrosine with tyrosyl-tRNA synthetase was observed in the trichloroacetic acid precipitation assay; therefore, it was not possible to demonstrate the aminoacylation unequivocally by this means. In a second assay an aliquot of the tRNA fraction, after aminoacylation, was digested with pancreatic ribonuclease and the presence of ['4C]tyrosyl-2'(3')-adenosine was detected by cellulose thin-layer electrophoresis (Table 1).

Efficiency of the Aminoacyl-tRNA . EF-Tu ' GTP Ternury Complex Formation with Correctly Aminoucylated and Misaminoacyluted Aminoucyl-tRNA Species

The effect of EF-Tu . GTP on the rate of spon- taneous hydrolysis of the amino acid from aminoacyl-

tRNA [21] was measured using correctly aminoacylated and misaminoacylated tRNA species. The rate of hydrolysis of the amino acid ester linkage of Phe- tRNAPh' at 37°C and pH 7.5 is markedly decreased in the presence of EF-Tu . GTP as compared to the rate in the absence of EF-Tu . GTP. This protection is dependent upon the concentration of EF-Tu . GTP in the reaction mixture (Fig. 1 B). With Tyr-tRNAPhe the effect is even more pronounced. The misaminoacylated tRNAPhe species is nearly completely stable over a period of more than 2 h at 37 "C when EF- Tu . GTP is present at a concentration of 1.32 pM (Fig. 1 A). Under similar conditions more than 60 % of the Phe-tRNAPhe is hydrolysed (Fig. 1 B). Thus this experiment indicates that the nature of the amino acid may influence the stability of an aminoacyl- tRNA . EF-Tu . GTP ternary complex. This is de- monstrated even more clearly by the experiment where L ~ s - ~ R N A ' , ~ ~ and Phe-tRNALY' are compared in the EF-Tu . GTP-mediated hydrolysis protection assay (Fig.2). The rate of hydrolysis of both aminoacyl- tRNALys species is identical in the absence of EF- Tu . GTP. In a 0.5 pM EF-Tu . GTP solution containing 0.09 pM aminoacyl-tRNALYs species (Fig. 2 B) protection was observed for the misaminoacylated Phe-tRNALy5. In contrast, the rate of the lysine hydrolysis from Lys-tRNALYs in the presence of EF- Tu . GTP remained unchanged. This implies that therc is no interaction of Lys-tRNALYS with EF-Tu . GTP under these conditions. Increasing the concentration of the aminoacyl-tRNAsLYs to 0.45 pM and that of EF-Tu . GTP to 0.9 pM (Fig. 2A) results in protection in both cases. Protection is still, however, more pronounced in the case of Phe-tRNALYs than Lys-tRNALYS.

The stronger interaction of Phe-tRNALYS with EF- Tu . GTP as compared to Lys-tRNALYs could be shown directly by gel filtration of the ternary complexes [6]. If a solution 1.5 pM in both aminoacyl- tRNAs',YS and 14 pM EF-Tu. GTP is subjected to a filtration the radioactivity corresponding to Phe- tRNALyS appears in earlier fractions than the radioactivity corresponding to Lys-tRNALYS (Fig. 3). This indicates that more ternary complex is formed with the misaminoacylated Phe-tRNALY". Although both

218 Aminoacyl-tRNA EF-Tu . GTP Complex

- c 0.3 5 0 .- 5 0.4 + c Y

5 0.2 4 z LL +, 0.2 Y

0 01 ._ E 4 0 1 - I

0 0

Time (min)

Fig. 1. Rate of hydrolysis of the aminoacyl ester linkage of ( A ) Tyr-tRNAPIce and ( B ) Phe-tRNAP'" from E. coli in the absence (0) and in the presence of 0.44 p M (B) or 1.32 p M EF-Tu . GTP (A). See Materials and Methods for reaction conditions

0 100 0 100

"0 103 Time (min)

0.10

0.05

0

Fig. 2. Hydrolysis of the aminoacyl ester linkage of Lys-tRNAL"" (O,.) and Phe-tRNALyS (0, B) f rom E. coli. The initial concentrations of the aminoacyl-tRNALYs were (A) 0.45 pM and (B) 0.09 pM. The hydrolysis reaction was examined in the absence (0,O) and in the presence (0, B) of (A) 0.9 pM and (B) 0.5 pM EF-Tu . GTP. The experiment was otherwise identical to that shown in Fig. 1

aminoacyl-tRNA species interact with EF-Tu . GTP clearly Lys-tRNALYS dissociates more easily from EF-Tu . GTP than Phe-tRNALYs.

