Báo cáo Y học: tRNA-dependent amino acid discrimination by yeast seryl-tRNA synthetase pdf

tRNA-dependent amino acid discrimination by yeast seryl-tRNA synthetase Ita Gruic-Sovulj 1,2 , Irena Landeka 1,2 , Dieter So¨ll 3 and Ivana Weygand-Durasevic 1,2 1 Department of Chemistry, Faculty of Science, University of Zagreb, Croatia; 2 Rudjer Boskovic Institute, Zagreb, Croatia; 3 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA The ability of aminoacyl-tRNA synthetases to distinguish betweensimilaraminoacidsiscrucialforaccuratetrans- lation of the genetic code. Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) employs tRNA-dependent recognition of its cognate amino acid serine [Lenhard, B., Filipic, S., Landeka, I., Skrtic, I., So ¨ ll, D. & Weygand- Durasevic, I. (1997) J. Biol. Chem. 272, 1136–1141]. Here we show that dimeric SerRS enzyme complexed with one molecule of tRNA Ser is more specific and more efficient in catalyzing seryl-adenylate formation than the apoenzyme alone. Sequence-specific tRNA–protein interactions enhance discrimination of the amino acid substrate by yeast SerRS and diminish the misactivation of the structurally similar noncognate threonine. This may proceed via a tRNA-induced conformational change in the enzyme’s act- ive site. The 3¢-terminal adenosine of tRNA Ser is not important in effecting the rearrangement of the serine binding site. Our results do not provide an indication for a readjustment of ATP binding in a tRNA-assisted manner. The stoichiometric analyses of the complexes between the enzyme and tRNA Ser revealed that two cognate tRNA molecules can be bound to dimeric SerRS, however, with very different affinities. Keywords:tRNA Ser ÆSerRS complexes; tRNA-dependent amino acid recognition; amino acid selection; tRNA bind- ing; covalent cross-linking. Accurate translation of genetic information is dependent on the high fidelity of several molecular processes. Different quality control mechanisms are adapted to prevent or correct naturally occurring mistakes [1]. Measurements of the selectivity of aminoacyl-tRNA synthetases in vitro indicate that the upper limit for error in the selection of correct amino acid for protein synthesis is in the range of 10 )4 )10 )5 . The frequency of errors involving noncognate tRNA aminoacylation is, in most cases, 10 )6 or lower [2]. While the selection of the correct tRNA is assumed to occur as a result of preferential reaction kinetics for the formation of cognate proteinÆRNA complexes, the differences of the side chains of amino acids are often sufficient to allow their specific binding [3]. The basic challenge in achieving high specificity for the amino acid substrates lies in the rejection of smaller substrates with similar side chain chemistry or isosteric amino acids [2]. A number of different pathways have been proposed to correct misactivation of amino acids in vivo. The noncognate aminoacyl-adenylate can be hydrolyzed by several class I synthetases by tRNA- dependent or tRNA-independent Ôpre-transferÕ editing, whereas in the Ôpost-transferÕ pathway a mischarged tRNA is rapidly deacylated in a synthetase-dependent manner [4–12]. The editing reactions of class II synthetases have been studied much less than those of class I. Class II phenylalanyl-tRNA synthetase (PheRS) specifically deacy- lates Ile–tRNA Phe [13], and alanyl-tRNA synthetase (AlaRS) has been shown to hydrolyze misactivated serine and glycine [14]. Escherichia coli lysyl-tRNA synthetase (LysRS) hydrolyzes misactivated homocysteine, homoser- ine, cysteine, threonine and alanine [15]. Recent reports on threonyl-(ThrRS) [16,17] and prolyl-tRNA synthetase (ProRS) [18–20] show clearly that these enzymes misactivate smaller noncognate amino acids, serine and alanine, respectively, and therefore require proofreading activity. However, during the editing process, some of the correct products are also destroyed [21]. This makes corrective pathways energetically costly, and they are disfavored in the cell. Alternatively, the quality of aminoacyl-tRNA synthesis in the cell can be improved by the tRNA-mediated mechanisms that enhance the accuracy of amino acid discrimination. These are based on conformational changes in the enzymes, induced by the formation of macromolec- ular complex [22–24] or possibly by the interaction with a nonsynthetase protein [25,26]. The active site of class II aaRSs contains the motif 2 loop which is involved in binding of ATP, an amino acid, and the acceptor end of tRNA. Our earlier investigation revealed that accurate seryl-tRNA synthesis in Saccharomyces cere- visiae is accomplished via tRNA-assisted optimization of amino acid binding to the enzyme active site [27]. To understand the mechanism by which this occurs, we have generated a number of SerRS mutants with altered motif 2 Correspondence to I. Weygand-Durasevic, Department of Chemistry, Faculty of Science, University of Zagreb, Strossmayerov trg 14, 10000 Zagreb, Croatia. Fax: + 385 1 4561177, Tel.: +385 1 4561197, E-mail: weygand@rudjer.irb.hr Abbreviations: aaRS, aminoacyl-tRNA synthetase with amino acid representingAla,Cys,Gln,Lys,Phe,Pro,SerandThr,thusfor alanyl- (EC 6.1.1.7), cysteinyl- (EC 6.1.1.16), glutaminyl- (EC 6.1.1.18), lysyl- (EC 6.1.1.6), phenylalanyl- (EC 6.1.1.20), prolyl- (EC 6.1.1.15), seryl- (EC 6.1.1.11) and threonyl-tRNA synthetase (EC 6.1.1.3); aaRSÆtRNA and aaRS–tRNA, noncovalent and covalent complexes between synthetase and tRNA, respectively. (Received 27 May 2002, revised 9 August 2002, accepted 9 September 2002) Eur. J. Biochem. 