A peculiarity of the reaction of tRNA aminoacylation catalyzed by phenylalanyl-tRNA synthetase from the extreme thermophile Thermus thermophilus

Biochimica et Biophysica Acta 1386 (1998) 1^15

A peculiarity of the reaction of tRNA aminoacylation catalyzed by phenylalanyl-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 by Thermus thermophilus phenylalanyl-tRNA synthetase is a result of enzymatic synthesis of tRNA bearing two bound phenylalanyl residues (bisphenylalanyl-tRNA). The efficiency of bisphenylalanyl-tRNA formation was shown to be quite low : the second phenylalanyl residue is attached to tRNA approximately 50 times more slowly than the first one. The thermophilic synthetase can aminoacylate twice not only T. thermophilus tRNAPhe but also Escherichia coli tRNAPhe and E. 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 free phenylalanine. Under these conditions phenylalanine is released from bisphenylalanyl-tRNA with almost the same rate as from 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 bisphenylalanyltRNA under normal growth of culture. A possible mechanism of bisphenylalanyl-tRNA synthesis is discussed. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Phenylalanyl-tRNA synthetase; tRNAPhe ; tRNA aminoacylation mechanism

1. Introduction Aminoacyl-tRNA synthetases belong to a numerous group of enzymes participating in the realization

of genetic information. The mechanism of tRNA aminoacylation is generally accepted to include two main steps: (a) An aminoacyl-tRNA synthetase (aaRS) con-

Abbreviations: PheRS, phenylalanyl-tRNA synthetase (EC 6.1.1.20); [A76-3 H]tRNAPhe (r), tRNA with tritium-labelled 3P-terminal nucleotide obtained by restoration of tRNA lacking A76 in the presence of CTP (ATP):tRNA nucleotidyltransferase and [3 H]ATP; [A76-3 H]tRNAPhe (ex), tRNA with tritium-labelled 3P-terminal nucleotide obtained by CTP (ATP):tRNA nucleotidyltransferase promoted reaction of A76 exchange in the presence of [3 H]ATP; HEPPS, N-[2-hydroxyethyl]piperazine-NP-[2-propanesulfonic acid]; MOPS, 3-[Nmorpholino]propanesulfonic acid; Tricine, N-tris[hydroxymethyl]methylglycine ; PPi , inorganic pyrophosphate * Corresponding author. Fax: +7 (383) 2-33-36-77; E-mail: [email protected], [email protected] 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

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verts a speci¢c amino acid (aa) to aminoacyladenylate (activation step) aaRS ‡ ATP ‡ aa ! aaRSc…aaVAMP† ‡ PPi

…1†

(b) The amino acid is attached to tRNA complexed with the enzyme yielding aminoacyl-tRNA (transfer step) aaRSc…aaVAMP† ‡ tRNAaa ! aaRSc…aaVtRNAaa † ‡AMP ! aaRS ‡ AMP ‡ aaVtRNAaa

…2†

To measure the quantity of aminoacyl-tRNA formed in the course of tRNA aminoacylation the labelled amino acid with known speci¢c radioactivity is added to the reaction mixture. Precipitation of aminoacyl-tRNA with trichloroacetic acid (TCA) makes it possible to separate it from free amino acid. Thus, it is possible to determine a portion of tRNA converted to aminoacyl-tRNA by juxtaposition of radioactivity of TCA precipitate and initial concentration of tRNA in the mixture. Usually the concentrations of both ATP and amino acid in aminoacylation assay mixtures are signi¢cantly higher than that of tRNA. Therefore the plateau of the reaction is determined by the concentration of tRNA which is the limiting substrate. From the general scheme of the reaction mechanism it follows that the plateau in this case corresponds to the situation when all tRNA molecules bear one amino acid residue. In reality the maximal percentage of aminoacylated tRNA may be lower than 100% due to instability of the ester bond between the aminoacyl residue and tRNA in conditions favorable for aminoacyl-tRNA synthetases functioning (pH 7^9). In this case the reaction plateau is determined by a dynamic balance between synthetase-catalyzed tRNA charging and non-enzymatic hydrolysis of aminoacyl-tRNA [1^4]. However, a few years ago it was found that the catalytic mechanism of phenylalanyl-tRNA synthetase from the extremely thermophilic eubacterium Thermus thermophilus cannot be described in terms of this common scheme [5]. The total amount of labelled phenylalanine associated with TCA-insoluble material corresponded to a tRNAPhe charging level higher than 100%. The assumption was made that the population of tRNAPhe aminoacylated with thermophilic synthetase contains not only monophen-

ylalanyl-tRNA (the usual product of tRNA aminoacylation) but also bisphenylalanyl-tRNA (tRNA with two covalently bound phenylalanyl residues). However, the possibility of synthetase-catalyzed attachment of more than one amino acid molecule to tRNA was never discussed in articles dedicated to the mechanism of tRNA aminoacylation. The main target of this work was to verify thoroughly our hypothesis about the cause of the anomalously high level of tRNA charging and to investigate the nature and regularities of this phenomenon. 2. Materials and methods 2.1. Materials Homogeneous phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 was puri¢ed as previously described [6]; its speci¢c activity was 210 U/ mg (37³C). Phenylalanyl-tRNA synthetase from Escherichia coli MRE-600 with speci¢c activity 310 U/ mg (37³C) was isolated as described in [7]. E. coli CTP (ATP):tRNA nucleotidyltransferase (speci¢c activity 30 U/mg at 37³C) was isolated according to [8]. E. coli tRNAPhe , T. thermophilus tRNAPhe (I) and tRNAPhe (II) (phenylalanine incorporation by E. 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. coli tRNAPhe transcript was synthesized and puri¢ed as described in [11] (phenylalanine incorporation by E. coli PheRS was 1930 pmol/A260 unit). Deoxyribonuclease I (RNase-free, 3000 U/mg) and lysozyme (50 000 U/mg) were from Sigma. 14 L-[U- C]Phenylalanine (325 Ci/mol) was from UVVVR (Czechoslovakia), L-phenyl-[2,3-3 H]phenylalanine (53 Ci/mmol) and [8-3 H]ATP (32 Ci/mmol) were products of St. Petersburg Institute of Applied Chemistry (Russia). 2.2. Aminoacylation of tRNAPhe The standard reaction mixture (50^1000 Wl) contained 5 mM ATP, 9 mM MgCl2 , 50 mM Tris-HCl or HEPPS-NaOH (pH 9.0 at 20³C), 20^50 WM L[14 C]phenylalanine, 0.5^2.5 WM tRNAPhe , 1^200 Wg/