Poly ( U ) and Poly(A)-dependent Synthesis of Polypeptides

The participation in synthetic messenger translation of native and misaminoacylated tRNAPhe or tRNALys species was investigated by protein-synthe- sizing system in vitro using either poly(U) or poly(A), 70-S ribosomes and purified elongation factors from E. coli. The data summarized in Table 2 reveal that there is an absolute requirement for both elongation factors in the purified ribosomal system. None of the elongation factors is able to catalyze the polypeptide synthesis by itself, which excludes the possibility of formation of oligopeptides by non-specific interactions of the aminoacyl-tRNA species with the 70-S ribosomes and the respective messenger. The extent of poly(Phe) synthesis is the same regardless of whether the misaminoacylated Phe-tRNALYS or the native Phe-

tRNAPhe is employed in the respective poly(A) or poly(U)-directed polymerisation reaction. There was no significant difference between the rates of poly(Phe) and poly(Tyr) formation when the synthesis of these polypeptides was measured on poly(U)-programmed ribosomes using Phe-tRNAPhe and Tyr-tRNAPhe respectively. Since the critical length necessary for oligo- peptide detection is smaller for oligo(Phe) peptides than for oligo(Tyr) peptides, the lower extent of poly(Tyr) formation suggested by the lower plateau value is most probably due to the trichloroacetic acid precipitation assay on which these experiments rely (Table 2).

It is also difficult to interpret the results of the experiment of Fig.4A. The measurement of poly(Phe) or poly(Lys) synthesis on poly(A)-programmed 70-S ribosomes, using Phe-tRNALYs or Lys-tRNALYs respectively, revealed significantly different rates of polymerisation in the first 'fast' phase of the reaction. This was followed by a slower increase in the yield of the polypeptides, which was similar in both cases. This difference at the beginning of the reaction could be


due to a slower initiation of the polymerisation process in the case of Lys-tRNALys. However, an electrophoretic analysis of the products after alkaline treatment of the reaction mixture indicated that the amount of unpolymerized amino acids in the solution is similar for both Phe-tRNALYS and Lys-tRNALys. The differences in the polymerisation rates are, therefore, only apparent and result from the different procedures employed to detect the synthesized polypeptides. In the case of the oligolysine the peptides formed in the first phase of the reaction are smaller than that critical length necessary for detection by the applied method. In contrast the oligophenylalanines of corre-

10

a b 8 - I I

I I I I I I

C A

10 20 30 40 Fraction number

Fig. 3. Gel filtration of u mixture of 45.0pmol [3H]Lys-tRNA1,ys (0) and 45.9pmol ['4C]Phe-tRNALY" (a) in (A) the absence and in (B) the presence of 420pmol EF-Tu. GTP. (a) The position of the ternary complex; (b) the position of the aminoacyl-tRNA and (c) the position of the amino acid in the elution profile. The conditions are described in Materials and Methods

sponding lengths can already be detected during this first phase of synthesis. Experiments of Fig. 4 do not necessarily indicate a difference in the efficiency of translation of Phe-tRNALYs and Lys-tRNALYs on poly(A)-programmed ribosomes.