269, 5271–5279 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03241.x loop residues and showed that these mutations affect tRNA-dependent amino acid recognition, possibly by interfering with the flexibility of the motif 2 loop [27]. The same mode of conformational adjustment has been recently proposed for maize organellar SerRS [28]. To get better insight into contribution of tRNA in the amino acid selection, we have generated nonchargeable, 3¢-truncated tRNA analogs, which retain the ability of complex forma- tion with SerRS. These tRNAs significantly influence enzyme affinity for serine, as revealed by steady-state kinetic studies in the reaction of pyrophosphate exchange and decrease the misactivation of threonine by yeast seryl-tRNA synthetase. The stoichiometric analyses of the complexes between the enzyme and tRNA Ser revealed that two cognate tRNA molecules can be bound to dimeric SerRS, however, with very different affinities. The binding of the first tRNA Ser is sufficient for complete readjustment of serine binding site(s). EXPERIMENTAL PROCEDURES The overexpression and purification of S. cerevisiae wild- type and mutant (E281D, G291A) SerRS has been described [27]. [ 14 C]Serine (166.1 mCiÆmmol )1 )andtetra- sodium [ 32 P]pyrophosphate (2.44 CiÆmmol )1 )were purchased from DuPont NEN. The amino acids were from Sigma. The purity of threonine was checked by ESI-Ion Trap mass spectrometry. The signal at m/z value that corresponds to serine was not recorded. tRNAs Yeast tRNA Ser and tRNA Tyr , purified from total brewer’s yeast tRNA as described previously [29], accepted 1.2 nmol of serine and 1.4 nmol of tyrosine per A 260 unit of tRNA, respectively. tRNA integrity was checked by MALDI-TOF mass spectrometry. A mass resolution m/Dm ¼ 220 (for tRNA Tyr )and170(fortRNA Ser ) was achieved in the linear mode of operation [30]. Nonchargeable substrate analogs were prepared by stepwise modifications of yeast tRNA Ser at the 3¢-end. Intact tRNA Ser CCA was first denatured to enable better exposition of the 3¢-adenosine diol groups and then oxidized by periodate treatment essentially as described [31], except that the concentration of periodate was increased five-fold. As the oxidized product is known as synthetase inhibitor, tRNA Ser ox was extensively dialyzed prior to removal of 3¢-terminal nucleoside by b-elimination with lysine (pH 8.0), followed by alkaline phosphatase treatment [32]. The acceptor activity of truncated tRNA Ser CC was not detectable in the standard aminoacylation assay [27]. All tRNA substrates were carefully renatured prior the use in the kinetic assays and complex formation experiments by heating to 80 °C and the temperature was then allowed to decrease to 50 °C. Then, MgCl 2 was added, to a final concentration of 10 m M , and the tRNA sample was cooled to 30 °C. E. coli tRNA Ser 1 was purchased from Subriden RNA (Rolling Bay, WA, USA). Synthesis of seryl-adenylate The extent of adenylate formation was measured by ATP- PP i exchange at 30 °C [27]. Standard reaction mixtures contained 100 m M Hepes/KOH pH 7.2, 10 m M MgCl 2 , 0.5 m M [ 32 P]PP i (4–7 c.p.m.Æpmol )1 ), 10 m M KF. For the K m determination of serine the concentration varied from 50 l M to 900 l M , while ATP–MgCl 2 waskeptconstantat 2m M . The concentrations of wild-type and mutant enzymes were between 50 and 100 n M , and nonchargeable tRNAs were in the range of 100–500 n M .TheK m for ATP was determined in the standard reaction mixture with ATP– MgCl 2 varied from 0.4 l M to 50 l M , and serine was kept constant at 900 l M . Misactivation To study the misactivation of threonine by wild-type and mutated SerRS (141 n M ), 0.5 m M )50 m M threonine was substituted for serine in the standard reaction mixture for the pyrophosphate exchange. In the experiment designed to determine the ability of tRNA Ser CC to suppress threonine misactivation, SerRS and tRNA Ser CC were 100 n M . Inactivation of SerRS by oxidized tRNA Ser The inactivation of the SerRS pyrophosphate exchange activity by tRNA Ser ox was studied with 100 n M enzyme and tRNA Ser ox varied from 0 to 1500 n M . In order to preform the macromolecular complex before binding of small substrates, SerRS and tRNA Ser were preincubated under conditions favorable for noncovalent complex formation (5 min, 30 °C) and the activation reaction was started by addition of serine and ATP in the standard reaction