tmRNA from Thermus thermophilus Interaction with alanyl-tRNA synthetase and elongation factor Tu Victor G. Stepanov and Jens Nyborg Institute of Molecular and Structural Biology, University of Aarhus, Denmark The interaction of a Thermus thermophilus tmRNA tran- script with alanyl-tRNA synthetase and elongation factor Tu has been studied. The synthetic tmRNA was found to be stable up to 70 °C. The thermal optimum of tmRNA alanylation was determined to be around 50 °C. At 50 °C, tmRNA transcript was aminoacylated by alanyl-tRNA synthetase with 5.9 times lower efficiency (k cat /K m ) than tRNA Ala , primarily because of the difference in turnover numbers (k cat ). Studies on EF-Tu protection of Ala$tmRNA against alkaline hydrolysis revealed the existence of at least two different binding sites for EF-Tu on charged tmRNA. The possible nature of these binding sites is discussed. Keywords:tmRNA;elevatedtemperatures;alanyl-tRNA synthetase; EF-Tu. The transfer-messenger RNA (tmRNA) is a small stable bacterial RNA that is an object of considerable interest because of its obvious structural and functional dualism. This molecule possesses both mRNA and tRNA activities and contains easily recognizable mRNA-like and tRNA- like modules [1]. The latter is formed by converging-3¢-and 5¢-termini of the 300–400 nucleotide-long chain. The main biological function of tmRNA is to relieve ribosomes that remain for a long time in complex with mRNA without elongating the polypeptide chain. Such a situation arises upon translation of truncated mRNA deprived of stop- codon, or intact mRNA with clustered rare codons. The intervention of tmRNA may also take place in the case when the ribosomes idle at the mRNA stop-codon awaiting proper termination of translation [2]. As a first step of the tmRNA-assisted ribosome rescue (called trans-translation), the aminoacylated tRNA-like module of the tmRNA binds to the A-site of stalled 70S ribosomes with peptidyl-tRNA in the P-site. The polypep- tide chain is transferred onto the 3¢-end of the tmRNA in the course of the transpeptidation reaction. Then the tRNA- like module, now carrying the polypeptide, moves into the ribosomal P-site. At the same time, the first codon of the mRNA-like part of the tmRNA enters the A-site and the reprogrammed ribosome resumes the polypeptide chain elongation by adding approximately 10 aminoacyl residues to the synthesized protein. When the stop-codon of the mRNA-like module is reached, the translation is terminated in the usual way [3]. Thus, the trans-translation results both in release of the arrested ribosome and in labelling the newly formed protein with a standard C-terminal peptide tag that serves as a signal for degradation by specific proteases. During its functioning, tmRNA interacts with a number of proteins. The identity determinants of the tRNA-like module of the tmRNA are equivalent to those of tRNA Ala , so that tmRNA can be charged with alanine by alanyl- tRNA synthetase [4,5]. EF-Tu*GTP has been shown to form a complex with alanylated tmRNA, in which the ester bond between the alanyl residue and the 3¢-terminal adenosine of tmRNA is protected against hydrolysis as in the canonical ternary complex between EF-Tu, GTP and aminoacyl-tRNA [5,6]. Two other proteins, S1 and SmpB, are indispensable for the proper interaction of the tmRNA with the ribosome. S1 binds near the mRNA-like module and probably assists the entrance of the tag-encoding tmRNA part into the ribosome [7]. SmpB can bind to the tRNA-mimicking domain simultaneously with EF-Tu and presumably stabilizes the active conformation of this tmRNA region [8]. The significant stimulative effect of SmpB on the efficiency of tmRNA aminoacylation [9] makes it likely that this protein is an integral part of a tmRNA-based ribosome rescue complex in vivo. In contrast, SmpB was found to inhibit the tRNA Ala aminoacylation reaction [8]. Some other proteins, RNase R, SAF and phosphoribosyl phosphorylase, were also observed to form