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ml PheRS from T. thermophilus or E. coli. The reaction was carried out at 40³C. The velocity of the esteri¢cation reaction was measured by the rate of [14 C]phenylalanine incorporation into tRNA. At the appropriate times aliquots were spotted onto FN-11 paper ¢lters impregnated with TCA. Then the ¢lters were extensively washed with ice-cold 5% TCA to remove free amino acid. TCA-insoluble radioactivity was measured by liquid scintillation counting. 2.3. Separation of bisphenylalanyl-tRNA from uncharged tRNA and monophenylalanyl-tRNA Bisphenylalanyl-tRNA was separated from tRNA and monophenylalanyl-tRNA by a chromatography on a column with C18 reversed-phase sorbent (LiChrosorb RP-18, Merck) coated with methyltrioctylammonium chloride (Adogen 464, Serva). The sorbent was prepared according to [10]. Solution containing 0.5^2.5 nmol of aminoacylated tRNAPhe was mixed with 2.5 M sodium acetate (pH 5.0) to achieve a ¢nal pH value of 5^5.5 and loaded onto the 49U5 mm column. The elution was performed with a linear gradient of bu¡er B (6 M ammonium acetate, 10 mM MgCl2 , 1 mM Na2 EDTA, pH 5.7) in bu¡er A (0.5 M ammonium acetate, 10 mM MgCl2 , 1 mM Na2 EDTA, pH 4.5) with a £ow rate of 0.5 ml/ min. All chromatographies were run on a Pharmacia FPLC system at room temperature. It should be mentioned that the last step of puri¢cation of all tRNAPhe s was the chromatography on the LiChrosorb RP-18/Adogen 464 column. Therefore the retention of admixtures corresponds to that of uncharged tRNAPhe and they do not interfere with peaks of both species of aminoacylated tRNA. 2.4. Determination of protein in chromatographic fractions after separation of components of the aminoacylation reaction mixture on the LiChrosorb RP-18/Adogen 464 column Chromatographic fractions containing bisphenylalanyl-tRNA were collected and concentrated with the use of Ultrafree-MC 100000 NMWL Filter Units (Sigma) from 3 ml to a ¢nal volume of 5^10 Wl, then diluted with 50 mM potassium phosphate (pH 8.0) to 300 Wl and concentrated again. The last procedure was repeated twice. Finally the volume of solution

3

was adjusted with 20 mM potassium phosphate (pH 8.0) to 50 Wl and the presence of protein was checked after addition of Bradford's reagent (1 ml) according to [12]. Control solutions containing 12^250 Wg of T. thermophilus PheRS in an appropriate mixture of bu¡ers A and B were subjected to the same procedure. Absorbance at 595 nm of the samples was measured on spectrophotometer SF-46 against `blank' solution (50 Wl of 20 mM potassium phosphate (pH 8.0) mixed with 1 ml of Bradford's reagent). 2.5. Preparation of 3 H-labelled tRNAPhe (A) The 3P-terminal nucleotide of T. thermophilus tRNAPhe (I) was removed by the successive treatment with sodium periodate, aniline and alkaline phosphatase according to [13]. The native 3P-end was restored in a reaction mixture containing 60 WM [8-3 H]ATP, 10 mM MgCl2 , 50 mM TrisHCl (pH 9.0), 0.5 mM Na2 EDTA, 15 WM truncated tRNAPhe and 40 Wg/ml CTP (ATP):tRNA nucleotidyltransferase. The reaction was stopped after 1 h incubation at 37³C by addition of sodium acetate (pH 5.0) to a ¢nal concentration of 0.3 M. The enzyme was removed by phenol extraction. tRNA was separated from low-molecular-weight components of the reaction mixture by gel ¢ltration on a TSK-Gel Toyopearl HW-40F column equilibrated with 50 mM sodium acetate (pH 5.0). The labelled tRNAPhe prepared by this way was designated [A763 H]tRNAPhe (r). Finally [A76-3 H]tRNAPhe (r) was preparatively aminoacylated under standard conditions with E. coli PheRS and phenylalanyl-tRNA was separated from non-aminoacylated tRNA material on a LiChrosorb RP-18/Adogen 464 column. After complete deacylation followed by two successive ethanol precipitations from 0.1 M sodium acetate (pH 5.0) the phenylalanine acceptance of [A763 H]tRNAPhe (r) was 1750 pmol/A260 unit. (B) tRNAPhe with labelled A76 was also prepared by CTP (ATP):tRNA nucleotidyltransferase-catalyzed pyrophosphate exchange reaction in the presence of [8-3 H]ATP. The reaction was carried out at 37³C in a mixture containing 18 WM [8-3 H]ATP, 0.4 mM ATP, 20 mM MgCl2 , 20 mM Tris-HCl (pH 8.1), 1 mM Na2 P2 O7 , 15 WM T. thermophilus tRNAPhe (I) and 60 Wg/ml CTP (ATP):tRNA nucle-

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otidyltransferase. The reaction was stopped after 6 h incubation by addition of sodium acetate (pH 5.0) to a ¢nal concentration of 0.3 M. The enzyme was removed by phenol extraction. tRNA was puri¢ed on a LiChrosorb RP-18/Adogen 464 column under the conditions described above. This type of labelled tRNA, designated [A76-3 H]tRNAPhe (ex), had a charging capacity of 1120 pmol/A260 unit. The e¤ciency of tRNA labelling was estimated after analytical separation of [A76-3 H]tRNAPhe (ex) aminoacylated with non-radioactive phenylalanine from uncharged tRNA on a LiChrosorb RP-18/Adogen 464 column. 14% of native tRNA molecules were found to bear [3 H]adenosine on the 3P-end. 2.6. Hydrolysis of aminoacylated tRNA Non-enzymatic deacylation of aminoacylated tRNA was performed in a solution containing 2.5^ 3 WM mono- or bisphenylalanyl-tRNA, 50 mM HEPPS-NaOH (pH 9.0 at 20³C) and 9 mM MgCl2 . The reaction was initiated by the addition of the bu¡er preincubated at 40³C to dry precipitate of aminoacylated tRNA. Deacylation rate was measured from the time dependence of the residual amount of [14 C]phenylalanine coupled with tRNA. Enzyme-assisted deacylation of bisphenylalanyltRNA was monitored as follows: a dry pellet of bis[14 C]phenylalanyl-tRNA was rapidly mixed with a mixture containing 5 mM ATP, 9 mM MgCl2 , 50 mM HEPPS-NaOH (pH 9.0 at 20³C), 1 mM non-radioactive phenylalanine and 50 Wg/ml T. thermophilus PheRS at 40³C. The non-radioactive phenylalanine was added to prevent back-incorporation of [14 C]phenylalanine into tRNA after deacylation. The rate of [14 C]phenylalanine release from TCA-insoluble material is conditioned by both enzymatic and non-enzymatic deacylation of overcharged tRNAPhe . 2.7. Isolation of total tRNA from T. thermophilus under acidic conditions T. thermophilus HB8 cells were cultured at 72³C for 7 h in a medium described by Oshima and Baba [14]. The cells were harvested at the mid-log phase by centrifugation at 2³C and resuspended in a solution containing 20 mM sodium acetate (pH

