A peculiarity of the reaction of tRNA aminoacylation catalyzed byphenylalanyl-tRNA synthetase from the extreme thermophile

Thermus thermophilus

Victor G. Stepanov, Nina A. Moor, Valentina N. Ankilova, Inna A. Vasil'eva,Maria V. Sukhanova, Ol'ga I. Lavrik *

Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, 630090, prospect Lavrentiev 8,Novosibirsk, Russia

Received 9 December 1997; revised 18 March 1998; accepted 25 March 1998

Abstract

It was confirmed unambiguously that the anomalously high plateau in the tRNA aminoacylation reaction catalyzed byThermus thermophilus phenylalanyl-tRNA synthetase is a result of enzymatic synthesis of tRNA bearing two boundphenylalanyl residues (bisphenylalanyl-tRNA). The efficiency of bisphenylalanyl-tRNA formation was shown to be quitelow: the second phenylalanyl residue is attached to tRNA approximately 50 times more slowly than the first one. Thethermophilic synthetase can aminoacylate twice not only T. thermophilus tRNAPhe but also Escherichia coli tRNAPhe andE. coli tRNAPhe transcript, indicating that the presence of modified nucleotides is not necessary for tRNAPhe overcharging.Bisphenylalanyl-tRNA is stable in acidic solution, but it decomposes in alkaline medium yielding finally tRNA and freephenylalanine. Under these conditions phenylalanine is released from bisphenylalanyl-tRNA with almost the same rate asfrom monophenylalanyl-tRNA. In the presence of the enzyme the rate of bisphenylalanyl-tRNA deacylation increases.Aminoacylated tRNAPhe isolated from T. thermophilus living cells was observed to contain no detectable bisphenylalanyl-tRNA under normal growth of culture. A possible mechanism of bisphenylalanyl-tRNA synthesis is discussed. ß 1998Elsevier Science B.V. All rights reserved.

Keywords: Phenylalanyl-tRNA synthetase; tRNAPhe ; tRNA aminoacylation mechanism

1. Introduction

Aminoacyl-tRNA synthetases belong to a numer-ous group of enzymes participating in the realization

of genetic information. The mechanism of tRNAaminoacylation is generally accepted to include twomain steps:

(a) An aminoacyl-tRNA synthetase (aaRS) con-

0167-4838 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 0 5 4 - 5

Abbreviations: PheRS, phenylalanyl-tRNA synthetase (EC 6.1.1.20); [A76-3H]tRNAPhe(r), tRNA with tritium-labelled 3P-terminalnucleotide obtained by restoration of tRNA lacking A76 in the presence of CTP (ATP):tRNA nucleotidyltransferase and [3H]ATP;[A76-3H]tRNAPhe(ex), tRNA with tritium-labelled 3P-terminal nucleotide obtained by CTP (ATP):tRNA nucleotidyltransferase promotedreaction of A76 exchange in the presence of [3H]ATP; HEPPS, N-[2-hydroxyethyl]piperazine-NP-[2-propanesulfonic acid] ; MOPS, 3-[N-morpholino]propanesulfonic acid; Tricine, N-tris[hydroxymethyl]methylglycine; PPi, inorganic pyrophosphate

* Corresponding author. Fax: +7 (383) 2-33-36-77; E-mail : lavrik@modul.bioch.nsk.su, lavrik@niboch.nsc.ru

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verts a speci¢c amino acid (aa) to aminoacyladenyl-ate (activation step)

aaRS�ATP� aa! aaRSc�aaVAMP� � PPi �1�(b) The amino acid is attached to tRNA com-

plexed with the enzyme yielding aminoacyl-tRNA(transfer step)

aaRSc�aaVAMP� � tRNAaa ! aaRSc�aaVtRNAaa�

�AMP! aaRS�AMP� aaVtRNAaa �2�

To measure the quantity of aminoacyl-tRNAformed in the course of tRNA aminoacylation thelabelled amino acid with known speci¢c radioactivityis added to the reaction mixture. Precipitation ofaminoacyl-tRNA with trichloroacetic acid (TCA)makes it possible to separate it from free aminoacid. Thus, it is possible to determine a portion oftRNA converted to aminoacyl-tRNA by juxtaposi-tion of radioactivity of TCA precipitate and initialconcentration of tRNA in the mixture.

Usually the concentrations of both ATP and ami-no acid in aminoacylation assay mixtures are signi¢-cantly higher than that of tRNA. Therefore the pla-teau of the reaction is determined by theconcentration of tRNA which is the limiting sub-strate. From the general scheme of the reactionmechanism it follows that the plateau in this casecorresponds to the situation when all tRNA mole-cules bear one amino acid residue. In reality themaximal percentage of aminoacylated tRNA maybe lower than 100% due to instability of the esterbond between the aminoacyl residue and tRNA inconditions favorable for aminoacyl-tRNA synthe-tases functioning (pH 7^9). In this case the reactionplateau is determined by a dynamic balance betweensynthetase-catalyzed tRNA charging and non-enzy-matic hydrolysis of aminoacyl-tRNA [1^4].

However, a few years ago it was found that thecatalytic mechanism of phenylalanyl-tRNA synthe-tase from the extremely thermophilic eubacteriumThermus thermophilus cannot be described in termsof this common scheme [5]. The total amount oflabelled phenylalanine associated with TCA-insolu-ble material corresponded to a tRNAPhe charginglevel higher than 100%. The assumption was madethat the population of tRNAPhe aminoacylated withthermophilic synthetase contains not only monophen-

ylalanyl-tRNA (the usual product of tRNA amino-acylation) but also bisphenylalanyl-tRNA (tRNAwith two covalently bound phenylalanyl residues).However, the possibility of synthetase-catalyzed at-tachment of more than one amino acid molecule totRNA was never discussed in articles dedicated to themechanism of tRNA aminoacylation. The main targetof this work was to verify thoroughly our hypothesisabout the cause of the anomalously high level oftRNA charging and to investigate the nature and reg-ularities of this phenomenon.

2. Materials and methods

2.1. Materials

Homogeneous phenylalanyl-tRNA synthetasefrom Thermus thermophilus HB8 was puri¢ed as pre-viously described [6] ; its speci¢c activity was 210 U/mg (37³C). Phenylalanyl-tRNA synthetase from Es-cherichia coli MRE-600 with speci¢c activity 310 U/mg (37³C) was isolated as described in [7]. E. coliCTP (ATP):tRNA nucleotidyltransferase (speci¢cactivity 30 U/mg at 37³C) was isolated according to[8]. E. coli tRNAPhe, T. thermophilus tRNAPhe (I)and tRNAPhe (II) (phenylalanine incorporation byE. coli PheRS was 1500, 1520 and 1400 pmol/A260

unit, respectively) were isolated as described in [9,10].Unless specially mentioned T. thermophilus tRNAPhe

(I) was used in all experiments. Wild-type E. colitRNAPhe transcript was synthesized and puri¢ed asdescribed in [11] (phenylalanine incorporation by E.coli PheRS was 1930 pmol/A260 unit). Deoxyribonu-clease I (RNase-free, 3000 U/mg) and lysozyme(50 000 U/mg) were from Sigma.