tight complexes with tmRNA, but their roles and the location of their binding sites on tmRNA remain elusive [10]. Thus, it is evident that tmRNA performance on the ribosome requires the assistance of numerous protein cofactors. Aminoacylation of tmRNA is an absolute prerequisite of its activity in trans-translation [11]. However, tmRNA charging with alanyl-tRNA synthetase in vitro in the absence of other proteins was found to be slow and inefficient in comparison with tRNA alanylation. SmpB improves significantly the substrate properties of tmRNA and induces a rise of the plateau of the tmRNA alanylation reaction [5,8]. An addition of EF-Tu*GTP to the reaction Correspondence to J. Nyborg, Institute of Molecular and Structural Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark. Fax: + 45 8612 3178, Tel.: + 45 8942 5257, E-mail: jnb@imsb.au.dk Abbreviations: AlaRSase, alanyl-tRNA synthetase; GDPNP, guano- sine 5¢-(b,c-imidotriphosphate) or 5¢-guanylylimidodiphosphate. Enzymes:alanyl-tRNAsynthetase,EC6.1.1.7;EF-Tu,(EC3.6.1.48). (Received 4 October 2002, revised 18 November 2002, accepted 27 November 2002) Eur. J. Biochem. 270, 463–475 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03401.x mixture was reported to increase further both the rate and the yield of tmRNA alanylation [5]. The observed stimu- lative effects of SmpB and EF-Tu on tmRNA charging were interpreted in terms of the dynamic interplay of synthetase- catalysed aminoacylation of tmRNA and spontaneous deacylation of Ala$tmRNA. The balance between these two processes changes when the catalytic efficiency of tmRNA alanylation (k cat /K m ) becomes higher under the influence of SmpB, or when synthesized Ala$tmRNA is trapped in a complex with EF-Tu*GTP and thus stabilized. As a result, the plateau of the tmRNA aminoacylation reaction could be increased to the biologically relevant level in the presence of these proteins. The major part of the above-mentioned features of the trans-translation mechanism has been revealed in experi- ments with Escherichia coli tmRNA and proteins. Studies on tmRNAs from other sources have been sporadic and have addressed only a limited number of special issues. In the context of our studies on the translation apparatus of Thermus species, we aimed to investigate Ala$tmRNA synthesis with alanyl-tRNA synthetase and its binding to elongation factor Tu. Taking into account the increased lability of the alanyl ester bond at high temperatures [12], the thermophile should encounter (and somehow overcome) the intense spontaneous deacylation of the Ala$tmRNA. A hot environment may in this way imprint the character of the specific interactions between the macromolecules involved in trans-translation in T. thermophilus.Herewe describe assays on thermophilic tmRNA, alanyl-tRNA synthetase and EF-Tu, related to their activity in the trans- translation reaction at elevated temperatures. Materials and methods Chemicals, RNAs and proteins L -[2,3– 3 H]Alanine (42.0 CiÆmmol )1 ) was from Amersham Life Science, GDPNP (5¢-guanylylimidodiphosphate), GMP, GTP, ATP, UTP, CTP, Spermidine*3HCl and Spermine*4HCl were products of Sigma, all other chemicals were from Fluka and AppliChem. T. thermophilus tRNA Ala with an amino-acid acceptance of 860 pmol per D 260 unit was purified by successive chromatographies on Sepharose 4B, BND-cellulose and DEAE-Sephadex A-50 columns. Alanyl-tRNA synthetase from T. thermophilus HB8 (M w 195 kDa) with a specific activity of 105 nmolÆmin )1 Æmg )1 (40 °C) was obtained generally according to Lechler et al. [13]. T. aquaticus EF-Tu (M w 45 kDa) was overproduced in Escherichia coli SCS1 carrying plasmid pTacTU2 with the tufA gene and purified as described in [14]. T7 RNA polymerase was overproduced in E. coli BL21 carrying plasmid pAR1219 and purified as described in [15]. Calf liver alkaline phosphatase immobilized on agarose beads was from Sigma. All restriction enzymes, Taq DNA polymerase and T4 DNA ligase were from New England