5.0), 5 mM MgCl2 , 10% sucrose, 0.3 M KCl and 1 mg/ml lysozyme. All subsequent steps were carried out at 8³C. The cell suspension was gently stirred for 30 min and Nonidet P-40 and DNase were added to ¢nal concentrations of 1.6% and 0.01 mg/ml, respectively. After 1 h stirring DNase action was stopped by the addition of 10 mM Na2 EDTA and insoluble material was removed by centrifugation. The supernatant was applied to a DEAE-cellulose column and tRNA was eluted with a linear gradient of NaCl from 0 to 1 M in 0.1 M sodium acetate (pH 5.0). tRNA-containing fractions were collected, tRNA was precipitated by ethanol and dried. We checked that bisphenylalanyl-tRNA was stable during all these procedures by subjecting the control solution of this compound to the same treatments. The presence of tRNAPhe or its aminoacylated forms in chromatographic fractions was established by synthetase-catalyzed [14 C] or [3 H]phenylalanine incorporation into tRNA under standard aminoacylation conditions. The lability of the ester bond between the amino acid and tRNA at pH 9 permitted us to introduce the labelled amino acid in tRNA esteri¢ed primarily with non-radioactive phenylalanine, i.e. to reaminoacylate tRNA. The aliquots from each fraction were taken out, added to the standard aminoacylation mixture with 0.02 mg/ml of T. thermophilus PheRS and after 20 min incubation at 40³C the amount of labelled phenylalanine incorporated into tRNA was measured. 3. Results The anomalous behavior of T. thermophilus PheRS can be clearly demonstrated by the following experiment (Fig. 1). The tRNAPhe aminoacylation is carried out at ¢rst with E. coli PheRS and the reaction reaches a plateau when each tRNAPhe molecule bears one phenylalanyl residue. Then the thermophilic synthetase is added to the reaction mixture. Further incorporation of extra [14 C]phenylalanine into TCAinsoluble material interpreted as bisphenylalanyltRNA synthesis [15] results in a new plateau corresponding to 1.4^1.8 mol phenylalanine bound per mol tRNA. This e¡ect is speci¢c for T. thermophilus PheRS because its E. coli counterpart was not observed to overcharge tRNA in a broad range of ex-

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

perimental conditions (for example, the plateau of tRNA aminoacylation reaction did not change upon variation of the enzyme concentration from 2 to 200 Wg/ml). Therefore we often used the E. coli enzyme in control experiments and for determination of true charging capacity of di¡erent tRNAPhe s. The successive tRNAPhe aminoacylation by the two synthetases in one tube makes it possible to estimate accurately the quantity of extra phenylalanine bound to tRNA by thermophilic PheRS even for low amounts of tRNAPhe with unknown charging capacity. It is easy also to monitor the process of tRNA overcharging in its pure state, severed from the normal synthesis of monophenylalanyl-tRNA. When tRNA aminoacylation is carried out in the presence of only T. thermophilus PheRS, the same plateau is achieved as in the case of tRNA charging by the mixture of T. thermophilus and E. coli PheRSs. Thus, the ¢nal yield of bisphenylalanyltRNA synthesized by the thermophilic enzyme does not depend on the presence or absence of the E. coli synthetase in the same reaction mixture. It should be mentioned that T. thermophilus and E. coli PheRSs can aminoacylate with similar e¤ciency

5

both T. thermophilus and E. coli tRNAPhe . All peculiarities of tRNA aminoacylation observed when T. thermophilus tRNAPhe was used as a substrate were also valid for E. coli tRNAPhe . The mode of tRNA charging by each synthetase did not depend on whether T. thermophilus or E. coli tRNAPhe was used. Therefore we describe below only experiments performed with T. thermophilus tRNAPhe (I `isoacceptor') except when the structural di¡erence between the tRNAPhe s was important. To isolate bisphenylalanyl-tRNA from the reaction mixture a procedure of its separation from free tRNA and monophenylalanyl-tRNA has been developed. It is well known that some tRNAs being esteri¢ed with amino acid interact with hydrophobic sorbents more strongly than uncharged ones [16]. We supposed that bisphenylalanyl-tRNAPhe , monophenylalanyl-tRNAPhe and free tRNAPhe could be resolved in the same way. Chromatography on the mixed-mode ionic-hydrophobic matrix designated LiChrosorb RP-18/Adogen 464 (C18 resin covered by methyltrioctylammonium chloride) was used for this purpose. Two aliquots were taken from the reaction mixture at di¡erent times; one of them contained tRNAPhe aminoacylated by E. coli PheRS only, the other one contained tRNAPhe charged in the presence of both E. coli and T. thermophilus enzymes (Fig. 2). Both these samples were chromatographed on the LiChrosorb RP-18/Adogen 464 column. Low-molecularweight components of the reaction mixture (ATP, AMP, PPi , [14 C]Phe) were slightly retained and washed o¡ as a large peak by isocratic elution with pure bu¡er A. The following small satellite peak corresponded to the same set of compounds (as estimated by TLC analysis). Perhaps its appearance was caused by accelerated coming o¡ the tail of the major 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 the optical density pro¢le corresponded to that of uncharged tRNAPhe , this material seems to be an admixture presenting in the initial tRNAPhe preparation (see Section 2). The following peak containing [14 C]phenylalanine was identi¢ed as monophenylalanyl-tRNA according to the ratio 14 C radioactivity/A260 . These two peaks were present on the optical density pro¢les of both chromatographies. However,

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Fig. 2. Chromatographic analysis of the aminoacylation reaction mixtures containing normally charged and overcharged T. thermophilus tRNAPhe . (a) Successive tRNA aminoacylation with 46 Wg/ml E. coli PheRS and 67 Wg/ml T. thermophilus PheRS. The reaction was initiated by E. coli enzyme and after 21 min of tRNA charging the T. thermophilus enzyme was added, the dilution of the reaction mixture being equal to 0.5% of the ¢nal volume. The reaction mixture contained 1.4 WM tRNAPhe , 50 mM HEPPS-NaOH (pH 9.0), 5 mM ATP, 9 mM MgCl2 and 20 WM [14 C]Phe. At the times indicated by the arrows aliquots were withdrawn for chromatographic analysis. (b) Chromatographic resolution of the aliquot with tRNA charged with only E. coli PheRS. The aliquot containing 1 nmol of [14 C]phenylalanylated tRNA was mixed with an equal volume of bu¡er A, applied onto the LiChrosorb RP-18/Adogen 464 column and chromatographed as described in Section 2. Fractions of 0.5 ml were collected and assayed for 14 C radioactivity. (c) Chromatographic resolution of the aliquot with tRNA charged with the mixture of E. coli and T. thermophilus PheRSs. The aliquot containing 1 nmol of [14 C]phenylalanylated tRNA was resolved as in (b).