L-[U-14C]Phenylalanine (325 Ci/mol) was fromUVVVR (Czechoslovakia), L-phenyl-[2,3-3H]phenyl-alanine (53 Ci/mmol) and [8-3H]ATP (32 Ci/mmol)were products of St. Petersburg Institute of AppliedChemistry (Russia).

2.2. Aminoacylation of tRNAPhe

The standard reaction mixture (50^1000 Wl) con-tained 5 mM ATP, 9 mM MgCl2, 50 mM Tris-HClor HEPPS-NaOH (pH 9.0 at 20³C), 20^50 WM L-[14C]phenylalanine, 0.5^2.5 WM tRNAPhe, 1^200 Wg/

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ml PheRS from T. thermophilus or E. coli. The reac-tion was carried out at 40³C. The velocity of theesteri¢cation reaction was measured by the rate of[14C]phenylalanine incorporation into tRNA. At theappropriate times aliquots were spotted onto FN-11paper ¢lters impregnated with TCA. Then the ¢lterswere extensively washed with ice-cold 5% TCA toremove free amino acid. TCA-insoluble radioactivitywas measured by liquid scintillation counting.

2.3. Separation of bisphenylalanyl-tRNA fromuncharged tRNA and monophenylalanyl-tRNA

Bisphenylalanyl-tRNA was separated from tRNAand monophenylalanyl-tRNA by a chromatographyon a column with C18 reversed-phase sorbent (Li-Chrosorb RP-18, Merck) coated with methyltrioctyl-ammonium chloride (Adogen 464, Serva). The sorb-ent was prepared according to [10]. Solutioncontaining 0.5^2.5 nmol of aminoacylated tRNAPhe

was mixed with 2.5 M sodium acetate (pH 5.0) toachieve a ¢nal pH value of 5^5.5 and loaded onto the49U5 mm column. The elution was performed witha linear gradient of bu¡er B (6 M ammonium ace-tate, 10 mM MgCl2, 1 mM Na2EDTA, pH 5.7) inbu¡er A (0.5 M ammonium acetate, 10 mM MgCl2,1 mM Na2EDTA, pH 4.5) with a £ow rate of 0.5 ml/min. All chromatographies were run on a PharmaciaFPLC system at room temperature.

It should be mentioned that the last step of puri-¢cation of all tRNAPhes was the chromatography onthe LiChrosorb RP-18/Adogen 464 column. There-fore the retention of admixtures corresponds tothat of uncharged tRNAPhe and they do not interferewith peaks of both species of aminoacylated tRNA.

2.4. Determination of protein in chromatographicfractions after separation of components ofthe aminoacylation reaction mixture on theLiChrosorb RP-18/Adogen 464 column

Chromatographic fractions containing bisphenyl-alanyl-tRNA were collected and concentrated withthe use of Ultrafree-MC 100000 NMWL Filter Units(Sigma) from 3 ml to a ¢nal volume of 5^10 Wl, thendiluted with 50 mM potassium phosphate (pH 8.0) to300 Wl and concentrated again. The last procedurewas repeated twice. Finally the volume of solution

was adjusted with 20 mM potassium phosphate (pH8.0) to 50 Wl and the presence of protein was checkedafter addition of Bradford's reagent (1 ml) accordingto [12]. Control solutions containing 12^250 Wg of T.thermophilus PheRS in an appropriate mixture ofbu¡ers A and B were subjected to the same proce-dure. Absorbance at 595 nm of the samples wasmeasured on spectrophotometer SF-46 against`blank' solution (50 Wl of 20 mM potassium phos-phate (pH 8.0) mixed with 1 ml of Bradford's re-agent).

2.5. Preparation of 3H-labelled tRNAPhe

(A) The 3P-terminal nucleotide of T. thermophilustRNAPhe (I) was removed by the successive treat-ment with sodium periodate, aniline and alkalinephosphatase according to [13]. The native 3P-endwas restored in a reaction mixture containing60 WM [8-3H]ATP, 10 mM MgCl2, 50 mM Tris-HCl (pH 9.0), 0.5 mM Na2EDTA, 15 WM truncatedtRNAPhe and 40 Wg/ml CTP (ATP):tRNA nucleoti-dyltransferase. The reaction was stopped after 1 hincubation at 37³C by addition of sodium acetate(pH 5.0) to a ¢nal concentration of 0.3 M. The en-zyme was removed by phenol extraction. tRNA wasseparated from low-molecular-weight components ofthe reaction mixture by gel ¢ltration on a TSK-GelToyopearl HW-40F column equilibrated with 50 mMsodium acetate (pH 5.0). The labelled tRNAPhe pre-pared by this way was designated [A76-3H]tRNAPhe(r). Finally [A76-3H]tRNAPhe(r) waspreparatively aminoacylated under standard condi-tions with E. coli PheRS and phenylalanyl-tRNAwas separated from non-aminoacylated tRNA mate-rial on a LiChrosorb RP-18/Adogen 464 column.After complete deacylation followed by two succes-sive ethanol precipitations from 0.1 M sodium ace-tate (pH 5.0) the phenylalanine acceptance of [A76-3H]tRNAPhe(r) was 1750 pmol/A260 unit.

(B) tRNAPhe with labelled A76 was also preparedby CTP (ATP):tRNA nucleotidyltransferase-cata-lyzed pyrophosphate exchange reaction in the pres-ence of [8-3H]ATP. The reaction was carried out at37³C in a mixture containing 18 WM [8-3H]ATP,0.4 mM ATP, 20 mM MgCl2, 20 mM Tris-HCl(pH 8.1), 1 mM Na2P2O7, 15 WM T. thermophilustRNAPhe (I) and 60 Wg/ml CTP (ATP):tRNA nucle-

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otidyltransferase. The reaction was stopped after 6 hincubation by addition of sodium acetate (pH 5.0) toa ¢nal concentration of 0.3 M. The enzyme was re-moved by phenol extraction. tRNA was puri¢ed on aLiChrosorb RP-18/Adogen 464 column under theconditions described above. This type of labelledtRNA, designated [A76-3H]tRNAPhe(ex), had acharging capacity of 1120 pmol/A260 unit.

The e¤ciency of tRNA labelling was estimatedafter analytical separation of [A76-3H]tRNAPhe(ex)aminoacylated with non-radioactive phenylalaninefrom uncharged tRNA on a LiChrosorb RP-18/Ado-gen 464 column. 14% of native tRNA molecules werefound to bear [3H]adenosine on the 3P-end.

2.6. Hydrolysis of aminoacylated tRNA

Non-enzymatic deacylation of aminoacylatedtRNA was performed in a solution containing 2.5^3 WM mono- or bisphenylalanyl-tRNA, 50 mMHEPPS-NaOH (pH 9.0 at 20³C) and 9 mM MgCl2.The reaction was initiated by the addition of thebu¡er preincubated at 40³C to dry precipitate ofaminoacylated tRNA. Deacylation rate was meas-ured from the time dependence of the residualamount of [14C]phenylalanine coupled with tRNA.