Biolabs. Construction of a recombinant plasmid harbouring the tmRNA gene The wild-type tmRNA gene, ssrA, was amplified from T. thermophilus HB8 genomic DNA by a Taq DNA polymerase-promoted polymerase chain reaction with the first primer 5¢-CgaattcTAATACGACTCACTATAGGG GGTGAAACGGTCTCG-3¢, containing the sense strand sequence of the tmRNA 5¢-end and the T7 promoter, and the second primer 5¢-CGTGAATTCATGCATGGTGGA GGTGGGGGGAG-3¢, containing the antisense strand sequence of the tmRNA-3¢-end and a NsiI restriction site (underlined). The obtained DNA fragment without any additional treatment was ligated to the linear pCR2.1 vector for TA cloning (Invitrogen). E. coli B843 (DE3) cells transformed with the resulting plasmid were plated onto Luria–Bertani plates with 75 lgÆmL )1 ampicillin and grown for 10 h at 37 °C. All colonies contained the plasmid with the ssrA-insert. The nucleotide sequence of the isolated recombinant plasmids was checked by the dideoxy method on both strands. In the obtained constructs, the ssrA-insert was found in two different orientations in relation to the body of the pCR2.1 vector. The variant designated pCR2.1- A1L3 (Fig. 1) was selected for further studies. Synthesis and purification of the tmRNA transcript The pCR2.1-A1L3 plasmid was isolated from 10 g of transformed E. coli cells. Prior to use, the plasmid was treatedwiththeNsiI restriction enzyme, so that the 423 bp DNA fragment containing the tmRNA-encoding sequence under the T7 promoter was cut out of the pCR2.1-A1L3 construct. The 3¢-overhangs of the obtained DNA duplexes were removed by treatment with E. coli exonuclease I. The 423-bp DNA fragment was separated from the rest of the plasmid by size-exclusion chromatography on Sephacryl S- 500 H (Pharmacia) and used as a template for T7 RNA polymerase-catalysed run-off transcription. The tmRNA synthesis was performed at 37 °C in a reaction mixture containing 40 m M Tris/HCl (pH 8.0), 26 m M MgCl 2 ,5m M dithiothreitol, 0.5 m M Spermine, 0.5 m M Spermidine, 0.01% (v/v) Triton X100, 4 m M ATP, 4 m M UTP, 8 m M GTP, 8 m M CTP, 30 m M GMP, 80 mgÆmL )1 PEG 8000, 75–100 lgÆmL )1 DNA template and 100 lgÆmL )1 T7 RNA polymerase. After 6 h of incubation the reaction mixture Fig. 1. Construction of the pCR2.1-A1L3 plasmid carrying the T. thermophilus tmRNA-encoding sequence (striped arrow) under the T7 promoter. Orientation of the T7 promoters is shown by triangles. 464 V. G. Stepanov and J. Nyborg (Eur. J. Biochem. 270) Ó FEBS 2003 was phenol extracted and RNA was purified by HPLC on a mixed-mode ionic-hydrophobic sorbent, methyltrioctylam- ine-coated LiChrosorb RP-18 matix [16], followed by preparative gel-electrophoresis in 7% polyacrylamide gel with 7.8 M urea. Usually the tmRNA transcript was annealed prior to use by quick heating to 80 °Cin50m M Hepes/NaOH (pH 7.6), 1 m M MgCl 2 , followed by slow cooling down to 20 °C. Aminoacylation assays Unless otherwise mentioned, the aminoacylation reaction mixture contained 2.5 m M ATP, 12 m M MgCl 2 ,50m M Hepes/NaOH (pH 7.6 at 20 °C), 15 l ML -[ 3 H]alanine, 0.5 m M Spermine, 0.05–2 l M chargeable RNA and 1– 10 lg/mL of alanyl-tRNA synthetase (referred to as standard aminoacylation conditions). The velocity of the aminoacylation was measured by the rate of the L -[ 3 H]ala- nine covalent attachment to RNA. At appropriate times aliquots were taken out of the reaction mixture by a lambda pipette and spotted onto Whatman 3MM paper filters impregnated with trichloroacetic acid. Then the filters were extensively washed with ice-cold 5% trichloroacetic acid to remove free amino acid. Trichloroacetic acid-insoluble radioactivity was measured by liquid scintillation counting. Structural analysis tmRNA melting curves were recorded in a Varian Cary 50 spectrophotometer equipped with a thermocontrolled cuvette holder. Measurements were per- formed in 50 m M Hepes/NaOH (pH 7.6), 1 m M MgCl 2 , 0.1 m M Na 2 -EDTA. The temperature was increased at a rate of 0.34 °CÆmin )1 in the range 18–90 °C. The experiment was performed in duplicate. Ala$tmRNA and Ala$tRNA deacylation protection assays The protective effect of EF-Tu against spontaneous hydro- lysis of the Ala$tmRNA or Ala$tRNA ester bond was studied upon quick dissolution of the dry pellet of purified [ 3 H]Ala$tmRNA or [ 3 H]Ala$tRNA in a EF-Tu*GDPNP- containing mixture, preincubated for 10 min at the appro- priate temperature. The EF-Tu*GDPNP complex was prepared as described in [14]. [ 3 H]Ala$tmRNA was synthesized under standard conditions, treated with phenol, separated from low-molecular-mass components of the reaction mixture by gel-filtration on Sephadex G-25 (Phar- macia) in 50 m M sodium acetate (pH 5.0), and from uncharged tmRNA by chromatography on acetylated DBAE–cellulose (Serva) at 4 °C [17]. Alanylated tmRNA was collected into a tube with ice-cold 0.5 M sodium acetate (pH 5.0), quantified by radioactivity, divided into appro- priate portions and precipitated with 3 vol. of ethanol. The pellet was dried using SpeedVac. Alanylated tRNA was passed through the same procedure. The deacylation reaction mixtures contained 0.38–4.52 l M EF-Tu*GDPNP, 35 n M [ 3 H]Ala$tmRNA or 16 n M [ 3 H]Ala$tRNA, 2.0 m M GDPNP, 90 m M Hepes/NaOH (pH 7.6), 10 m M MgCl 2 , 10 m M NH 4 Cl, 0.3 m M Spermine, 0.5 m M dithiothreitol, 0.25 m M Na 2 -EDTA. The time course of the [ 3 H]Ala$tmRNA and [ 3 H]Ala$tRNA hydrolysis was monitored by the filter technique. All kinetics of the decay reaction were characterized by 11 datapoint each. Lambda pipettes were used to take out aliquots from the reaction mixtures. In all cases, [ 3 H]Ala$tmRNA and [ 3 H]Ala$tRNA Ala decay could be described by pseudo- first order kinetics characterized by the corresponding apparent rate constant k app and by the initial deacylation rate k app [Ala$tmRNA] t ¼ 0 or k app [Ala$tRNA Ala ] t ¼ 0 . Gel mobility shift assays The standard mixture for mobility shift assays (10 lL) contained 3–12 l M EF-Tu*GDPNP, 1.0 D 260 units per mL of uncharged tmRNA transcript, 1.5–4.5 m M GDPNP, 100 m M Hepes/NaOH (pH 7.6), 10 m M MgCl 2 ,10m M NH 4 Cl, 0.3 m M Spermine, 0.5 m M dithiothreitol, 0.25 m M Na 2 -EDTA, 10% (v/v) glycerol. After 10 min of incubation at 30 °C the solution was kept on ice for another 10 min and then subjected to electrophoresis in nondenaturing 6% polyacrylamide gel, with 25 m M Tris-Borate (pH 8.3), 1.0 m M magnesium acetate as gel and running buffer, at room temperature and 12 VÆcm )1 for 2.5 h. The experi- ments were performed in duplicate for separate RNA and protein visualization. In order to visualize RNA only, the gels were stained with pyronin Y [18] with consecutive silver enhancement according to Blum et al. [19]. The location of EF-Tu bands on the gels was revealed by staining with Coomassie Brilliant Blue R-250. Mathematical treatment of the kinetic data General numerical analysis of the kinetic data and simula- tion studies on the model reaction networks were performed with the use of the DYNAFIT program generally according to the DYNAFIT Reference Manual and [20,21]. Unless other- wise mentioned, the desirable kinetic parameters were determined within a 95% confidence interval by a least- squares regression procedure based on the Levenberg– Marquardt fitting algorithm. Evaluation of the apparent rate constants, k app values, from the kinetics of the Ala$tmRNA and Ala$tRNA hydrolytic decay was per- formed with the use of the ÔLSW Data Analysis ToolboxÕ add-in (MDL Information Systems, Inc) for Microsoft EXCEL . Results The sequence of the ssrA gene of T. thermophilus HB8 determined in this study differs in a single base (G 310 instead of A 310 ) from the previously reported complete ssrA sequences of T. thermophilus strains HB8 [22] and HB27 (database of T. thermophilus HB27 genomic sequences at Go ¨ ttingen Genomics Laboratory website, http:// www.g2l.bio.uni-goettingen.de). Guanine in position 310 was also found by Martindale and Williams in a partial sequence of the ssrA gene from strain HB8 (T. thermophilus tmRNA sequence, version 2, deposited 04/11/2000 at The tmRNA website, http://www.indiana.edu/$tmrna). This minor difference can possibly be explained by an intraspe- cific genomic variation. The presumed secondary structure of T. thermophilus tmRNA resembles that of E. coli tmRNA (Fig. 2). T. thermophilus tmRNA was synthesized by run-off transcription with the ssrA gene under the T7 promoter as Ó FEBS 2003 tmRNA from Thermus thermophilus (Eur. J. Biochem. 