C

the resolution of tRNAPhe aminoacylated by the mixture of E. coli and T. thermophilus synthetases resulted in the appearance of an additional peak eluted after monoaminoacyl-tRNA (Fig. 2c). The UV spectrum of this compound was identical to that of tRNA. The ratio of 14 C radioactivity to absorbance at 260 nm was estimated to correspond to 1.85 mol Phe per mol tRNA. The compound can be precipitated by the addition of ethanol or TCA. Thus, the phenomenon of the anomalously high plateau in the T. thermophilus PheRS-catalyzed tRNA aminoacylation can be explained by the enzymatic synthesis of this unusual product, supposed to be bisphenylalanyl-tRNA. However, the ¢nal yield of bisphenylalanyl-tRNA calculated from the aminoacylation kinetic (67% of aminoacylated tRNAPhe ) shows signi¢cant discrepancy with its percentage evaluated from the chromatographic data (41% of aminoacylated tRNAPhe ). To establish the cause of this divergence both species of aminoacylated tRNAPhe were collected separately and rechromatographed under the same conditions. Monophenylalanyl-tRNA was found to be stable during this procedure. At the same time approximately 40% of bisphenylalanyl-tRNA loaded onto the column was decomposed yielding monophenylalanyl-tRNA and free amino acid. Taking this observation into account we should multiply the quantity of bisphenyl-

alanyl-tRNA found after elution from the column by a factor 1.67 to obtain its true content in the reaction mixture. Such low stability of this compound set us testing an alternative hypothesis about its nature. T. thermophilus PheRS is supposed to be a functional dimer [6], i.e. to have two identical active sites. The compound interpreted as bisphenylalanyl-tRNA might in fact be a stable complex consisting of one molecule of the enzyme, one molecule of phenylalanyl-tRNA and one molecule of phenylalanine (or phenylalanyladenylate). The UV spectrum of such a complex would be determined almost completely by the tRNA moiety, all other components would make

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7

only a minor contribution to its absorbance at 260 nm. At the same time the ratio of [14 C]phenylalanine to tRNA would be 2:1. This model satisfactorily explains all experimental data presented above as well as the bisphenylalanyl-tRNA concept. To check the assumption the compound containing more than 1 mol Phe per mol tRNA was tested for the presence of the protein. The products of T. thermophilus PheRS-catalyzed tRNA aminoacylation were separated on the LiChrosorb RP-18/Adogen 464 column (Fig. 3). To measure accurately the tRNA amount in the chromatographic fractions we used a tritium-labelled tRNAPhe ([A763 H]tRNAPhe (ex)) obtained by CTP (ATP):tRNA nucleotidyltransferase-promoted exchange reaction of the tRNA 3P-terminal nucleotide. The expected quantity of protein in the last eluted compound was calculated from the ratio 1 mol of enzyme per mol of [A76-3 H]tRNAPhe (ex) (or 1 mol of enzyme per 2 mol of [14 C]phenylalanine) in the hypothetical complex. It should be approximately 180 Wg. To concentrate the pooled fractions and to change acetate bu¡er with potassium phosphate, ultra¢ltration through Ultrafree-MC 100000 NMWL membrane micro¢lter was used. The control mixtures containing

Fig. 4. AMP/PPi inhibition of tRNAPhe overcharging. 2.1 WM T. thermophilus tRNAPhe was aminoacylated at 40³C for 15 min with 50 Wg/ml E. coli PheRS (F). Then the reaction mixture was divided into three equal portions and tRNA charging was continued after addition of T. thermophilus PheRS to a concentration of 80 Wg/ml alone (b) or together with AMP and PPi (to ¢nal concentrations of 100 WM) (a). As a control AMP and PPi (to ¢nal concentrations of 100 WM) (S) were added to the third portion to check their in£uence on the E. coli enzymecontrolled plateau. The reaction mixture contained 50 mM Tris-HCl (pH 9.0), 5 mM ATP, 8 mM MgCl2 and 20 WM [14 C]Phe. The dilution of the reaction mixtures upon the enzyme additions did not exceed 2% of the ¢nal volume. The amount of [14 C]Phe bound to tRNA was determined by ¢lter assay.

Fig. 3. Characterization of the anomalous product of tRNAPhe aminoacylation. 0.46 WM tRNAPhe was aminoacylated at 40³C for 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 [14 C]Phe. The reaction was stopped by addition of an equal volume of bu¡er A, the mixture was applied on the LiChrosorb RP-18/Adogen 464 column and chromatographed as described in Section 2. Fractions of 0.4 ml were collected and [14 C]Phe and [A76-3 H]tRNAPhe (ex) amounts were established by separate counting of 14 C and 3 H radioactivity, respectively. The last eluted compound was tested for the presence of protein by Bradford's technique as described in Section 2.

12^250 Wg of T. thermophilus PheRS were subjected to the same procedure. The lower limit of the method is approximately 1 Wg of protein per sample and the enzyme in the control solutions was revealed reliably in the whole range of concentrations. However, the material collected after chromatography did not contain any detectable amount of protein, therefore the assumption about the existence of a stable enzymec phenylalanyl-tRNAcphenylalanine (or phenylalanyladenylate) complex was recognized to be inadequate. The ¢nal choice was made in favor of the bisphenylalanyl-tRNA concept. We had serious reasons to suppose that the attachment of the second phenylalanine molecule to tRNA proceeds via phenylalanyladenylate formation. Both rate and yield of bisphenylalanyl-tRNA synthesis are reduced after the addition of AMP and PPi to the reaction mixture (Fig. 4). The e¡ect can be interpreted as an inhibition of tRNA overcharging at the amino acid activation step by the reaction products.

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V.G. Stepanov et al. / Biochimica et Biophysica Acta 1386 (1998) 1^15 Table 1 The 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 Tricine-HCl Diethanolamine-HCl Tris-HCl Tris-HCl Tris-HCl MOPS-NaOH

9.0 9.0 9.0 9.0 8.5 8.1 7.0

1.51 þ 0.09 1.32 þ 0.08 1.26 þ 0.09 1.21 þ 0.07 1.40 þ 0.05 1.25 þ 0.06 1.04 þ 0.05

The reaction was carried out at 40³C in a mixture containing 100 Wg/ml PheRS, 1.3 WM tRNAPhe , 5 mM ATP, 9 mM MgCl2 and 10 WM [14 C]Phe. a Values were normalized to the level of tRNAPhe charging by E. coli PheRS, which was determined to be identical in di¡erent bu¡ers (HEPPS-NaOH (pH 9.0), Tricine-HCl (pH 9.0), TrisHCl (pH 9.0)). Fig. 5. Dependence of the aminoacylation level on the concentrations of T. thermophilus phenylalanyl-tRNA synthetase and tRNAPhe in the reaction mixture. The normalized values of tRNA charging level (mol Phe/mol tRNA) are presented in boxes. Each value averages eight plateau points. The control level of charging was measured at 57 Wg/ml E. coli PheRS. The reaction was carried out at 40³C. The mixtures contained 50 mM HEPPS-NaOH (pH 9.0), 5 mM ATP, 9 mM MgCl2 and 20 WM [14 C]Phe. The time course of tRNA aminoacylation was monitored by ¢lter assay.