Enzyme-assisted deacylation of bisphenylalanyl-tRNA was monitored as follows: a dry pellet ofbis[14C]phenylalanyl-tRNA was rapidly mixed witha mixture containing 5 mM ATP, 9 mM MgCl2,50 mM HEPPS-NaOH (pH 9.0 at 20³C), 1 mMnon-radioactive phenylalanine and 50 Wg/ml T. ther-mophilus PheRS at 40³C. The non-radioactive phen-ylalanine was added to prevent back-incorporationof [14C]phenylalanine into tRNA after deacylation.The rate of [14C]phenylalanine release from TCA-in-soluble material is conditioned by both enzymaticand non-enzymatic deacylation of overchargedtRNAPhe.

2.7. Isolation of total tRNA from T. thermophilusunder acidic conditions

T. thermophilus HB8 cells were cultured at 72³Cfor 7 h in a medium described by Oshima andBaba [14]. The cells were harvested at the mid-logphase by centrifugation at 2³C and resuspended ina solution containing 20 mM sodium acetate (pH

5.0), 5 mM MgCl2, 10% sucrose, 0.3 M KCl and1 mg/ml lysozyme. All subsequent steps were carriedout at 8³C. The cell suspension was gently stirred for30 min and Nonidet P-40 and DNase were added to¢nal concentrations of 1.6% and 0.01 mg/ml, respec-tively. After 1 h stirring DNase action was stoppedby the addition of 10 mM Na2EDTA and insolublematerial was removed by centrifugation. The super-natant was applied to a DEAE-cellulose column andtRNA was eluted with a linear gradient of NaClfrom 0 to 1 M in 0.1 M sodium acetate (pH 5.0).tRNA-containing fractions were collected, tRNAwas precipitated by ethanol and dried. We checkedthat bisphenylalanyl-tRNA was stable during allthese procedures by subjecting the control solutionof this compound to the same treatments.

The presence of tRNAPhe or its aminoacylatedforms in chromatographic fractions was establishedby synthetase-catalyzed [14C] or [3H]phenylalanineincorporation into tRNA under standard aminoacyl-ation conditions. The lability of the ester bond be-tween the amino acid and tRNA at pH 9 permittedus to introduce the labelled amino acid in tRNAesteri¢ed primarily with non-radioactive phenylala-nine, i.e. to reaminoacylate tRNA. The aliquotsfrom each fraction were taken out, added to thestandard aminoacylation mixture with 0.02 mg/mlof T. thermophilus PheRS and after 20 min incuba-tion at 40³C the amount of labelled phenylalanineincorporated into tRNA was measured.

3. Results

The anomalous behavior of T. thermophilus PheRScan be clearly demonstrated by the following experi-ment (Fig. 1). The tRNAPhe aminoacylation is car-ried out at ¢rst with E. coli PheRS and the reactionreaches a plateau when each tRNAPhe molecule bearsone phenylalanyl residue. Then the thermophilic syn-thetase is added to the reaction mixture. Further in-corporation of extra [14C]phenylalanine into TCA-insoluble material interpreted as bisphenylalanyl-tRNA synthesis [15] results in a new plateau corre-sponding to 1.4^1.8 mol phenylalanine bound permol tRNA. This e¡ect is speci¢c for T. thermophilusPheRS because its E. coli counterpart was not ob-served to overcharge tRNA in a broad range of ex-

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perimental conditions (for example, the plateau oftRNA aminoacylation reaction did not changeupon variation of the enzyme concentration from2 to 200 Wg/ml). Therefore we often used the E. colienzyme in control experiments and for determinationof true charging capacity of di¡erent tRNAPhes.

The successive tRNAPhe aminoacylation by thetwo synthetases in one tube makes it possible to es-timate accurately the quantity of extra phenylalaninebound to tRNA by thermophilic PheRS even for lowamounts of tRNAPhe with unknown charging ca-pacity. It is easy also to monitor the process oftRNA overcharging in its pure state, severed fromthe normal synthesis of monophenylalanyl-tRNA.When tRNA aminoacylation is carried out in thepresence of only T. thermophilus PheRS, the sameplateau is achieved as in the case of tRNA chargingby the mixture of T. thermophilus and E. coliPheRSs. Thus, the ¢nal yield of bisphenylalanyl-tRNA synthesized by the thermophilic enzyme doesnot depend on the presence or absence of the E. colisynthetase in the same reaction mixture.

It should be mentioned that T. thermophilus and E.coli PheRSs can aminoacylate with similar e¤ciency

both T. thermophilus and E. coli tRNAPhe. All pecu-liarities of tRNA aminoacylation observed when T.thermophilus tRNAPhe was used as a substrate werealso valid for E. coli tRNAPhe. The mode of tRNAcharging by each synthetase did not depend onwhether T. thermophilus or E. coli tRNAPhe wasused. Therefore we describe below only experimentsperformed with T. thermophilus tRNAPhe (I `isoac-ceptor') except when the structural di¡erence be-tween the tRNAPhes was important.

To isolate bisphenylalanyl-tRNA from the reac-tion mixture a procedure of its separation from freetRNA and monophenylalanyl-tRNA has been devel-oped. It is well known that some tRNAs being es-teri¢ed with amino acid interact with hydrophobicsorbents more strongly than uncharged ones [16].We supposed that bisphenylalanyl-tRNAPhe, mono-phenylalanyl-tRNAPhe and free tRNAPhe could beresolved in the same way. Chromatography on themixed-mode ionic-hydrophobic matrix designated Li-Chrosorb RP-18/Adogen 464 (C18 resin covered bymethyltrioctylammonium chloride) was used for thispurpose.

Two aliquots were taken from the reaction mixtureat di¡erent times; one of them contained tRNAPhe

aminoacylated by E. coli PheRS only, the other onecontained tRNAPhe charged in the presence of bothE. coli and T. thermophilus enzymes (Fig. 2). Boththese samples were chromatographed on the LiChro-sorb RP-18/Adogen 464 column. Low-molecular-weight components of the reaction mixture (ATP,AMP, PPi, [14C]Phe) were slightly retained andwashed o¡ as a large peak by isocratic elution withpure bu¡er A. The following small satellite peak cor-responded to the same set of compounds (as esti-mated by TLC analysis). Perhaps its appearancewas caused by accelerated coming o¡ the tail of themajor peak when the gradient elution was started.