mixture for pyrophosphate exchange. Gel mobility shift assay SerRSÆtRNA complexes were prepared by incubation of the enzyme with variable amounts of freshly renatured tRNA, for 5 min at 30 °C, in buffer containing 30 m M Hepes/KOH pH 7.0 (or Mes/KOH pH 6.0), 10 m M MgCl 2 and 1.6% glycerol, followed by cooling on ice. Glycerol was added to a final concentration of 7.5% and the preformed complexes were subjected to electrophoresis on 6% acrylamide/ bisacrylamide (40 : 1) gel containing 5% glycerol in elec- trophoresis buffer (25 m M Tris, 25 m M acetic acid, 10 m M magnesium acetate; pH 7.2, or 25 m M Mes/KOH, 10 m M magnesium acetate, pH 6.0). Electrophoresis was carried out at 4 °C for 3–4 h at 100 V, and the gels ware stained by standard silver staining procedure. Covalent SerRS–tRNA Ser complexes Cross-linking reactions were carried out essentially accord- ing to [33]. The reaction mixture contained 20 m M Hepes pH8.5, 10m M MgCl 2 , 1.7% (v/v) glycerol. SerRS was 0.65 l M ,andtRNA Ser ox /SerRS ratio was varied between 0.5 and 3.0. SerRS and freshly renatured tRNA Ser ox were incubated for 5 min at 30 °C to allow noncovalent complex formation and the cross-linking reaction was started with the addition of 1 lLNaCNBH 3 (0.5 m M ). The addition of NaCNBH 3 was repeated after 20 and 40 min. The final amount of NaCNBH 3 added was 1.5 nmol. The reaction was stopped after 60 min by the addition of 0.2 volumes of 4.8 m M NaBH 4 in 10 m M NaOH. After 10 min glycerol was added to a final concentration of 8%. The whole reaction mixture was loaded on the 6% nondenaturing 5272 I. Gruic-Sovulj et al. (Eur. J. Biochem. 269) Ó FEBS 2002 acrylamide/bisacrylamide gel (40 : 1) and the electrophor- esis took place at 4 °C(3h,100 V)with50m M Tris, 25 m M boric acid and 10 m M magnesium acetate, pH 8.0, as running buffer. Stoichiometric analysis Ferguson plot analysis on disc-PAGE [34] was used for mass determination of covalent SerRS–tRNA Ser ox complexes. Preformed covalent complexes between SerRS and tRNA Ser ox were separated on a series of gels containing 5, 6, 7, 8, 9 and 10% acrylamide/bisacrylamide (29 : 1) in 375 m M Tris, 5 m M MgCl 2 ; pH 8.0 alongside 1–10 lgof native protein molecular weight standards (Sigma). Stack- ing gel was 4% acryamide/bisacrylamide (40 : 1) in 62 m M Tris, 5 m M MgCl 2 pH 6.3. Electrophoresis was performed at 4 °C, 100 V with 50 m M Tris, 5 m M MgCl 2 pH8.0as anode buffer and 10 m M Tris, 90 m M glutamine pH 8.0 as cathode buffer. For each species, 100 [log(100l)] was determined and plotted against gel concentration, where l is equal to the mobility of the species relative to that of the bromophenol blue tracking dye. Logarithm of negative slope or retardation coefficient (K r ) was then plotted as a function of log molecular mass (kDa) for each protein standard, and from the evaluated retardation coefficients of covalent SerRS–tRNA Ser ox complexes their molecular mass was determined. RESULTS Complex formation with modified cognate and nonmodified heterologous tRNAs The integrity of the truncated tRNA Ser CC was first checked by high-resolution polyacrylamide gel electrophoresis (Fig. 1). Modified tRNA Ser CC appeared intact and its migration, with respect to mature tRNA Ser CCA , was in satisfying agreement with the removal of one nucleotide. Additional bands of lower intensity, visible in both lanes, are probably due to the presence of other tRNA Ser isoacceptors of different length. In order to determine the ability of modified tRNAs to participate in complex formation with the cognate synthe- tase, preformed complexes of SerRS with either tRNA Ser CCA , tRNA Ser ox or tRNA Ser CC were analyzed by the gel mobility shift assay (Fig. 2). Under the conditions described in the Experimental procedures, native, oxidized and truncated tRNA Ser species form complexes of equal mobility (lanes 1–3, respectively). Although the input ratio of enzyme to tRNA was the same (1 : 2) in all three cases, the shifted band that corresponds to the enzymeÆtRNA complex is somewhat weaker in lane 3. This may suggest that the removal of 3¢-terminal nucleotide slightly influences the complex stability. The oxidation of 3¢-terminal adenosine in tRNA Ser does not interfere with complex formation and its stability, in agreement with previously shown results for the tyrosyl–tRNA synthetase system [31]. When SerRS was mixed with an excess amount of E. coli tRNA Ser and electrophoresis performed as above (Fig. 2, lane 4), the gel mobility shift analysis revealed a lack of low mobility bands. This indicates that heterologous complex was either not formed or its low stability did not allow detection under the conditions used. This is an interesting finding, as E. coli tRNA Ser contains a conserved long extra arm which is considered to be the main recognition element for all cytosolic SerRS enzymes [35]. Furthermore, this tRNA is recognized by yeast SerRS in vivo and in vitro [36], showing that some recognition elements in tRNA