270) 465 a template. The transcript was purified by HPLC on a mixed-mode ionic-hydrophobic matrix followed by prepar- ative urea-PAGE. The obtained RNA was annealed in presence of 1 m M MgCl 2 and analysed by gel-electrophor- esis (Fig. 3). The tmRNA transcript migrated as a single band during separation under denaturing conditions. At the same time, nondenaturing gel-electrophoresis in agarose revealed few faint satellite bands following the main one. The major RNA species was isolated from the agarose gel and reannealed. However, when it was subjected again to the electrophoretic separation under identical conditions, the presence of the same high-molecular-mass admixtures was observed. Such a heterogeneity of the tmRNA transcript is thus likely to be caused by the reversible RNA oligomerization. As a macromolecule with numerous self-complementary stretches, tmRNA may be prone to form intermolecular contacts instead of the equivalent intramolecular ones. Taking into consideration that the presumed oligomers account for a relatively small fraction of the transcript population, we used the obtained RNA without further purification. The distinctive feature of the T. thermophilus tmRNA is its anomalously high GC content even in comparison with tmRNAs from the more extreme thermophiles, Thermotoga maritima and Aquifex aeolicus (Table 1). The percentage of GC pairs in predicted double-stranded regions is equal to 84.3% of the total number of base pairs (in the case of E. coli tmRNA this parameter amounts to only 57.5%). Therefore, it was natural to expect a high resistance of T. thermophilus tmRNA to thermoinduced unfolding. Indeed, tmRNA melting experiments revealed no structural changes in the temperature range from 18 °Cto70°C. Noticeable transitions were registered only above 73 °C (Fig. 4). Under similar conditions (1 m M MgCl 2 ,near- neutral pH), the melting profiles of E. coli tmRNA exhi- bited two peaks, around 25 °Cand57°C, and the interval of structural constancy was only from 30 °Cto45 °C[23]or even more narrow [5]. Remarkably, even in the absence of any stabilizing protein cofactors the unmodified T. thermo- philus tmRNA transcript can sustain heating up to the temperatures compatible with the efficient growth of this thermophilic bacterium. The apparent initial rate of the tmRNA aminoacylation with T. thermophilus alanyl-tRNA synthetase was found to be maximal at 50 °C. A similar activity profile was observed inthecaseoftRNA Ala charging (Fig. 5). This is somewhat lower than the optimal temperature of tRNA aminoacyla- tion reported for cloned T. thermophilus alanyl-tRNA synthetase (% 60 °C) [13]. Other Thermus synthetases exhibit maximal activity at even higher temperatures: glutamyl-tRNA synthetase at 65 °C [24], isoleucyl-tRNA synthetase at 70 °C [25], phenylalanyl-tRNA synthetase at 70 °C[25]or78°C [26]. Therefore we checked whether the decline of the tmRNA alanylation rate above 50 °Cis caused by irreversible degradation of any of the components of the aminoacylation reaction mixture. tmRNA was charged at 30 °C until a stable plateau was reached, then the tube with the reaction mixture was incubated at 80 °C for 20 min and transferred back to 30 °C.Thetimecourse of the Ala$tmRNA synthesis upon these temperature alterations is shown on Fig. 6. Heating the reaction mixture to 80 °C resulted in quick decrease of the Ala$tmRNA concentration to almost zero level. However when it was cooled back to 30 °C, recharging of tmRNA occured with almost the same rate and to the same extent as it was before the thermal jump. This indicates that all the initial Fig. 2. Secondary structure of Thermus thermophilus tmRNA. Four pseudoknots are labelled pK1, pK2, pK3 and pK4. Trinucleo- tides that encode amino acids of the tag- peptide are boxed. Helix numbering is given according to Zwieb et al. (1999) [34]. The ambiguous nucleotide at position 310 is marked by the arrow. 