All tRNAPhe species were eluted in a range of 20^60% bu¡er B. The position of the ¢rst peak on theoptical density pro¢le corresponded to that of un-charged tRNAPhe, this material seems to be an ad-mixture presenting in the initial tRNAPhe preparation(see Section 2). The following peak containing[14C]phenylalanine was identi¢ed as monophenyl-alanyl-tRNA according to the ratio 14C radioactiv-ity/A260. These two peaks were present on the opticaldensity pro¢les of both chromatographies. However,

Fig. 1. Aminoacylation of T. thermophilus tRNAPhe with E. coliand T. thermophilus phenylalanyl-tRNA synthetases. 1.2 WMtRNAPhe was aminoacylated at 40³C with 5 Wg/ml E. coli (a)or 70 Wg/ml T. thermophilus (E) PheRS. After 16 min of tRNAcharging by E. coli enzyme the reaction mixture was dividedinto two equal portions. Then T. thermophilus (8) or E. coli(S) PheRS was added to each of these portions to a concentra-tion of 70 Wg/ml, the dilution of the reaction mixture beingequal to 2% of the ¢nal volume. The reaction was done in 50mM Tris-HCl (pH 9.0), 5 mM ATP, 9 mM MgCl2 and 50 WM[14C]Phe. The amount of Phe bound to tRNA was estimated by¢lter assay.

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the resolution of tRNAPhe aminoacylated by the mix-ture of E. coli and T. thermophilus synthetases re-sulted in the appearance of an additional peak elutedafter monoaminoacyl-tRNA (Fig. 2c).

The UV spectrum of this compound was identicalto that of tRNA. The ratio of 14C radioactivity toabsorbance at 260 nm was estimated to correspondto 1.85 mol Phe per mol tRNA. The compound canbe precipitated by the addition of ethanol or TCA.Thus, the phenomenon of the anomalously high pla-teau in the T. thermophilus PheRS-catalyzed tRNAaminoacylation can be explained by the enzymaticsynthesis of this unusual product, supposed to bebisphenylalanyl-tRNA. However, the ¢nal yield ofbisphenylalanyl-tRNA calculated from the amino-acylation kinetic (67% of aminoacylated tRNAPhe)shows signi¢cant discrepancy with its percentageevaluated from the chromatographic data (41% ofaminoacylated tRNAPhe). To establish the cause ofthis divergence both species of aminoacylatedtRNAPhe were collected separately and rechromato-graphed under the same conditions. Monophenyl-alanyl-tRNA was found to be stable during this pro-cedure. At the same time approximately 40% ofbisphenylalanyl-tRNA loaded onto the column wasdecomposed yielding monophenylalanyl-tRNA andfree amino acid. Taking this observation into ac-count we should multiply the quantity of bisphenyl-

alanyl-tRNA found after elution from the column bya factor 1.67 to obtain its true content in the reactionmixture.

Such low stability of this compound set us testingan alternative hypothesis about its nature. T. thermo-philus PheRS is supposed to be a functional dimer[6], i.e. to have two identical active sites. The com-pound interpreted as bisphenylalanyl-tRNA might infact be a stable complex consisting of one moleculeof the enzyme, one molecule of phenylalanyl-tRNAand one molecule of phenylalanine (or phenylalanyl-adenylate). The UV spectrum of such a complexwould be determined almost completely by thetRNA moiety, all other components would make

Fig. 2. Chromatographic analysis of the aminoacylation reac-tion mixtures containing normally charged and overcharged T.thermophilus tRNAPhe. (a) Successive tRNA aminoacylationwith 46 Wg/ml E. coli PheRS and 67 Wg/ml T. thermophilusPheRS. The reaction was initiated by E. coli enzyme and after21 min of tRNA charging the T. thermophilus enzyme wasadded, the dilution of the reaction mixture being equal to 0.5%of the ¢nal volume. The reaction mixture contained 1.4 WMtRNAPhe, 50 mM HEPPS-NaOH (pH 9.0), 5 mM ATP, 9 mMMgCl2 and 20 WM [14C]Phe. At the times indicated by the ar-rows aliquots were withdrawn for chromatographic analysis. (b)Chromatographic resolution of the aliquot with tRNA chargedwith only E. coli PheRS. The aliquot containing 1 nmol of[14C]phenylalanylated tRNA was mixed with an equal volumeof bu¡er A, applied onto the LiChrosorb RP-18/Adogen464 column and chromatographed as described in Section 2.Fractions of 0.5 ml were collected and assayed for 14C radio-activity. (c) Chromatographic resolution of the aliquot withtRNA charged with the mixture of E. coli and T. thermophilusPheRSs. The aliquot containing 1 nmol of [14C]phenylalanyl-ated tRNA was resolved as in (b).

C

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only a minor contribution to its absorbance at260 nm. At the same time the ratio of [14C]phenyl-alanine to tRNA would be 2:1. This model satisfac-torily explains all experimental data presented aboveas well as the bisphenylalanyl-tRNA concept. Tocheck the assumption the compound containingmore than 1 mol Phe per mol tRNA was tested forthe presence of the protein. The products of T. ther-mophilus PheRS-catalyzed tRNA aminoacylationwere separated on the LiChrosorb RP-18/Adogen464 column (Fig. 3). To measure accurately thetRNA amount in the chromatographic fractionswe used a tritium-labelled tRNAPhe ([A76-3H]tRNAPhe(ex)) obtained by CTP (ATP):tRNA nu-cleotidyltransferase-promoted exchange reaction ofthe tRNA 3P-terminal nucleotide. The expectedquantity of protein in the last eluted compoundwas calculated from the ratio 1 mol of enzyme permol of [A76-3H]tRNAPhe(ex) (or 1 mol of enzymeper 2 mol of [14C]phenylalanine) in the hypotheticalcomplex. It should be approximately 180 Wg. To con-centrate the pooled fractions and to change acetatebu¡er with potassium phosphate, ultra¢ltrationthrough Ultrafree-MC 100000 NMWL membranemicro¢lter was used. The control mixtures containing

12^250 Wg of T. thermophilus PheRS were subjectedto the same procedure. The lower limit of the methodis approximately 1 Wg of protein per sample and theenzyme in the control solutions was revealed reliablyin the whole range of concentrations. However, thematerial collected after chromatography did not con-tain any detectable amount of protein, therefore theassumption about the existence of a stable enzymecphenylalanyl-tRNAcphenylalanine (or phenylalanyl-adenylate) complex was recognized to be inadequate.The ¢nal choice was made in favor of the bisphen-ylalanyl-tRNA concept.

We had serious reasons to suppose that the attach-ment of the second phenylalanine molecule to tRNAproceeds via phenylalanyladenylate formation. Bothrate and yield of bisphenylalanyl-tRNA synthesis arereduced after the addition of AMP and PPi to thereaction mixture (Fig. 4). The e¡ect can be inter-preted as an inhibition of tRNA overcharging atthe amino acid activation step by the reaction prod-ucts.