To de¢ne optimal conditions for bisphenylalanyltRNA synthesis by the thermophilic synthetase we investigated the dependence of tRNA phenylalanylation level on the concentrations of enzyme and tRNAPhe (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 is probably caused by a change of the balance between

enzyme-catalyzed tRNA aminoacylation and non-enzymatic hydrolysis of the charged tRNA. The higher tRNA content in the reaction mixture can also provoke the stronger product inhibition at the late stage of tRNA aminoacylation. Our interest in the e¡ect of bu¡er on bisphenylalanyl-tRNA yield was caused by the observation of reproducible degradation of the aminoacylation plateau in Tris-containing reaction mixtures (see, for example, Fig. 4 and Fig. 7). The plateau decrease was found to be not due to synthetase inactivation, tRNAPhe damage, ATP expenditure or AMP/PPi inhibitory e¡ect. The amino acid was observed to form a covalent adduct with Tris. Thus, tRNA aminoacylation slows down owing to the decrease of phenylalanine concentration in the mixture. The reaction takes place in the presence of E. coli PheRS as well

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

thermophilus tRNAPhe (I) thermophilus tRNAPhe (II) coli tRNAPhe coli tRNAPhe transcript

Aminoacylation levela (mol Phe/mol tRNA) by chromatographic analysis

by ¢lter assay

1.84 1.82 ^ 1.35

1.84 ^ 1.59 ^

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 [14 C]Phe. a For each tRNA the value was normalized to the level of charging by E. coli PheRS, measured under the same conditions.

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Fig. 6. Aminoacylation of [A76-3 H]tRNAPhe (r) with T. thermophilus phenylalanyl-tRNA synthetase. (a) 2.2 WM [A763 H]tRNAPhe (r) was aminoacylated with 100 Wg/ml T. thermophilus PheRS at 40³C. The reaction mixture contained 50 mM Tris-HCl (pH 9.0), 5 mM ATP, 9 mM MgCl2 and 25 WM [14 C]Phe. 15 min after initiation, when the reaction reached a plateau, an aliquot with 2.5 nmol of aminoacylated tRNA was taken out, mixed with an equal volume of bu¡er A and applied on the LiChrosorb RP-18/Adogen 464 column. The separation was done as described in Section 2. Fractions of 0.46 ml were collected and [14 C]Phe and [A76-3 H]tRNAPhe (r) amounts were established by separate counting of 14 C and 3 H radioactivity, respectively. (b) The mixture of 1.45 WM native T. thermophilus tRNAPhe and 0.26 WM [A76-3 H]tRNAPhe (r) was aminoacylated with 100 Wg/ml T. thermophilus PheRS at 40³C. The conditions of aminoacylation and chromatographic separation were the same as in (a).

as in the case of the thermophilic enzyme, but it can be detected only at high enzyme concentrations (data not shown; this reaction is now under investigation). The transfer of phenylalanyl residues on tRNAPhe may compete with the phenylalanine attachment to a Tris molecule. The aminoacylation plateau is stable when pH is maintained by HEPPS, Tricine or diethanolamine bu¡ers. The highest level of tRNAPhe charging by T. thermophilus PheRS was achieved when HEPPS was used for the reaction mixture bu¡ering (see Table 1). T. thermophilus PheRS can use E. coli, yeast and human tRNAPhe s as substrates [17]. The enzyme also aminoacylates unmodi¢ed tRNAPhe transcript with high e¤ciency [11]. This synthetase seems to have

9

Fig. 7. Time course of bisphenylalanyl-tRNA synthesis. (a) The extra [14 C]Phe was attached to mono[14 C]phenylalanyl-tRNA obtained in situ with 50 Wg/ml E. coli PheRS. The reaction of tRNA overcharging was initiated by addition of T. thermophilus PheRS to the concentration 83 (E), 96 (b) and 152 (O) Wg/ml. The aminoacylation assays were performed at 40³C using ¢lter technique. The reaction mixtures contained 1.8 WM tRNAPhe , 50 mM Tris-HCl (pH 9.0), 5 mM ATP, 8 mM MgCl2 and 25 WM [14 C]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 TrisHCl (pH 9.0), 5 mM ATP, 9 mM MgCl2 and 100 WM [14 C]Phe. At the appropriate times aliquots were taken out from the reaction mixture and analyzed on the LiChrosorb RP-18/Adogen 464 column as described in Section 2. The amounts of monoand bisphenylalanyl-tRNA were calculated from the total 14 C radioactivity in the corresponding peaks. The experimental values were corrected taking into account the deacylation of 40% of bisphenylalanyl-tRNA to monophenylalanyl-tRNA during the chromatography.

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very limited structural requirements for proper interaction with tRNAPhe . Therefore it is not surprising that the enzyme has been found to be capable of overcharging E. coli tRNAPhe and unmodi¢ed E. coli tRNAPhe transcript (Table 2). However, the aminoacylation of T. thermophilus [A76-3 H]tRNAPhe (r) (obtained by successive periodate oxidation, basepromoted 3P-terminal adenosine removal, phosphatase treatment and 3P-end repairing by CTP (ATP):tRNA nucleotidyltransferase in the presence of [3 H]ATP) resulted in monophenylalanyl-tRNA formation only. The synthesis of bisphenylalanyl[A76-3 H]tRNAPhe (r) was strongly impaired (Fig. 6a). To prove that the e¡ect is caused by the tRNA treatment but not by some inhibitory impurity in the [A76-3 H]tRNAPhe (r) preparation, a mixture of [A76-3 H]tRNAPhe (r) and native (untreated) T. thermophilus tRNAPhe with a molar ratio of 1:5.6 was aminoacylated (Fig. 6b). The thermophilic enzyme gives native tRNAPhe a pronounced preference over [A76-3 H]tRNAPhe (r) as follows from the ratio 3 H radioactivity/A260 in the peaks corresponding to

Fig. 8. Deacylation of [14 C]phenylalanylated tRNAPhe at 40³C. (W) Deacylation of bis[14 C]phenylalanyl-tRNA in 0.1 M sodium acetate (pH 6.2). (a) Non-enzymatic deacylation of mono[14 C]phenylalanyl-tRNA at pH 9.0. (O) Non-enzymatic deacylation of bis[14 C]phenylalanyl-tRNA at pH 9.0. (E) Enzyme-assisted deacylation of bis[14 C]phenylalanyl-tRNA.