The effect of the concentration of EF-Tu . GTP on the rate of the poly(A)-dependent synthesis of poly(Phe) using misaminoacylated Phe-tRNALYS is shown in Fig.4B. As the concentration of Phe-tRNALY5 . EF-Tu . GTP ternary complexes decreases lower initial rates of poly(Phe) synthesis as well as lower plateau values of formed poly(Phe) are observed. This results from the rapid hydrolysis of phenylalanine from Phe-tRNALY", since there is decreased protection at low concentration of EF-Tu . GTP. A direct com- parison of the extent of poly(Lys) and of poly(Phe) synthesis at a concentration of 0.61 pM EF-Tu . GTP indicates clearly that, while the Phe-tRNALYs is still protected and participates in poly(Phe) synthesis in form of the Phe-tRNALYS. EF-Tu . GTP complex, this concentration of EF-Tu . GTP is not sufficient for an interaction with Lys-tRNALys. Even by considering

Table 2. Polypeptide synthesis using misaminoacylated tRNAs

System Incorporation of Incorporation of amino acid by poly(A) with poly(U) with

Lys- Phe- Phe Tyr- tRNALyS tRNALys tRNAPhe tRNAPhe into into into into

amino acid by

POlY(LYS) PolYPhe) PolY(Phe) poly(Tyr)

pmol/A260 unit ribosomes ~-

Without EF 0.5 1.2 0.3 1.2 + EF-Tu. GTP 1 .o 1.2 0.3 1 .0 + EF-G 0.8 1 .o 0.5 1 .o With EF 40.2 64.9 62.7 50.8

5 B

a A

3 - - 2

- 1

2 I l- I n

0 10 20 x) 40 -0 10 20 30 Time (rnin)

Fig.4. ( A ) Poly(A)-directed synthesis of poly(Lys) using Lys-tRNALy' (a) and of poly(Phe) using Phe-tRNAL"" (W). ( B ) Kinetics of the poly(A)-directed synthesis of poly(Pke) with Phe-tRNALYs at EF-Tu . GTP concentrations of 0.61 p M (A). 0.31 p M (O), 0.12 p M (a) und 0.06 p M (m). (A) The concentration of the aminoacyl-tRNA species was 1.0 pM, that of EF-Tu . GTP 7.5 pM. For the detection of the respective polypeptides, the described methods were used. (B) In a control experiment the synthesis of poly(Lys) with Lys-tRNALYS at 0.61 pM EF-Tu . GTP was measured (A). The initial concentration of the aminoacyl-tRNALYs was 0.64 pM

220 Atninoacyl-tRNA . EF-Tu GTP Complex

the fact that the oligolysines of short chain lengths are not detected by the applied analytical method the lysine polymerisation stops at a severalfold lower level as compared to the plateau value of poly(Phe) synthesis.

DISCUSSION

From the presented data it is evident that the side- chain of the aminoacyl residue attached to aminoacyl-tRNA contributes significantly to the stability of the complex formed by aminoacyl-tRNA and EF-Tu . GTP. Pingoud et al. [21] reported recently that different aminoacyl-tRNA species differ in their ability to bind to EF-Tu . GTP. He observed that the association constants of Escherichiu coli Ser-tRNASc‘ and Phe- tRNAPhe when interacting with EF-Tu from E. coli differ by about one order of magnitude. This difference may result either from the different primary structures of the respective tRNAs or from, what was considered to be more unlikely, the nature of the side-chain of the aminoacyl residue of the aminoacyl-tRNA. We have now shown in experiments using misaminoacylated E. coli tRNA species that the side-chain of the amino acid contributes significantiy to the stability of the aminoacyl-tRNA . EF-Tu . GTP ternary complex. For example, Lys-tRNALYs forms a relatively unstable ternary complex with EF-Tu . GTP. If, however, this tRNALys carries a phenylalanine on its 3’ end, its complex with EF-Tu . GTP has stability comparable with that of the homologous Phe-tRNAPhe. A similar effect by the amino acid was observed in the case of misaminoacylated Tyr-tRNAPh‘ from E. coli. The difference in this case, in which complex formation of Phe-tRNAPh‘ or Tyr-tRNAPhe with EF-Tu . GTP was examined, was less pronounced. The replacement of an amino acid with a polar side-chain (lysine) by one with an aromatic side-chain (phenylalanine) has therefore a more dramatic effect on the stability of an aminoacyl-tRNA . EF-Tu . GTP ternary complex than the replacement of phenylalanine for the struc- turally similar tyrosine.