are shared between bacteria and yeast. Nonchargeable yeast tRNA Ser affects serine activation by SerRS Kinetic parameters for serine were determined in pyrophos- phate exchange reaction, catalyzed by yeast SerRS (Table 1). Under standard conditions in the absence of tRNA, kinetic parameters are in a very good agreement with previously reported data [27]. The addition of modified Fig. 1. Preparation of 3¢-truncated tRNA Ser . The size difference between intact tRNA Ser CCA and truncated tRNA Ser CC was confirmed by gel electrophoresis (left). The reaction scheme is shown on the right. Fig. 2. Gel mobility shift assay of SerRS complexes with different tRNAs. SerRS (9 pmol) was incubated with 18 pmol of tRNA Ser CCA , tRNA Ser ox ,tRNA Ser CC ,andE. coli tRNA Ser CCA and subjected to poly- acrylamide gel under native conditions (lanes 1, 2, 3, and 4, respect- ively). Noncomplexed tRNA Ser (5 pmol) was loaded onto the gel as electrophoretic mobility marker (lane 5). Full line arrow, noncom- plexed tRNA; dashed line arrow, SerRSÆtRNA complexes. The bands migrating between noncomplexed tRNAs and SerRSÆtRNA com- plexes could be assigned to tRNA oligomers and they also appear in tRNA lane when a higher amount is loaded (see Fig. 6A, lane 1). This additional tRNA band appeared when complex formation and elec- trophoresis were performed at pH 7.0 and above. Ó FEBS 2002 tRNA-dependent amino acid discrimination (Eur. J. Biochem. 269) 5273 yeast tRNA Ser species, which cannot be charged, influences the kinetics with respect to serine. tRNA Ser ox decreases the K m for serine twofold. The reduction of k cat may reflect the interference between the tRNA’s modified 3¢-terminal adenosine with other substrates in the active site. In the presence of tRNA Ser CC , which lacks the 3¢-end nucleotide, the binding of serine is significantly enhanced, as revealed by a K m value decreased by almost an order of magnitude. Moreover, the K m value of 6.0 · 10 )5 M is very close to that obtained in the aminoacylation reaction with wild-type SerRS (6.3 · 10 )5 M ) [27]. An order of magnitude increased affinity toward serine in the presence of truncated tRNA Ser CC suggests that the 3¢-terminal adenosine of tRNA Ser is not important in effecting the rearrangement of the serine binding site. It is worth noting that an equimolar concen- tration of tRNA per dimeric enzyme is capable of inducing a complete rearrangement of amino acid binding sites, as judged by full range change of the K m value for serine. This suggests that a 2 Æ(tRNA Ser ) 1 is a functional complex in seryl- tRNA formation. The effect of 3¢-truncated tRNA Ser on the activation of serine by the mutated yeast SerRS, bearing two amino acid changes in the motif 2 loop of the active site (E281D; G291A), is much less pronounced. The effect of heterologous tRNA Ser on serine binding Despite the observation that E. coli tRNA Ser 1 neither is charged well by yeast enzyme [36], nor forms a stable noncovalent complex with yeast SerRS (Fig. 2, lane 4), it affects serine binding properties of yeast synthetase. In the presence of E. coli tRNA Ser 1 ,theK m for serine is decreased two-fold and the velocity of seryl-adenylate synthesis is elevated 2.5-fold (Table 1). Thus, interaction of yeast SerRS with heterologous tRNA Ser , that contains the long extra arm identity element, improves the overall catalytic prop- erties of the RNAÆsynthetase complex in the pyrophosphate exchange reaction. Accordingly, even though the binding of tRNA Ser analogs slightly decreased the k cat value, which may be due to the interference of 3¢-CCA ox and 3¢-CC ends with the accessibility of the active site, an observed increase in k cat in the presence of E. coli tRNA suggests that the tRNAÆSerRS noncovalent complex is catalytically more efficient in seryl-tRNA formation than noncomplexed SerRS. On the other hand, the increase in k cat may be consistent with reduced stability of the heterologous tRNAÆSerRS complex and consequently facilitates substrate turnover, as previously observed in other systems [23,37]. It is also important to note that bacterial tRNA Ser does not fully optimize the serine binding site of yeast SerRS, as the K m for serine never reaches the value of 6.5 · 10 )5 M , characteristic for aminoacylation of yeast tRNA Ser . Never- theless, heterologous tRNA Ser ÆSerRS interactions are suffi- cient to promote conformational change of SerRS, leading to increased k cat /K m value. Thus, tRNA optimized amino acid activation can occur irrespectively of aminoacylation, as shown previously for GlnRS [37]. This finding can be explained by the fact that discrimination between cognate and noncognate tRNA occurs predominantly during the transfer reaction [24]. Binding of ATP to yeast SerRS is not mediated by tRNA The analysis of kinetic parameters for ATP in seryl- adenylate formation did not reveal considerable differences between the K m values for ATP in the presence and absence of tRNAs Ser (Table 