monophenylalanyl-tRNA and bisphenylalanyltRNA. PheRS attaches only one phenylalanyl residue to [A76-3 H]tRNAPhe (r) but native tRNAPhe is overcharged in the same reaction mixture. Therefore the procedure of the removal and the restoration of 3P-terminal nucleotide was concluded to be accompanied by some changes in tRNA structure. These changes do not prevent monophenylalanyl-tRNA synthesis but strongly suppress the transfer of the second phenylalanyl residue to tRNA. In fact, the most signi¢cant modi¢cations of tRNAPhe are the oxidation of thiouridine at position 8 and a partial removal of 5P-terminal phosphate. It is doubtful that these two fragments participate directly in bisphenylalanyl-tRNA formation as acceptors of the extra phenylalanyl residue. Most probably, they are involved in the maintenance of the correct orientation of the extra phenylalanine-accepting group toward the amino acid activation site of the enzyme. The rate of bisphenylalanyl-tRNA synthesis was evaluated by two di¡erent ways. Firstly, we measured the initial rate of extra phenylalanine attachment to tRNAPhe preaminoacylated by E. coli PheRS (Fig. 7a). This approach is based on the assumption that the E. coli enzyme does not interfere at all with bisphenylalanyl-tRNA formation. Another way to measure separately the rates of mono- and bisphenylalanyl-tRNA synthesis includes the chromatographic resolution of aliquots withdrawn from the reaction mixture at di¡erent times and the calculation of mono- and bisphenylalanyl-tRNA quantities from the optical density pro¢les or 14 C label distributions, taking into account the bisphenylalanyl-tRNA instability during chromatography (Fig. 7b). The values of initial rates of bisphenylalanyltRNA synthesis at 40³C, obtained by both methods, were found to be quite close: 4.4 nmol/mg/min according to the ¢rst one and 5.5 nmol/mg/min according to the second one. The initial rate of monophenylalanyl-tRNA synthesis was determined by the measurement of the amount of [14 C]phenylalanine bound to tRNAPhe before the tRNA charging level exceeded 50%, i.e. when bisphenylalanyl-tRNA production was negligible. It was found to be equal to 240 nmol/mg/min under the same reaction conditions. Thus, the ¢rst amino acyl residue is transferred to tRNA 44^54 times faster than the second one. One of the characteristic properties of aminoacyl-

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tRNA synthetases is their high speci¢city toward the cognate amino acids. In spite of its unusual catalytic behavior T. thermophilus PheRS also possesses high selectivity during tRNA aminoacylation. We failed to detect incorporation of L-[14 C]tyrosine, L[14 C]leucine, L-[14 C]isoleucine and L-[14 C]valine into tRNAPhe at 40³C and 100 Wg/ml of enzyme (data not shown). We have investigated extensively the stability of bisphenylalanyl-tRNA under di¡erent conditions to elucidate the nature of the bond between the extra phenylalanyl residue and tRNA. It is well known that the aminoacyl-tRNA ester bond is labile at pH greater than 6 and the rate of amino acid release is strongly dependent on hydroxide ion concentration. In the presence of aminoacyl-tRNA synthetase the aminoacyl-tRNA deacylation proceeds faster, but it is unclear whether this enzyme-catalyzed hydrolysis has some biological signi¢cance [18]. Bisphenylalanyl-tRNA was found to be stable at pH 5^6. The rate of base-induced phenylalanine release from bisphenylalanyl-tRNA at pH 9 is slightly greater than that from monophenylalanyl-tRNA (Fig. 8). Taking into account the similar stability of mono- and bisphenylalanyl-tRNA in alkaline solution, the bond between the extra phenylalanyl residue and tRNA was supposed to be covalent but quite labile. In the presence of T. thermophilus PheRS the overcharged tRNA is deacylated more intensively. The only radioactive product of bisphenylalanyl-tRNA deacylation is free phenylalanine (according to HPLC and TLC identi¢cation tests). To establish whether bisphenylalanyl-tRNA is synthesized in vivo or not we studied the tRNAPhe aminoacylation products isolated from T. thermophilus living cells. The total tRNA pool was isolated under acidic conditions to avoid hydrolysis of the bond between the amino acid and tRNA. This material was resolved on the LiChrosorb RP-18/Adogen 464 column. The aliquots from each fraction were then subjected to reaminoacylation in the presence of labelled phenylalanine (see Section 2). Three tRNAPhe containing peaks were detected (Fig. 9a). The main di¤culty in identi¢cation of free tRNAPhe and its aminoacylated forms is the existence of two so-called isoacceptors of T. thermophilus tRNAPhe , tRNAPhe (I) and tRNAPhe (II) (two distinct products of tRNAPhe posttranscriptional mod-

11

Fig. 9. Identi¢cation of tRNAPhe aminoacylation products formed in T. thermophilus living cells. The chromatographies were done on the LiChrosorb RP-18/Adogen 464 column as described in Section 2. Fractionation of total tRNA isolated from T. thermophilus under acidic conditions. The presence of tRNAPhe or its aminoacylated forms in 100 Wl aliquots from each fraction was estimated by synthetase-catalyzed [14 C]phenylalanine incorporation into tRNA as described in Section 2. The peaks are numbered according to the order of elution. Chromatography of material from peak 3 (indicated by black bars in a) subjected to complete base-catalyzed deacylation. tRNA detection was as in previous case but [3 H]phenylalanine was used for tRNA aminoacylation. Separation of mono[14 C]phenylalanyl-tRNAPhe (I) and bis[14 C]phenylalanyl-tRNAPhe (I). The arrow shows the elution volume for uncharged tRNAPhe (I). Separation of mono[14 C]phenylalanyltRNAPhe (II) and bis[14 C]phenylalanyl-tRNAPhe (II). The arrow shows the elution volume for uncharged tRNAPhe (II).

i¢cation at position 37 [19]), having a di¡erent chromatographic behavior (Fig. 9c,d). Therefore, the ¢rst peak is tRNAPhe (I) and the second one is the mixture of tRNAPhe (II) and monophenylalanyltRNAPhe (I). The third peak might be identi¢ed as bisphenylalanyl-tRNAPhe (I) but monophenylalanyl-

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tRNAPhe (II) has the same chromatographic mobility. To determine the nature of tRNA in the third peak we subjected this material to complete basecatalyzed deacylation (pH 9.0, 40³C, 60 min) and rechromatography (Fig. 9b). Only tRNAPhe (II) was found. Therefore, the third peak corresponds to monophenylalanyl-tRNAPhe (II) and the total tRNAPhe pool from T. thermophilus does not contain a detectable amount of bisphenylalanyl-tRNA. The calculated threshold of bisphenylalanyl-tRNA registration was equal to 50 pmol (approx. 0.3% of the total amount of phenylalanine-accepting tRNA isolated from the cells). Thus, we should conclude that the content of bisphenylalanyl-tRNA in the pool of aminoacylated tRNAPhe formed under normal growth of T. thermophilus culture could not exceed this value. 4. Discussion In our previous studies on T. thermophilus PheRS the tRNAPhe aminoacylation reaction catalyzed by this enzyme was shown to result in an anomalously high plateau corresponding to more than one amino acyl residue attachment to the tRNA molecule [5,15]. The e¡ect has been observed at synthetase concentrations of 5^50 Wg/ml in a temperature range of 25^ 80³C. We have explained this phenomenon by the speci¢c ability of the thermophilic enzyme to form bisphenylalanyl-tRNA. We have suggested that these two phenylalanyl residues are bound to 2P- and 3Phydroxyl groups of the 3P-terminal adenosine. The assumption was based on the fact that one of the [14 C]phenylalanine-containing compounds appearing after RNase A hydrolysis of overcharged tRNA had a chromatographic mobility close to that of chemically synthesized bis-2P,3P-O-phenylalanyladenosine. However, this coincidence might be accidental and could not be considered a ¢nal proof of bisphenylalanyl-tRNA structure. Moreover, even the existence of bisphenylalanyl-tRNA remained hypothetical because we had no method to isolate it from an aminoacylation reaction mixture separately from monophenylalanyl-tRNA. Therefore the pure bisphenylalanyl-tRNA was not characterized and we could not exclude another explanation of the observed anomaly.