The reported observation may have some impli- cations on the models describing the interaction of aminoacyl-tRNA with EF-Tu . GTP. There is evidence, derived from chemical modification of aminoacyl-tRNA, which suggests that the acceptor stem [22] with its C-C-A terminal and the 5‘-terminal phosphate [23] of the aminoacyl-tRNA are the major regions of contact between the tRNA and the protein. Further- more the ester linkage and the a-amino group of the attached amino acid are evidently involved in the interaction with the protein [24]. Based on our present results we suggest that in addition to these interactions, the side-chain of the amino acid residue of aminoacyl-tRNA is also placed in a binding pocket

of the elongation factor Tu. The interaction of the side-chain of the amino acid with EF-Tu is probably more efficient in the case of an aromatic amino acid. This is in agreement with the observation that aminoacyl-tRNAs with hydrophilic amino acid residues, such as Ser-tRNASer or Lys-tRNALYS, have lower association constants than those with hydrophobic amino acid residues, such as Phe-tRNAPhe or Tyr-tRNATYr [21,25].

An important consequence of our observations is the fact that EF-Tu . GTP does not appear able to select between the correctly aminoacylated and a misaminoacylated tRNA, since in our experiments the misaminoacylated Phe-tRNALYs is more strongly bound to EF-Tu . GTP than the correctly aminoacylated Lys-tRNALY”. The possibility has been pre- viously discussed in the literature that a misaminoacylation of tRNA can be corrected by the action of the cognate aminoacyl-tRNA synthetase, which would result in hydrolysis of the incorrect amino acid [26]. Our experiments provide evidence against this hypothesis. Since EF-Tu . GTP is present in the cell in significantly higher concentration than a particular synthetase [2], it should trap any misaminoacylated tRNA. The correction of a misaminnacylation must, therefore, take place prior to the release of the aminoacyl-tRNA from the synthetase [27,28].

The study of the activity of aminoacylated oligo- nucleotide analogues of the C-C-A end of tRNA in the ribosomal fragment assay revealed that the amino- acid’s side-chain is also interacting with the donor as well as the acceptor site of the ribosomal peptidyl transferase center [29]. The activity of the fragments carrying aromatic amino acids is higher than the activity of fragments linked with hydrophilic amino acid residues.

By investigating the efficiency of the poly(A)- dependent synthesis of poly(Lys) and poly(Phe) using the respective Lys-tRNALYs and Phe-tRNALYS, we were not able to detect any significant changes in the rate of the polymerisation reactions. We therefore suggest that the contribution of the side-chain of the amino acid to the binding of an aminoacyl-tRNA to ribosomes is minimal compared to the sum of the other interactions of aminoacyl-tRNA with the ribosomal binding sites. Consequently, the misaminoacylated tRNA is translated with the same efficiency as the correctly aminoacylated species. The differences observed in the plateau values of poly(Phe) and poly- (Lys) synthesis using a poly(A)-dependent ribosomal system (see Fig.4) are related to the fact that the binding efficiencies of Phe-tRNALYS and Lys-tRNALYs to EF-Tu . GTP differ considerably. At low concentrations of aminoacyl-tRNA and EF-Tu . GTP, the species with the lower association constant, Lys- tRNALy’, is predominantly in a free form, i.e. not associated with EF-Tu . GTP. This leads to a rapid

T. Wagner and M. Sprinzl

hydrolysis of the aminoacyl residue from the tRNALys and consequently to the termination of the polymerisation reaction.

We thank Prof. Dr F. Cramer for his continuous support and encouragement, Drs H. Faulhammer, F. von der Haar, and E. Holler for their kind supply of aminoacyl-tRNA synthetases, Dr D. GauR and Dr L. McLaughlin lor the critical reading of the manuscript. The experl technical assistance of E. Graeser and M. Kucharzew- ski is highly appreciated. We are indebted to Dr F. von der Haar for his suggestions concerning the ATPase activity of phenylalanyl- tRNA synthetase from yeast.

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the complex formation between escherichia coli aminoacyl-trna, elongation factor tu and gtp : the...

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