466 V. G. Stepanov and J. Nyborg (Eur. J. Biochem. 270) Ó FEBS 2003 ingredients of the aminoacylation reaction mixture remain undamaged upon prolonged incubation at the highest temperature used in our study. The decreased thermal optimum of the alanyl-tRNA synthetase activity observed in our experiments may be explained considering that the monitored accumulation of charged RNA in solution is determined by the balance between enzyme-catalysed aminoacylation of RNA and spontaneous deacylation of aminoacyl-RNA. Studies on aminoacyl-tRNA stability revealed the alanyl ester bond to be one of the most susceptible to hydrolytic cleavage. Therefore, alanyl-tRNA synthetase encounters more intense deacylation of charged RNA than synthetases of other specificities. As a result, the measured maximum of the apparent initial rate of RNA alanylation is shifted towards lower temperatures and may float depending on the concentration of alanyl-tRNA synthetase in the reaction mixtures and on its specific activity in different buffers. In order to characterize the substrate properties of the tmRNA transcript, we attempted to estimate the kinetic parameters of the tmRNA alanylation. A standard approach based on the Michaelis–Menten scheme of the enzyme-catalysed reaction was considered inadequate at the conditions of our experiments. The corresponding constants k cat and K m are usually calculated from the dependence of Fig. 3. Analysis of the synthetic transcript of T. thermophilus tmRNA. (A) Non-denaturing 2% agarose gel stained with ethidium bromide. 0.004 D 260 units (lane 1) and 0.001 D 260 units (lane 3) of the tmRNA transcript were separated on the gel in the presence of DNA markers [lane 2, 100 bp DNA ladder (New England Biolabs)]. (B) Denaturing 8% polyacrylamide gel stained with pyronin Y. Lanes 1 and 2 show separation patterns of the samples equivalent to 0.1 and 0.01 lL, respectively, of the standard transcription reaction mixture after the tmRNA synthesis was completed. Table 1. Correlation between growth temperature and tmRNA GC-content for selected bacterial species. Bacterial species Growth temperature, °C tmRNA total GC content (%) Optimum Maximum Aquifex aeolicus 85 95 66.8 Thermotoga maritima 80 90 62.1 Thermus thermophilus 72 85 70.5 Bacillus stearothermophilus 60 75 59.6 Escherichia coli 37 45 52.9 Fig. 4. UV-absorbance melting curve of the purified T. thermophilus tmRNA transcript. Fig. 5. Temperature dependence of apparent initial rate of aminoacy- lation of the T. thermophilus tmRNA transcript (black circles) and tRNA Ala (grey squares) by the homologous alanyl-tRNA synthetase. The dependence is expressed as the relative aminoacylation activity, with 100% corresponding to the maximal observed initial reaction rate. Ó FEBS 2003 tmRNA from Thermus thermophilus (Eur. J. Biochem. 270) 467 the initial reaction rates on the substrate concentration. However, at elevated temperatures fast spontaneous Ala$tmRNA hydrolysis disguised the real velocity of tmRNA charging and shortened the linear part of amino- acylation kinetics to the level, where correct measurement of the initial reaction rate was barely possible. Another serious problem was associated with the uncertainty of the molar concentration of chargeable transcript in the reaction mixtures. The extent of tmRNA aminoacylation was varying dramatically depending on the reaction conditions (temperature, buffer composition, enzyme concentration), the maximal observed level being about 45 pmol Ala/D 260 unit of tmRNA transcript. Therefore, the estimates of the total tmRNA concentration