Fig. 4. AMP/PPi inhibition of tRNAPhe overcharging. 2.1 WMT. thermophilus tRNAPhe was aminoacylated at 40³C for 15 minwith 50 Wg/ml E. coli PheRS (F). Then the reaction mixturewas divided into three equal portions and tRNA charging wascontinued after addition of T. thermophilus PheRS to a concen-tration of 80 Wg/ml alone (b) or together with AMP and PPi

(to ¢nal concentrations of 100 WM) (a). As a control AMP andPPi (to ¢nal concentrations of 100 WM) (S) were added to thethird portion to check their in£uence on the E. coli enzyme-controlled plateau. The reaction mixture contained 50 mMTris-HCl (pH 9.0), 5 mM ATP, 8 mM MgCl2 and 20 WM[14C]Phe. The dilution of the reaction mixtures upon the en-zyme additions did not exceed 2% of the ¢nal volume. Theamount of [14C]Phe bound to tRNA was determined by ¢lterassay.

Fig. 3. Characterization of the anomalous product of tRNAPhe

aminoacylation. 0.46 WM tRNAPhe was aminoacylated at 40³Cfor 50 min with T. thermophilus PheRS. The reaction mixture(7 ml) contained 50 mM HEPPS-NaOH (pH 9.0), 5 mM ATP,9 mM MgCl2 and 20 WM [14C]Phe. The reaction was stoppedby addition of an equal volume of bu¡er A, the mixture wasapplied on the LiChrosorb RP-18/Adogen 464 column andchromatographed as described in Section 2. Fractions of 0.4 mlwere collected and [14C]Phe and [A76-3H]tRNAPhe(ex) amountswere established by separate counting of 14C and 3H radioactiv-ity, respectively. The last eluted compound was tested for thepresence of protein by Bradford's technique as described in Sec-tion 2.

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To de¢ne optimal conditions for bisphenylalanyl-tRNA synthesis by the thermophilic synthetase weinvestigated the dependence of tRNA phenylalanyla-tion level on the concentrations of enzyme andtRNAPhe (Fig. 5), pH and type of bu¡er (Table 1).The lower the tRNAPhe concentration, the more e¤-ciently bisphenylalanyl-tRNA is formed. The e¡ect isprobably caused by a change of the balance between

enzyme-catalyzed tRNA aminoacylation and non-en-zymatic hydrolysis of the charged tRNA. The highertRNA content in the reaction mixture can also pro-voke the stronger product inhibition at the late stageof tRNA aminoacylation.

Our interest in the e¡ect of bu¡er on bisphenyl-alanyl-tRNA yield was caused by the observation ofreproducible degradation of the aminoacylation pla-teau in Tris-containing reaction mixtures (see, forexample, Fig. 4 and Fig. 7). The plateau decreasewas found to be not due to synthetase inactivation,tRNAPhe damage, ATP expenditure or AMP/PPi in-hibitory e¡ect. The amino acid was observed to forma covalent adduct with Tris. Thus, tRNA aminoacyl-ation slows down owing to the decrease of phenyl-alanine concentration in the mixture. The reactiontakes place in the presence of E. coli PheRS as well

Table 2Aminoacylation of di¡erent phenylalanine-accepting tRNAs with T. thermophilus phenylalanyl-tRNA synthetase

tRNA Aminoacylation levela (mol Phe/mol tRNA)

by chromatographic analysis by ¢lter assay

T. thermophilus tRNAPhe (I) 1.84 1.84T. thermophilus tRNAPhe (II) 1.82 ^E. coli tRNAPhe ^ 1.59E. coli tRNAPhe transcript 1.35 ^

The reaction was carried out at 40³C in a mixture containing 100 Wg/ml PheRS, 0.75^0.9 WM tRNA, 50 mM Tris-HCl (pH 9.0),5 mM ATP, 9 mM MgCl2 and 25 WM [14C]Phe.aFor each tRNA the value was normalized to the level of charging by E. coli PheRS, measured under the same conditions.

Table 1The in£uence of bu¡er on the level of tRNAPhe charging by T.thermophilus phenylalanyl-tRNA synthetase

Bu¡er pH at 20³C Aminoacylation levela

(mol Phe/mol tRNA)

HEPPS-NaOH 9.0 1.51 þ 0.09Tricine-HCl 9.0 1.32 þ 0.08Diethanolamine-HCl 9.0 1.26 þ 0.09Tris-HCl 9.0 1.21 þ 0.07Tris-HCl 8.5 1.40 þ 0.05Tris-HCl 8.1 1.25 þ 0.06MOPS-NaOH 7.0 1.04 þ 0.05

The reaction was carried out at 40³C in a mixture containing100 Wg/ml PheRS, 1.3 WM tRNAPhe, 5 mM ATP, 9 mM MgCl2and 10 WM [14C]Phe.aValues were normalized to the level of tRNAPhe charging byE. coli PheRS, which was determined to be identical in di¡erentbu¡ers (HEPPS-NaOH (pH 9.0), Tricine-HCl (pH 9.0), Tris-HCl (pH 9.0)).

Fig. 5. Dependence of the aminoacylation level on the concen-trations of T. thermophilus phenylalanyl-tRNA synthetase andtRNAPhe in the reaction mixture. The normalized values oftRNA charging level (mol Phe/mol tRNA) are presented inboxes. Each value averages eight plateau points. The controllevel of charging was measured at 57 Wg/ml E. coli PheRS. Thereaction was carried out at 40³C. The mixtures contained 50mM HEPPS-NaOH (pH 9.0), 5 mM ATP, 9 mM MgCl2 and20 WM [14C]Phe. The time course of tRNA aminoacylation wasmonitored by ¢lter assay.

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as in the case of the thermophilic enzyme, but it canbe detected only at high enzyme concentrations (datanot shown; this reaction is now under investigation).The transfer of phenylalanyl residues on tRNAPhe

may compete with the phenylalanine attachment toa Tris molecule. The aminoacylation plateau is stablewhen pH is maintained by HEPPS, Tricine or dieth-anolamine bu¡ers. The highest level of tRNAPhe

charging by T. thermophilus PheRS was achievedwhen HEPPS was used for the reaction mixture bu¡-ering (see Table 1).

T. thermophilus PheRS can use E. coli, yeast andhuman tRNAPhes as substrates [17]. The enzyme alsoaminoacylates unmodi¢ed tRNAPhe transcript withhigh e¤ciency [11]. This synthetase seems to have

Fig. 7. Time course of bisphenylalanyl-tRNA synthesis. (a) Theextra [14C]Phe was attached to mono[14C]phenylalanyl-tRNAobtained in situ with 50 Wg/ml E. coli PheRS. The reaction oftRNA overcharging was initiated by addition of T. thermophilusPheRS to the concentration 83 (E), 96 (b) and 152 (O) Wg/ml.The aminoacylation assays were performed at 40³C using ¢ltertechnique. The reaction mixtures contained 1.8 WM tRNAPhe,50 mM Tris-HCl (pH 9.0), 5 mM ATP, 8 mM MgCl2 and25 WM [14C]Phe. (b) Changes of monophenylalanyl-tRNA (E)and bisphenylalanyl-tRNA (b) concentrations upon tRNAPhe

aminoacylation with 65 Wg/ml T. thermophilus PheRS at 40³C.The reaction mixture contained 1.3 WM tRNAPhe, 50 mM Tris-HCl (pH 9.0), 5 mM ATP, 9 mM MgCl2 and 100 WM [14C]Phe.At the appropriate times aliquots were taken out from the reac-tion mixture and analyzed on the LiChrosorb RP-18/Adogen464 column as described in Section 2. The amounts of mono-and bisphenylalanyl-tRNA were calculated from the total 14Cradioactivity in the corresponding peaks. The experimental val-ues were corrected taking into account the deacylation of 40%of bisphenylalanyl-tRNA to monophenylalanyl-tRNA duringthe chromatography.