1). Furthermore, the nature of non- chargeable or heterologous tRNA Ser did not affect the ATP binding properties of yeast SerRS. Thus, contrary to the observation based on crystallographic studies of Thermus thermophilus SerRS complexes [38], our biochemical experi- ments do not provide indications for tRNA-assisted rear- rangement of ATP binding site. We have previously observed a 6.3-fold decrease in the apparent affinity of ATP for the yeast enzyme in the aminoacylation reaction compared to the PP i exchange [27]. This could be explained by possible interference of correctly positioned Ser–tRNA Ser with ATP binding. If so, the transfer of acyl moiety to tRNA decreases the affinity of SerRS toward ATP. Misactivation of threonine by wild-type and mutant SerRS Slight misactivation of threonine by wild-type yeast SerRS (about 0.4%, based on the comparison of turnover numbers for cognate and noncognate amino acid) has been observed in the pyrophosphate exchange reaction, in which 0.5– 50 m M threonine was substituted for serine (Fig. 3). Table 1. The influence of nonchargeable tRNAs Ser and heterologous tRNAs on seryl-adenylate synthesis by yeast SerRS. Kinetic parameters were determined in the PP i exchange reaction. Numbers in parenthesis denote the concentration of tRNA. The enzymes were at a concentration of 100 n M . K m for ATP (l M ) K m for Ser (l M ) k cat (s )1 ) k cat /K m, Ser (sÆl M ) )1 SerRS wt Without tRNA 14 500 3.9 8 · 10 )3 With tRNA Ser ox (500 n M ) 13 250 0.94 4 · 10 )3 With tRNA Ser CC (100 n M ) 16 70 4.9 70 · 10 )3 With tRNA Ser CC 20 60 1.8 30 · 10 )3 With tRNA Ser CCA E. coli (100 n M ) ND 260 10 39 · 10 )3 With tRNA Ser CCA E. coli (500 n M ) 9 300 10 33 · 10 )3 SerRS (E281D; G291A) Without tRNA 40 850 0.056 6.59 · 10 )5 With tRNA Ser CC (100 n M ) ND 490 0.030 6.12 · 10 )5 With tRNA Ser CC (500 n M ) ND 350 0.028 8.00 · 10 )5 5274 I. Gruic-Sovulj et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Although threonine concentration used in vitro corresponds to approximately 1–100 · K m for serine, these data may be relevant in vivo, as the average amino acid concentrations in yeast cells are in the millimolar range [39]. Glycine and alanine showed no detectable activation in PP i exchange assay. Mutated SerRS, bearing the alterations at two amino acid positions in the motif 2 loop of the active site (E281D; G291A) exhibits more pronounced misactivation with threonine (Fig. 3, inset). This was expected, as the kinetic analysis of the aminoacylation and amino acid activation reactions revealed that substitution of the class II invariant glycine has dramatic effect on seryl-adenylate formation, resulting in severe reduction of catalytic efficiency [27]. The other altered amino acid, E281, is conserved in the active site of seryl-tRNA synthetases and also has an important functional role in yeast SerRS [27]. Based on our experi- mental data, amino acid substitutions in the active site of mutant SerRS (E281D; G291A) decrease the ability of the synthetase to discriminate against noncognate substrates. The addition of tRNA Ser CC decreases the misactivation by yeast SerRS Figure 3 compares the extent of threonyl-adenylate synthe- sis by yeast SerRS with and without tRNA addition. The decreased level of misactivation observed in the presence of cognate, but nonchargeable tRNA, could be due to hydrolytic editing as described for several other aaRSs [16,18,40,41] or the consequence of tRNA-optimized amino acid discrimination. For the reasons discussed below, we favor the latter hypothesis. Nevertheless, our results point out the importance of tRNAÆsynthetase complex formation in the accuracy of amino acid selection, which may contribute to fidelity of translation in vivo. Analysis of the complexes between SerRS and tRNA Ser Our kinetic experiments show that equimolar concentration of tRNA per dimeric enzyme induces a complete rearrange- ment of amino acid binding sites, as judged by full range change of the K m value for serine (Table 1). This is an indication that a 2 Æ(tRNA Ser ) 1 is a functional complex in seryl- tRNA formation. In order to determine whether SerRS can bind more than one cognate tRNA molecule under various conditions, several experimental approaches were employed. (a) Inactivation of SerRS by oxidized tRNA Ser .The dependence of the velocity of the pyrophosphate exchange reaction on the concentration of the tRNA Ser ox is shown in Fig. 4. The concentration of oxidized tRNA Ser was from 0 to 1500 n M , while the other substrates were held at constant concentration (2 m M ATP and 1 m M serine), in the standard pyrophosphate exchange reaction. The velocity decreased to 17% when the concentration of the tRNA Ser ox reached the value of about 1200 n M . As the active site titration performed without tRNA [42] showed that dimeric enzyme possesses two active sites at which the activation of serine occurs, almost total inactivation of the a 2 -dimeric SerRS with a nonchargeable tRNA Ser