The data presented in this paper allow us to conclude that bisphenylalanyl-tRNA really exists as one of the products of T. thermophilus PheRS-catalyzed tRNAPhe aminoacylation. We have succeeded in separating this compound from other components of the reaction mixture and a precise ratio of Phe:tRNA close to 2:1 has been estimated. However, the site of the extra phenylalanyl residue attachment to tRNAPhe is still unknown. The fact that tRNAPhe transcript can be overcharged speaks against the necessity of tRNA posttranscriptional modi¢cation for the second amino acid to be accepted. Taking into account the observed lability of bisphenylalanyltRNA we suppose that the ribose hydroxyl groups are the most likely candidates to be esteri¢ed with the second amino acid. The extra phenylalanine may be transferred to one of the 2P-hydroxyl groups of nucleotides 1^75. Moreover, although the thermophilic synthetase can acylate only the 2P-hydroxyl group of adenosine-76 [15], the spontaneous 2PC3P migration of the aminoacyl residue followed by the repeated aminoacylation of the liberated 2P-hydroxyl group might result in the placement of both phenylalanines on the neighboring hydroxyl groups of tRNA 3P-terminal ribose. The absence of detectable [14 C]phenylalanine incorporation into a tRNAPhe analog with 2P-deoxyadenosine instead of natural adenosine at position 76 described in our previous paper [15] cannot be a decisive argument against aminoacylation of 2P-hydroxyl groups of nucleotides 1^75. Really, this tRNA analog was obtained by successive periodate, aniline and alkaline phosphatase treatments followed by a CTP (ATP):tRNA nucleotidyltransferase-catalyzed 3P-terminus reconstruction in the presence of 2P-dATP. However, the procedure was found to reduce drastically the ability of tRNAPhe to accept the extra amino acid even if the tRNA 3P-end is restored to the natural CCA sequence. Perhaps, we have failed to detect overcharging of another tested tRNAPhe analog bearing on its 3P-end an adenosine with C2P-C3P opened ribose cycle (tRNAoxÿred ) for the same reason. The most probable T. thermophilus tRNAPhe damage caused by the procedures of 3P-terminal nucleotide removal or modi¢cation is the periodate-induced oxidation of 4-thiouridine at position 8 to uridine sulfonate [20]. Furthermore, a partial tRNA 5P-dephosphorylation takes place under phos-

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13

Fig. 10. Comparison of phenylalanyl-tRNA synthetase primary sequences relating to the amino acid activation domain (K subunits) or mimicking it (L subunits). Conserved residues in each sequence 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 (mitochondrial); S. ce. (cyt), Saccharomyces cerevisiae (cytoplasmic); M. ca., Mycoplasma capricolum; B. su., Bacillus subtilis; E. co., Escherichia coli; T. th., Thermus thermophilus. Sequence alignments are given essentially according to [22,31,32] with modi¢cations. The known sequence of the L subunit of Saccharomyces cerevisiae PheRS is not considered here because it shows a little homology to the bacterial L subunits [32].

6

phatase treatment. The in£uence of these structural changes on the extra phenylalanine incorporation may be mediated by an enzyme-tRNA interaction:

the bisphenylalanyl-tRNA synthesis slows down owing to unfavorable mutual orientation of synthetase and damaged tRNA in catalytic complex. We can draw only preliminary conclusions about the mechanism of bisphenylalanyl-tRNA synthesis on the base of data presented. It is obvious that the ¢rst phenylalanine is transferred to the 2P-hydroxyl group of adenosine-76 resulting in monophenylalanyl-tRNA formation. An attachment of the second amino acid to tRNA proceeds approximately 50 times more slowly. Therefore bisphenylalanyl-tRNA synthesis becomes detectable long after complete tRNA conversion to monophenylalanyl-tRNA is ¢nished. We cannot say whether these two acts of amino acid transfer to tRNA are strictly coordinated in time or are independent but proceed with very di¡erent rates. The second amino acid is supposed to be activated through aminoacyladenylate formation like the ¢rst one. The amino acid selection for tRNA aminoacylation is under the same severe control as in the case of other PheRSs: the thermophilic synthetase cannot transfer to tRNAPhe amino acids other than phenylalanine. The enzyme seems to be able to bind reversibly bisphenylalanyl-tRNA and to destabilize selectively the bond between the extra phenylalanyl residue and tRNA as follows from the higher lability of bisphenylalanyl-tRNA in the presence of the enzyme. Abnormal T. thermophilus PheRS functioning points to a singularity of its structural organization and made us look for the principal di¡erence between the thermophilic enzyme and PheRSs from other sources. The T. thermophilus PheRS molecule consists of two small (K) subunits (Mr 39 000, 350 amino acid residues) and two large (L) subunits

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(Mr 86 500, 785 amino acid residues), the KL heterodimer being topological and, probably, a functional unit of this tetrameric enzyme [21]. Identi¢cation of the amino acid activation domain within the K subunit fragment 101^327 is based on the location of three signature sequence motifs [22] and a speci¢c spatial fold [21,23] characteristic for active site domains of class II aminoacyl-tRNA synthetases. tRNAPhe was found to contact generally di¡erent parts of the L subunit [24]. Anticodon recognition is carried out by the C-terminal domain of the L subunit, interactions with the K subunit amino acid activation domain have been observed only for adenosine-76. The impression is evoked that the K subunit mainly ensures an amino acid activation and the L subunit recognizes tRNAPhe and poses it properly upon binding, i.e. both subunits of the KL dimer are functionally specialized. However, the L subunit has been found to contain a structural module with a spatial fold typical of the amino acid activation domain of class II synthetases. Moreover, this so-called catalytic-like module (residues 483^678) demonstrates a sequence homology to the K subunit, highly conserved motifs presumably forming the amino acid activation site (Fig. 10). Such a homology between K and L subunits is observed only for T. thermophilus PheRS but not for other bacterial PheRSs with known primary structure. It would be very attractive to explain the unique catalytic property of the thermophilic enzyme by the existence of two separate amino acid activation sites on KL functional unit. However, is the `catalytic-like module' really capable of aminoacyladenylate formation? Its structural di¡erence from a true active site domain is still signi¢cant and this explanation of bisphenylalanyl-tRNA synthesis remains very hypothetical. On the other hand, the opinion that T. thermophilus PheRS has only two amino acid activation domains per K2 L2 tetramer [23] is not based on direct experimental data obtained for this enzyme. The only reason for this statement is the analogy with E. coli and yeast PheRSs [25^30]. It is still unclear whether this phenomenon plays any role in metabolism of the thermophilic bacteria. Bisphenylalanyl-tRNA has not been observed in T. thermophilus growing cells. However, the compound may be tightly associated with insoluble cellular fragments or decompose under cell disruption proce-