Fig. 6. Aminoacylation of [A76-3H]tRNAPhe(r) with T. thermo-philus phenylalanyl-tRNA synthetase. (a) 2.2 WM [A76-3H]tRNAPhe(r) was aminoacylated with 100 Wg/ml T. thermophi-lus PheRS at 40³C. The reaction mixture contained 50 mMTris-HCl (pH 9.0), 5 mM ATP, 9 mM MgCl2 and 25 WM[14C]Phe. 15 min after initiation, when the reaction reached aplateau, an aliquot with 2.5 nmol of aminoacylated tRNA wastaken out, mixed with an equal volume of bu¡er A and appliedon the LiChrosorb RP-18/Adogen 464 column. The separationwas done as described in Section 2. Fractions of 0.46 ml werecollected and [14C]Phe and [A76-3H]tRNAPhe(r) amounts wereestablished by separate counting of 14C and 3H radioactivity,respectively. (b) The mixture of 1.45 WM native T. thermophilustRNAPhe and 0.26 WM [A76-3H]tRNAPhe(r) was aminoacylatedwith 100 Wg/ml T. thermophilus PheRS at 40³C. The conditionsof aminoacylation and chromatographic separation were thesame as in (a).

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very limited structural requirements for proper inter-action with tRNAPhe. Therefore it is not surprisingthat the enzyme has been found to be capable ofovercharging E. coli tRNAPhe and unmodi¢ed E.coli tRNAPhe transcript (Table 2). However, the ami-noacylation of T. thermophilus [A76-3H]tRNAPhe(r)(obtained by successive periodate oxidation, base-promoted 3P-terminal adenosine removal, phospha-tase treatment and 3P-end repairing by CTP(ATP):tRNA nucleotidyltransferase in the presenceof [3H]ATP) resulted in monophenylalanyl-tRNAformation only. The synthesis of bisphenylalanyl-[A76-3H]tRNAPhe(r) was strongly impaired (Fig.6a). To prove that the e¡ect is caused by thetRNA treatment but not by some inhibitory impurityin the [A76-3H]tRNAPhe(r) preparation, a mixture of[A76-3H]tRNAPhe(r) and native (untreated) T. ther-mophilus tRNAPhe with a molar ratio of 1:5.6 wasaminoacylated (Fig. 6b). The thermophilic enzymegives native tRNAPhe a pronounced preference over[A76-3H]tRNAPhe(r) as follows from the ratio 3Hradioactivity/A260 in the peaks corresponding to

monophenylalanyl-tRNA and bisphenylalanyl-tRNA. PheRS attaches only one phenylalanyl resi-due to [A76-3H]tRNAPhe(r) but native tRNAPhe isovercharged in the same reaction mixture. Thereforethe procedure of the removal and the restoration of3P-terminal nucleotide was concluded to be accompa-nied by some changes in tRNA structure. Thesechanges do not prevent monophenylalanyl-tRNAsynthesis but strongly suppress the transfer of thesecond phenylalanyl residue to tRNA. In fact, themost signi¢cant modi¢cations of tRNAPhe are theoxidation of thiouridine at position 8 and a partialremoval of 5P-terminal phosphate. It is doubtful thatthese two fragments participate directly in bisphenyl-alanyl-tRNA formation as acceptors of the extraphenylalanyl residue. Most probably, they are in-volved in the maintenance of the correct orientationof the extra phenylalanine-accepting group towardthe amino acid activation site of the enzyme.

The rate of bisphenylalanyl-tRNA synthesis wasevaluated by two di¡erent ways. Firstly, we meas-ured the initial rate of extra phenylalanine attach-ment to tRNAPhe preaminoacylated by E. coli PheRS(Fig. 7a). This approach is based on the assumptionthat the E. coli enzyme does not interfere at all withbisphenylalanyl-tRNA formation. Another way tomeasure separately the rates of mono- and bisphenyl-alanyl-tRNA synthesis includes the chromatographicresolution of aliquots withdrawn from the reactionmixture at di¡erent times and the calculation ofmono- and bisphenylalanyl-tRNA quantities fromthe optical density pro¢les or 14C label distributions,taking into account the bisphenylalanyl-tRNA insta-bility during chromatography (Fig. 7b).

The values of initial rates of bisphenylalanyl-tRNA synthesis at 40³C, obtained by both methods,were found to be quite close: 4.4 nmol/mg/min ac-cording to the ¢rst one and 5.5 nmol/mg/min accord-ing to the second one. The initial rate of monophen-ylalanyl-tRNA synthesis was determined by themeasurement of the amount of [14C]phenylalaninebound to tRNAPhe before the tRNA charging levelexceeded 50%, i.e. when bisphenylalanyl-tRNA pro-duction was negligible. It was found to be equal to240 nmol/mg/min under the same reaction condi-tions. Thus, the ¢rst amino acyl residue is transferredto tRNA 44^54 times faster than the second one.

One of the characteristic properties of aminoacyl-

Fig. 8. Deacylation of [14C]phenylalanylated tRNAPhe at 40³C.(W) Deacylation of bis[14C]phenylalanyl-tRNA in 0.1 M so-dium acetate (pH 6.2). (a) Non-enzymatic deacylation ofmono[14C]phenylalanyl-tRNA at pH 9.0. (O) Non-enzymaticdeacylation of bis[14C]phenylalanyl-tRNA at pH 9.0. (E) En-zyme-assisted deacylation of bis[14C]phenylalanyl-tRNA.

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tRNA synthetases is their high speci¢city toward thecognate amino acids. In spite of its unusual catalyticbehavior T. thermophilus PheRS also possesses highselectivity during tRNA aminoacylation. We failedto detect incorporation of L-[14C]tyrosine, L-[14C]leucine, L-[14C]isoleucine and L-[14C]valine intotRNAPhe at 40³C and 100 Wg/ml of enzyme (datanot shown).

We have investigated extensively the stability ofbisphenylalanyl-tRNA under di¡erent conditions toelucidate the nature of the bond between the extraphenylalanyl residue and tRNA. It is well knownthat the aminoacyl-tRNA ester bond is labile at pHgreater than 6 and the rate of amino acid release isstrongly dependent on hydroxide ion concentration.In the presence of aminoacyl-tRNA synthetase theaminoacyl-tRNA deacylation proceeds faster, but itis unclear whether this enzyme-catalyzed hydrolysishas some biological signi¢cance [18]. Bisphenylalan-yl-tRNA was found to be stable at pH 5^6. The rateof base-induced phenylalanine release from bis-phenylalanyl-tRNA at pH 9 is slightly greater thanthat from monophenylalanyl-tRNA (Fig. 8). Takinginto account the similar stability of mono- and bis-phenylalanyl-tRNA in alkaline solution, the bondbetween the extra phenylalanyl residue and tRNAwas supposed to be covalent but quite labile. In thepresence of T. thermophilus PheRS the overchargedtRNA is deacylated more intensively. The only ra-dioactive product of bisphenylalanyl-tRNA deacyl-ation is free phenylalanine (according to HPLC andTLC identi¢cation tests).