suggests that two tRNAs could be simultaneously bound per dimeric protein. (b) Gel mobility shift assay. SerRS forms only one type of complex which was sufficiently stable to be detected by the gel mobility shift assay, as shown in Fig. 5. The complex of the same electrophoretic mobility was detected with SerRS/tRNA Ser ratios 2 : 1, 1 : 1, 1 : 2, 1 : 3, 1 : 4 and 1 : 5 (Fig. 5, lanes 2–7, respectively). Uncomplexed tRNA Ser was subjected on the same gel as electrophoretic mobility marker (Fig. 5, lane 1). SerRS in complex with two bound tRNA Ser was not detected by the mobility shift assay despite the broad variation in pH and ionic strength. To test the specificity of the method, Fig. 3. tRNA Ser CC decreases the misactivation of threonine by yeast SerRS. Threonyl-adenylate formation was compared with and without tRNA Ser CC . Noncognate aminoacyl-adenylate formation (pmol amino acid-AMP per pmol SerRS per minute) was measured at different amino acid concentrations (0.5–50 m M , which corresponds approximately to 1–100 · K m for serine) in a standard pyro- phosphate exchange reaction. Glycine and alanine showed no detectable activation. The inset shows misactivation of threonine by wild-type and mutant SerRS (E281D; G291A). Fig. 4. Dependence of seryl-adenylate formation velocity on the con- centration of tRNA Ser ox . To facilitate formation of noncovalent complex, SerRS and tRNA Ser ox were incubated for 5 min at 30 °Cpriorto addition to the reaction mixtures for pyrophosphate exchange. Ó FEBS 2002 tRNA-dependent amino acid discrimination (Eur. J. Biochem. 269) 5275 SerRS was incubated with the noncognate yeast tRNA Tyr present either in the equimolar amount or in high molar excess (Fig. 5, lanes 8 and 9, respectively), followed by the electrophoresis on the native polyacrylamide gel. No band of retarded electrophoretic mobility relative to noncomplexed tRNA Tyr was detected. (c) Covalent cross-linking. Oxidized tRNA possesses 3¢-terminal dialdehyde groups able to form Schiff bases with lysine side chain amino groups. Considering the nucleophilicity of the lysine side chain amino groups, cross-linking experiments were performed in the pH range 8.0–9.0. In all cases, two complexes, presumably SerRS– tRNA Ser ox and SerRS–(tRNA Ser ox ) 2 were detected (data not shown). The largest amount of both complexes was obtained at pH 8.5. SerRS and tRNA Ser ox wereincubatedinratios2 : 1, 1 : 1, 1 : 2 and 1 : 3 (Fig. 6A, lanes 2, 3, 4 and 5, respectively) under conditions described in the Experimental procedures. A higher mobility band, appearing in lanes with SerRS/ tRNA Ser ox ratios 1 : 1–1 : 3 (Fig. 6A, lanes 3–5) could be assigned to SerRS–(tRNA Ser ox ) 2 . Uncomplexed SerRS and tRNA Ser ox were used as mobility markers (Fig. 6A, lanes 6 and 1, respectively). Our results indicate that Schiff bases formed between yeast SerRS and tRNA Ser ox were stable even without reduction, as approximately the same amount of both cross- linked products were formed both with and without reducing reagents (NaCNBH 3 and NaBH 4 ) (data not shown). On the contrary, Madore et al. [33] could not detect the GlnRS– (tRNA Gln ) 2 complex without treatment with NaCNBH 3 . Stoichiometric analysis Ferguson plot analysis [34] was performed in order to confirm predicted stoichiometry of detected covalent com- plexes between SerRS and tRNA Ser ox . Relative mobilities of the protein standards and two covalent complexes were determined on a series of nondenaturing discontinuous gels of increasing polyacrylamide concentration having constant acrylamide/bisacrylamide ratio and their logarithm was plotted as a function of the gel concentration (Fig. 6B). The negative slope of each line [representing the retardation coefficient (K r ) for species] was defined and log K r was plotted against log molecular mass (kDa) (Fig. 6C). Deter- mined K r values for covalent complexes were 9.2 (lower mobility band) and 10.9 (higher mobility band), corres- ponding to molecular masses of 136 kDa and 183 kDa., i.e. to one and two tRNA Ser bound to SerRS dimer, respectively (Fig. 6C). Molecular masses of SerRSÆtRNA Ser and SerR- SÆ(tRNA Ser ) 2 complexes obtained by MALDI-MS analysis were 136 kDa and 163 kDa, respectively [29], in very good agreement with those determined by native electrophoresis. Percentage of error, calculated as (estimated molecular mass – reported molecular mass/reported molecular mass) · 100, was in the range of 0 and 12%, i.e. the same as in previously published experiments performed by this method [43]. DISCUSSION tRNA-dependent amino acid discrimination as a possible quality control mechanism in seryl-tRNA formation An order of magnitude of increased affinity toward serine in the presence of nonchargeable 3¢-truncated tRNA Ser con- firms our previous finding that SerRS modulates its affinity for serine in a tRNA-dependent manner [27]. We have showninthispaperthatcognatetRNAÆSerRS interactions increase the stringency of amino acid discrimination. This