dures. On the other hand, monophenylalanyl-tRNA being a substrate for bisphenylalanyl-tRNA synthesis is constantly consumed by the ribosomal machinery and this competition may be the reason for the absence of bisphenylalanyl-tRNA during normal growth of thermophilic cells. The favorable conditions for bisphenylalanyl-tRNA formation appear when monophenylalanyl-tRNA production exceeds its consumption (for example, when mRNA translation on the ribosome is blocked or PheRS is produced in excess, etc.). In this case bisphenylalanyltRNA might play a role of a signal compound formed in response to the disbalances in the system of peptide synthesis. However, we cannot exclude that bisphenylalanyl-tRNA is an arti¢cial product of tRNA aminoacylation reaction and its formation takes place only in vitro under conditions far from natural. The peculiarities of the cellular organization of extremely thermophilic bacteria allow them to exist at high temperature when the growth of other prokaryotes is suppressed. Up to now, the main e¡orts have been generally concentrated on the investigation of thermal stability of biopolymers isolated from thermophilic organisms. However, active life is possible only under coordinated functioning of all cell components. Therefore, the biochemical processes occurring in the cells of extremely thermophilic bacteria must also be adapted to a high temperature and their mechanisms can di¡er from the reaction pathways of mesophilic organisms. The unusual catalytic behavior of T. thermophilus PheRS may be a trace of such adaptation. Acknowledgements This study was supported by the Russian Foundation for Basic Research No. 96-04-50150 and by the High School Grant for Basic Research of the St. Petersburg University.

References [1] J. Bonnet, J.-P. Ebel, Eur. J. Biochem. 31 (1972) 335^344. [2] I. Svensson, Biochim. Biophys. Acta 167 (1968) 179^183. [3] D.J. Gusseck, Biochim. Biophys. Acta 477 (1977) 84^88.

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V.G. Stepanov et al. / Biochimica et Biophysica Acta 1386 (1998) 1^15 [4] F. Marashi, C.L. Harris, Arch. Biochem. Biophys. 182 (1977) 533^539. [5] V.G. Stepanov, N.A. Moor, V.N. Ankilova, O.I. Lavrik, FEBS Lett. 311 (1992) 192^194. [6] V.N. Ankilova, L.S. Reshetnikova, M.M. Chernaya, O.I. Lavrik, FEBS Lett. 227 (1988) 9^13. [7] V.N. Ankilova, O.I. Lavrik, S.N. Khodyreva, Prikl. Biokhim. Mikrobiol. 20 (1984) 208^216. [8] B.L. Alford, A.C. Chinault, S.O. Jolly, S.M. Hecht, Methods Enzymol. 59G (1979) 121^134. [9] K. Watanabe, T. Oshima, K. Iijima, Z. Yamaizumi, S. Nishimura, J. Biochem. 87 (1980) 1^13. [10] R. Bischo¡, L.W. McLaughlin, Anal. Biochem. 151 (1985) 526^533. [11] N.A. Moor, V.N. Ankilova, O.I. Lavrik, Eur. J. Biochem. 234 (1995) 897^902. [12] M.M. Bradford, Anal. Biochem. 72 (1976) 248^254. [13] J. Tal, M.P. Deutscher, U.Z. Littauer, Eur. J. Biochem. 28 (1972) 478^491. [14] T. Oshima, M. Baba, Biochem. Biophys. Res. Commun. 103 (1981) 156^160. [15] V.G. Stepanov, N.A. Moor, V.N. Ankilova, I.A. Vasil'eva, O.I. Lavrik, Mol. Biol. (Moscow) 29 (1995) 392^396. [16] R. Bischo¡, L.W. McLaughlin, in: K.M. Gooding, F.E. Regnier (Eds.), HPLC of Biological Macromolecules. Methods and Applications, Chromatographic Science Series, 51, Marcel Dekker, New York, 1990, pp. 641^668. [17] N. Moor, I. Nazarenko, V. Ankilova, S. Khodyreva, O. Lavrik, Biochimie 74 (1992) 353^356. [18] W. Freist, Biochemistry 28 (1989) 6787^6795.

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[19] U. Grawunder, A. Schon, M. Sprinzl, Nucleic Acids Res. 20 (1992) 137. [20] A. Favre, in: H. Morrison (Ed.), Bioorganic Photochemistry and the Nucleic Acids, Vol. 1, John Wiley and Sons, New York, 1990, pp. 380^425. [21] L. Mosyak, L. Reshetnikova, Y. Goldgur, M. Delarue, M.G. Safro, Nature Struct. Biol. 2 (1995) 537^547. [22] R. Kreutzer, V. Kruft, E. Bobkova, O. Lavrik, M. Sprinzl, Nucleic Acids Res. 20 (1992) 4173^4178. [23] L. Mosyak, M. Safro, Biochimie 75 (1993) 1091^1098. [24] Y. Goldgur, L. Mosyak, L. Reshetnikova, V. Ankilova, O. Lavrik, S. Khodyreva, M. Safro, Structure 5 (1997) 59^68. [25] S. Khodyreva, N. Moor, V. Ankilova, O. Lavrik, Biochim. Biophys. Acta 830 (1985) 206^212. [26] P. Bartmann, T. Hanke, E. Holler, J. Biol. Chem. 250 (1975) 7668^7674. [27] P. Dessen, A. Ducruix, C. Hountondji, R.P. May, S. Blanquet, Biochemistry 22 (1983) 281^284. [28] F. Fasiolo, J.-P. Ebel, M. Lazdunski, Eur. J. Biochem. 73 (1977) 7^15. [29] M. Baltzinger, S.X. Lin, P. Remy, Biochemistry 22 (1983) 675^681. [30] J.F. Lefevre, R. Ehrlich, P. Remy, Eur. J. Biochem. 103 (1980) 155^159. [31] T.J. Koerner, A.M. Myers, S. Lee, A. Tsaglo¡, J. Biol. Chem. 262 (1987) 3690^3696. [32] A.A. Brakhage, M. Wozny, H. Putzer, Biochimie 72 (1990) 725^734.

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