To establish whether bisphenylalanyl-tRNA is syn-thesized in vivo or not we studied the tRNAPhe ami-noacylation products isolated from T. thermophilusliving cells. The total tRNA pool was isolated underacidic conditions to avoid hydrolysis of the bondbetween the amino acid and tRNA. This materialwas resolved on the LiChrosorb RP-18/Adogen 464column. The aliquots from each fraction were thensubjected to reaminoacylation in the presence of la-belled phenylalanine (see Section 2). Three tRNAPhe-containing peaks were detected (Fig. 9a).

The main di¤culty in identi¢cation of freetRNAPhe and its aminoacylated forms is the existenceof two so-called isoacceptors of T. thermophilustRNAPhe, tRNAPhe (I) and tRNAPhe (II) (two dis-tinct products of tRNAPhe posttranscriptional mod-

i¢cation at position 37 [19]), having a di¡erent chro-matographic behavior (Fig. 9c,d). Therefore, the ¢rstpeak is tRNAPhe (I) and the second one is the mix-ture of tRNAPhe (II) and monophenylalanyl-tRNAPhe (I). The third peak might be identi¢ed asbisphenylalanyl-tRNAPhe (I) but monophenylalanyl-

Fig. 9. Identi¢cation of tRNAPhe aminoacylation productsformed in T. thermophilus living cells. The chromatographieswere done on the LiChrosorb RP-18/Adogen 464 column as de-scribed in Section 2. Fractionation of total tRNA isolated fromT. thermophilus under acidic conditions. The presence oftRNAPhe or its aminoacylated forms in 100 Wl aliquots fromeach fraction was estimated by synthetase-catalyzed[14C]phenylalanine incorporation into tRNA as described inSection 2. The peaks are numbered according to the order ofelution. Chromatography of material from peak 3 (indicatedby black bars in a) subjected to complete base-catalyzed de-acylation. tRNA detection was as in previous case but[3H]phenylalanine was used for tRNA aminoacylation. Separa-tion of mono[14C]phenylalanyl-tRNAPhe (I) and bis[14C]phenyl-alanyl-tRNAPhe (I). The arrow shows the elution volume foruncharged tRNAPhe (I). Separation of mono[14C]phenylalanyl-tRNAPhe (II) and bis[14C]phenylalanyl-tRNAPhe (II). The arrowshows the elution volume for uncharged tRNAPhe (II).

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tRNAPhe (II) has the same chromatographic mobil-ity. To determine the nature of tRNA in the thirdpeak we subjected this material to complete base-catalyzed deacylation (pH 9.0, 40³C, 60 min) andrechromatography (Fig. 9b). Only tRNAPhe (II)was found. Therefore, the third peak correspondsto monophenylalanyl-tRNAPhe (II) and the totaltRNAPhe pool from T. thermophilus does not containa detectable amount of bisphenylalanyl-tRNA. Thecalculated threshold of bisphenylalanyl-tRNA regis-tration was equal to 50 pmol (approx. 0.3% of thetotal amount of phenylalanine-accepting tRNA iso-lated from the cells). Thus, we should conclude thatthe content of bisphenylalanyl-tRNA in the pool ofaminoacylated tRNAPhe formed under normalgrowth of T. thermophilus culture could not exceedthis value.

4. Discussion

In our previous studies on T. thermophilus PheRSthe tRNAPhe aminoacylation reaction catalyzed bythis enzyme was shown to result in an anomalouslyhigh plateau corresponding to more than one aminoacyl residue attachment to the tRNA molecule [5,15].The e¡ect has been observed at synthetase concen-trations of 5^50 Wg/ml in a temperature range of 25^80³C. We have explained this phenomenon by thespeci¢c ability of the thermophilic enzyme to formbisphenylalanyl-tRNA. We have suggested that thesetwo phenylalanyl residues are bound to 2P- and 3P-hydroxyl groups of the 3P-terminal adenosine. Theassumption was based on the fact that one of the[14C]phenylalanine-containing compounds appearingafter RNase A hydrolysis of overcharged tRNA hada chromatographic mobility close to that of chemi-cally synthesized bis-2P,3P-O-phenylalanyladenosine.However, this coincidence might be accidental andcould not be considered a ¢nal proof of bisphenyl-alanyl-tRNA structure. Moreover, even the existenceof bisphenylalanyl-tRNA remained hypotheticalbecause we had no method to isolate it from anaminoacylation reaction mixture separately frommonophenylalanyl-tRNA. Therefore the pure bis-phenylalanyl-tRNA was not characterized and wecould not exclude another explanation of the ob-served anomaly.

The data presented in this paper allow us to con-clude that bisphenylalanyl-tRNA really exists as oneof the products of T. thermophilus PheRS-catalyzedtRNAPhe aminoacylation. We have succeeded in sep-arating this compound from other components of thereaction mixture and a precise ratio of Phe:tRNAclose to 2:1 has been estimated. However, the siteof the extra phenylalanyl residue attachment totRNAPhe is still unknown. The fact that tRNAPhe

transcript can be overcharged speaks against the ne-cessity of tRNA posttranscriptional modi¢cation forthe second amino acid to be accepted. Taking intoaccount the observed lability of bisphenylalanyl-tRNA we suppose that the ribose hydroxyl groupsare the most likely candidates to be esteri¢ed withthe second amino acid. The extra phenylalanine maybe transferred to one of the 2P-hydroxyl groups ofnucleotides 1^75. Moreover, although the thermo-philic synthetase can acylate only the 2P-hydroxylgroup of adenosine-76 [15], the spontaneous 2PC3Pmigration of the aminoacyl residue followed by therepeated aminoacylation of the liberated 2P-hydroxylgroup might result in the placement of both phenyl-alanines on the neighboring hydroxyl groups oftRNA 3P-terminal ribose.