may proceed via a tRNA-induced conformational change in the enzyme’s active site. In agreement with the observation of Cusack et al. [38] that the alteration of glycines may influence loop flexibility in T. thermophilus SerRS, our results revealed that the activation of serine by mutated yeast SerRS (E281D; G291A) was less significantly affected by 3¢-truncated tRNA Ser . This indicates that the flexibility of the motif 2 loop is important for structural readjustment of the amino acid binding site. Based on the structural and functional resemblance among seryl-tRNA synthetases [27], wesuggestthatinyeast,likeinT. thermophilus [44], docking interactions between the long variable arm of tRNA Ser and the N-terminal coiled coil of SerRS govern the positioning of the acceptor stem in the active site of the enzyme. Either these docking interactions or the interaction with the acceptor stem, induce conformational changes in the serine binding sites, which optimize the enzyme for seryl-adenylate formation. As a consequence, the misactivation of structur- ally similar noncognate amino acid is prevented. As recently shown for the aspartyl-system, hierarchical recognition of the synthetase and its cognate tRNA is crucial for formation of the active macromolecular complex [45]. In T. thermo- philus SerRS, serine specificity is guaranteed by two hydrogen bond interactions with the side chain hydroxyl group and by the size of the binding pocket [46]. Model building studies on this enzyme [46] revealed that binding of glycine and alanine would be unfavorable because of the absence of hydrogen bonding capacity, and many other amino acids would be too large. The hydroxyl group of structurally similar threonine also makes an important contribution to specificity of recognition by ThrRS [16,46]. Besides a protein side chain, a zinc ion serves as a specific recognition cofactor for the hydroxyl group of threonine in ThrRS. Although serine is weakly misactivated by ThrRS, the enzyme needs proofreading or editing activity to correct this misactivation. On the other hand, mechanistically analogous zinc ion-mediated amino acid discrimination by cysteinyl-tRNA synthetase (CysRS) fully assures specific Fig. 5. Gel mobility shift assay with different amounts of tRNA. SerRS (9 pmol) was incubated with 4.5, 9, 18, 27, 36 and 45 pmol of tRNA Ser andwith9and45pmolofyeasttRNA Tyr prior to electrophoresis on polyacrylamide gel under native conditions (lanes 2, 3, 4, 5, 6, 7 and 8, 9, respectively). Noncomplexed tRNA Ser was loaded on the gel as electrophoretic mobility marker (lane 1). Complex formation and electrophoresis were performed under pH 6.0. Full line arrow, non- complexed tRNA; dashed line arrow, SerRSÆtRNA complexes. 5276 I. Gruic-Sovulj et al. (Eur. J. Biochem. 269) Ó FEBS 2002 synthesis of cysteinyl-adenylate, without serine or threonine activation [47]. The discrimination against threonine by SerRS may rely partly on the fact that its methyl group would be in an unfavorable hydrophilic environment in the active site of enzyme [46]. However, this amino acid is still misactivated by yeast SerRS apoenzyme in vitro, especially when the enzyme is altered in the active site. The misacti- vation by wild-type enzyme is diminished in the presence of cognate tRNA. In the model, we suggested that such tRNA-optimized amino acid recognition may act as a quality control mechanism that lowers the extent of misactivation and thus editing would not be necessary. Another possibility, although much less likely, is that tRNA directs erroneous threonyl-adenylate from the active site to the center for editing. This needs to be further investigated. Thus far, the only experimental finding regarding the ability of SerRS to correct errors is related to pretransfer editing, which seems to be negligible with any amino acid tested, including threonine [2]. Additionally, SerRS displays AMP- and pyrophospate-independent deacylation of cog- nate aminoacyl-tRNA in the presence of thiols, which mimics editing of homocysteine [15]. Our experiments show that the 3¢-terminal adenosine of cognate tRNA Ser does not seem to be important in bringing about the rearrangement of the serine binding site. However, less specific noncovalent interactions between yeast enzyme and E. coli tRNA Ser induce less pronounced conformational change in the active site, revealing the importance of cognate complex formation for accurate amino acid recognition. Anticooperative binding of tRNA Ser The finding that a high molar surplus of yeast-oxidized tRNA Ser in a PP i exchange reaction deactivated more Fig. 6. Covalent complexes between SerRS and tRNA Ser ox . (A) Detection on the gel. SerRS (9 pmol) was incubated with 4.5, 9, 18 and 27 mol of tRNA Ser ox inthepresenceofNaCNBH 3 and subjected to polyacrylamide gel under native conditions (lanes 2, 3, 4, and 5, respectively). Non- complexed tRNA Ser ox (5 pmol) and SerRS (9 pmol) was loaded onto the gel as an electrophoretic mobility