The absence of detectable [14C]phenylalanine in-corporation into a tRNAPhe analog with 2P-deoxy-adenosine instead of natural adenosine at position76 described in our previous paper [15] cannot be adecisive argument against aminoacylation of 2P-hy-droxyl groups of nucleotides 1^75. Really, thistRNA analog was obtained by successive periodate,aniline and alkaline phosphatase treatments followedby a CTP (ATP):tRNA nucleotidyltransferase-cata-lyzed 3P-terminus reconstruction in the presence of2P-dATP. However, the procedure was found to re-duce drastically the ability of tRNAPhe to accept theextra amino acid even if the tRNA 3P-end is restoredto the natural CCA sequence. Perhaps, we havefailed to detect overcharging of another testedtRNAPhe analog bearing on its 3P-end an adenosinewith C2P-C3P opened ribose cycle (tRNAoxÿred) forthe same reason. The most probable T. thermophilustRNAPhe damage caused by the procedures of 3P-ter-minal nucleotide removal or modi¢cation is the per-iodate-induced oxidation of 4-thiouridine at position8 to uridine sulfonate [20]. Furthermore, a partialtRNA 5P-dephosphorylation takes place under phos-

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phatase treatment. The in£uence of these structuralchanges on the extra phenylalanine incorporationmay be mediated by an enzyme-tRNA interaction:

the bisphenylalanyl-tRNA synthesis slows down ow-ing to unfavorable mutual orientation of synthetaseand damaged tRNA in catalytic complex.

We can draw only preliminary conclusions aboutthe mechanism of bisphenylalanyl-tRNA synthesison the base of data presented. It is obvious thatthe ¢rst phenylalanine is transferred to the 2P-hy-droxyl group of adenosine-76 resulting in monophen-ylalanyl-tRNA formation. An attachment of the sec-ond amino acid to tRNA proceeds approximately 50times more slowly. Therefore bisphenylalanyl-tRNAsynthesis becomes detectable long after completetRNA conversion to monophenylalanyl-tRNA is ¢n-ished. We cannot say whether these two acts of ami-no acid transfer to tRNA are strictly coordinated intime or are independent but proceed with very di¡er-ent rates. The second amino acid is supposed to beactivated through aminoacyladenylate formation likethe ¢rst one. The amino acid selection for tRNAaminoacylation is under the same severe control asin the case of other PheRSs: the thermophilic syn-thetase cannot transfer to tRNAPhe amino acids oth-er than phenylalanine. The enzyme seems to be ableto bind reversibly bisphenylalanyl-tRNA and to de-stabilize selectively the bond between the extra phen-ylalanyl residue and tRNA as follows from the high-er lability of bisphenylalanyl-tRNA in the presenceof the enzyme.

Abnormal T. thermophilus PheRS functioningpoints to a singularity of its structural organizationand made us look for the principal di¡erence be-tween the thermophilic enzyme and PheRSs fromother sources. The T. thermophilus PheRS moleculeconsists of two small (K) subunits (Mr 39 000, 350amino acid residues) and two large (L) subunits

Fig. 10. Comparison of phenylalanyl-tRNA synthetase primarysequences relating to the amino acid activation domain (K sub-units) or mimicking it (L subunits). Conserved residues in eachsequence family are shown in bold type. Identical residues of T.thermophilus PheRS K and L subunits are marked by squares.Abbreviations used: S. ce. (mit), Saccharomyces cerevisiae (mi-tochondrial) ; S. ce. (cyt), Saccharomyces cerevisiae (cytoplas-mic) ; M. ca., Mycoplasma capricolum ; B. su., Bacillus subtilis ;E. co., Escherichia coli ; T. th., Thermus thermophilus. Sequencealignments are given essentially according to [22,31,32] withmodi¢cations. The known sequence of the L subunit of Saccha-romyces cerevisiae PheRS is not considered here because itshows a little homology to the bacterial L subunits [32].6

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(Mr 86 500, 785 amino acid residues), the KL hetero-dimer being topological and, probably, a functionalunit of this tetrameric enzyme [21]. Identi¢cation ofthe amino acid activation domain within the K sub-unit fragment 101^327 is based on the location ofthree signature sequence motifs [22] and a speci¢cspatial fold [21,23] characteristic for active site do-mains of class II aminoacyl-tRNA synthetases.

tRNAPhe was found to contact generally di¡erentparts of the L subunit [24]. Anticodon recognition iscarried out by the C-terminal domain of the L sub-unit, interactions with the K subunit amino acid ac-tivation domain have been observed only for adeno-sine-76. The impression is evoked that the K subunitmainly ensures an amino acid activation and the Lsubunit recognizes tRNAPhe and poses it properlyupon binding, i.e. both subunits of the KL dimerare functionally specialized. However, the L subunithas been found to contain a structural module with aspatial fold typical of the amino acid activation do-main of class II synthetases. Moreover, this so-calledcatalytic-like module (residues 483^678) demon-strates a sequence homology to the K subunit, highlyconserved motifs presumably forming the amino acidactivation site (Fig. 10). Such a homology between Kand L subunits is observed only for T. thermophilusPheRS but not for other bacterial PheRSs withknown primary structure.

It would be very attractive to explain the uniquecatalytic property of the thermophilic enzyme by theexistence of two separate amino acid activation siteson KL functional unit. However, is the `catalytic-likemodule' really capable of aminoacyladenylate forma-tion? Its structural di¡erence from a true active sitedomain is still signi¢cant and this explanation of bis-phenylalanyl-tRNA synthesis remains very hypo-thetical. On the other hand, the opinion that T. ther-mophilus PheRS has only two amino acid activationdomains per K2L2 tetramer [23] is not based on directexperimental data obtained for this enzyme. The onlyreason for this statement is the analogy with E. coliand yeast PheRSs [25^30].

It is still unclear whether this phenomenon playsany role in metabolism of the thermophilic bacteria.Bisphenylalanyl-tRNA has not been observed in T.thermophilus growing cells. However, the compoundmay be tightly associated with insoluble cellular frag-ments or decompose under cell disruption proce-

dures. On the other hand, monophenylalanyl-tRNAbeing a substrate for bisphenylalanyl-tRNA synthesisis constantly consumed by the ribosomal machineryand this competition may be the reason for the ab-sence of bisphenylalanyl-tRNA during normalgrowth of thermophilic cells. The favorable condi-tions for bisphenylalanyl-tRNA formation appearwhen monophenylalanyl-tRNA production exceedsits consumption (for example, when mRNA transla-tion on the ribosome is blocked or PheRS is pro-duced in excess, etc.). In this case bisphenylalanyl-tRNA might play a role of a signal compoundformed in response to the disbalances in the systemof peptide synthesis. However, we cannot excludethat bisphenylalanyl-tRNA is an arti¢cial productof tRNA aminoacylation reaction and its formationtakes place only in vitro under conditions far fromnatural.

The peculiarities of the cellular organization ofextremely thermophilic bacteria allow them to existat high temperature when the growth of other pro-karyotes is suppressed. Up to now, the main e¡ortshave been generally concentrated on the investigationof thermal stability of biopolymers isolated fromthermophilic organisms. However, active life is pos-sible only under coordinated functioning of all cellcomponents. Therefore, the biochemical processesoccurring in the cells of extremely thermophilic bac-teria must also be adapted to a high temperature andtheir mechanisms can di¡er from the reaction path-ways of mesophilic organisms. The unusual catalyticbehavior of T. thermophilus PheRS may be a trace ofsuch adaptation.

Acknowledgements

This study was supported by the Russian Founda-tion for Basic Research No. 96-04-50150 and by theHigh School Grant for Basic Research of the St.Petersburg University.

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