marker (lanes 1 and 6, respectively). Covalent cross-linking reactions were stopped by the addition of NaBH 4 . (B) Representative Ferguson analysis. A logarithmic function of relative mobility for each of the protein standards and two covalent complexes, SerRS–tRNA Ser ox and SerRS–(tRNA Ser ox ) 2 , was plotted against the poly- acrylamide concentration and fitted to the regression. Protein standards are indicated: (a) a-lactalbumin (molecular mass 14.2 kDa), (b and c) carbonic anhydrase (two charge isomers with molecular mass 29.0 kDa), (d) chicken egg albumin (molecular mass 45.0 kDa), (e and f) bovine serum albumin monomer (molecular mass 66 kDa) and dimer (molecular mass 132.0 kDa), respectively, (g and h) urease trimer (molecular mass 272.0 kDa) and hexamer (molecular mass 545.0 kDa), respectively. 1 : 1 and 1 : 2 denote SerRS–tRNA Ser ox and SerRS–(tRNA Ser ox ) 2 . R 2 values were in the range 0.9914–0.999 for all protein standards and both covalent complexes. (C) Representative plot of log K r vs. log of molecular mass (kDa). K r values for protein standards were derived by a Ferguson analysis and log K r was plotted as a function of log molecular mass (kDa). Protein standards (a–h) are as indicated in (B). Interpolations of log K r values derived for the SerRS–tRNA Ser ox and SerRS–(tRNA Ser ox ) 2 complexes (dashed lines) indicate molecular weights of 136 and 183 kDa, respectively. R 2 value was 0.993. Ó FEBS 2002 tRNA-dependent amino acid discrimination (Eur. J. Biochem. 269) 5277 than 80% of yeast SerRS activity led to the conclusion that two tRNA Ser ox molecules could be simultaneously bound to dimeric SerRS. This was confirmed by covalent cross-linking. However, SerRSÆ(tRNA Ser ) 2 complex was not stable enough to be detected by the gel mobility shift assay under the conditions of relatively broad pH and ionic strength ranges. This suggests that two tRNA Ser binding sites on yeast SerRS are nonequivalent, which is in agreement with previously reported studies of others which revealed that two binding sites for tRNA Ser differ by about two orders of magnitude in their binding constants [48,49]. Both, SerRSÆtRNA Ser and SerRSÆ(tRNA Ser ) 2 , complexes were also detected by MALDI-MS [29]. The specificity of the MALDI-MS measurements was confirmed in the competition assay. The SerRSÆ(tRNA Ser ) 2 complex was not detected on the control gel mobility shift assay conducted under the same conditions as the MALDI-MS measurements. This could be the consequence of increased electrostatic forces as the dielectric constant of the environ- ment is lowered during solvent evaporation and in vacuum. For prokaryotic systems, crystallographic studies showed that T. thermophilus SerRS form two types of noncovalent complexes with cognate tRNA Ser ;SerRSÆtRNA Ser and SerRSÆ(tRNA Ser ) 2 [44,50], while Escherichia coli SerRS forms only the SerRSÆ(tRNA Ser ) 2 complex [51]. Borel et al. [52] were able to detect both 1 : 1 and 1 : 2 E. coli SerRSÆ tRNA Ser complexes by zone interference gel electrophoreses and concluded that binding of the tRNA was positively cooperative. It is tempting to speculate as to why yeast SerRS binds two tRNA Ser molecules with nonequivalent affinity. Are these binding sites apriorinonequivalent, or does binding of the first tRNA leads to a conformational change which lessens the affinity towards tRNA at the second site? Results presented in this paper show undoubtedly that binding of the equimolar amount of tRNA Ser induces complete conformational optimization of the serine binding sites which results in efficient and accurate seryl-adenylate formation. The binding of one tRNA may promote structural rearrangement of SerRS which decreases the affinity for the second tRNA. ACKNOWLEDGMENTS We thank Sinisa Stipanicic for recording ESI-MS spectrum of the threonine sample and Tomislav Kamenski and Marko Mocibob for assistance in complex formation studies and Ferguson plot analysis. We are indebted to Professor Kucan for critical discussions. 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Borel, F., Vincent, C., Leberman, R. & Ha ¨ rtlein, M. (1994) Seryl- tRNA synthetase from Escherichia coli: implication of its N-terminal domain in aminoacylation activity and specificity. Nucleic Acids Res. 22, 2963–2969. Ó FEBS 2002 tRNA-dependent amino acid discrimination (Eur. J. Biochem. 269) 5279 . tRNA-dependent amino acid discrimination by yeast seryl-tRNA synthetase Ita Gruic-Sovulj 1,2 , Irena Landeka 1,2 , Dieter So¨ll 3 and Ivana Weygand-Durasevic 1,2 1 Department of Chemistry,. ability of aminoacyl-tRNA synthetases to distinguish betweensimilaraminoacidsiscrucialforaccuratetrans- lation of the genetic code. Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) employs tRNA-dependent recognition. aminoacyl-tRNA synthetase with amino acid representingAla,Cys,Gln,Lys,Phe,Pro,SerandThr,thusfor alanyl- (EC 6.1.1.7), cysteinyl- (EC 6.1.1.16), glutaminyl- (EC 6.1.1.18), lysyl- (EC 6.1.1.6), phenylalanyl-