based on the quantification of [ 3 H]Ala coupled with tmRNA at the reaction plateau were regarded as unreliable. To circumvent these difficulties, we determined the kinetic parameters of tmRNA aminoacylation by numerical analysis of a set of reaction curves obtained at different enzyme concentrations. A simplified scheme of the amino- acylation mechanism included the reversible reaction of Ala$tmRNA synthesis accompanied with the spontaneous Ala$tmRNA hydrolysis: E þ S ÀÀ* )ÀÀ k f k b ES ÀÀ* )ÀÀ k cat k rev E þ P ð1Þ P À! k b S ð2Þ where E, S and P represent alanyl-tRNA synthetase, tmRNA and Ala$tmRNA, respectively, and ES is a transient complex between the enzyme and tmRNA. The corresponding system of differential Eqns (3–6) contained five adjustable parameters, k f , k b , k cat , k rev and k h . d½E=dt ¼Àk f ½E½Sþk b ½ESþk cat ½ESÀk rev ½E½P ð3Þ d½S=dt ¼Àk f ½E½Sþk b ½ESþk h ½Pð4Þ d½ES=dt ¼ k f ½E½SÀk b ½ESÀk cat ½ESþk rev ½E½Pð5Þ d½P=dt ¼ k cat ½ESÀk rev ½E½PÀk h ½Pð6Þ Additionally, the total concentration of tmRNA (designa- ted S 0 ), which was the same in all the reaction mixtures, had to be searched for. Preliminary simulation studies on the above-mentioned kinetic model revealed some constraints on the possible organization of the kinetic experiment. The most important limitation was that in order to obtain maximally reliable estimates of the total concentration of functional tmRNA and of the affinity parameters K d and K m , the enzyme concentration should be varied in the same interval where S 0 , K d or K m are expected to be found (i.e. in the micromolar range). Also, the kinetic curve should be well sampled on different stages of the reaction progress. However, at comparable concentrations of alanyl-tRNA synthetase and tmRNA, the aminoacylation reaction rea- ches its plateau very quickly, and the raising part of the reaction curve is too short to be monitored accurately by the filter technique. Therefore, we measured the kinetics of tmRNA aminoacylation at an ATP concentration lowered to 20 l M . By that way the specific activity of alanyl-tRNA synthetase was decreased to the appropriate level, so that we could use the desirable high enzyme-to-substrate ratios. The proposed reaction mechanism was fitted to the experimental dataset (five kinetic curves with 12 points each measured at 50 °C) with the use of the DYNAFIT program. In a control experiment, T. thermophilus tRNA Ala was charged with alanine under the same conditions, and the kinetic param- eters of the reaction were determined by the same procedure as in the case of tmRNA (Fig. 7, Table 2). The obtained results reveal 5.9 times lower catalytic efficiency (k cat /K m )of alanyl-tRNA synthetase with tmRNA as a substrate than with tRNA Ala . The observed difference in substrate pro- perties of tmRNA and tRNA Ala should be attributed to a significantly lower k cat in the case of tmRNA alanylation. At the same time, alanyl-tRNA synthetase possesses slightly higher affinity towards tmRNA, mostly because of slower dissociation of the AlaRSase*tmRNA complex in compar- ison with the AlaRSase*tRNA Ala complex. With certain caution we can extrapolate some of our results to standard aminoacylation conditions, taking into account the fact that the decrease of ATP concentration from 2.5 m M to 20 l M in the reaction mixture results in a 345-fold drop of the specific activity of the enzyme at 40 °C (from 105 nmolÆmin )1 Æmg )1 to 0.304 nmolÆmin )1 Æmg )1 in thepresenceof4l M tRNA Ala ). If the same proportion is preserved at higher temperatures, the catalytic constant k cat for tRNA and tmRNA alanylation under standard reaction conditions and 50 °C should be close to 0.8 s )1 and 0.03 s )1 , respectively. This is to be compared with k cat values of 0.93 s )1 [27], 1.1 s )1 and 1.4 s )1 [28] determined for E. coli alanyl-tRNA synthetase and different isoacceptors of Fig. 6. Kinetics of tmRNA aminoacylation with [ 3 H]alanine by T. thermophilus alanyl-tRNA synthetase upon temperature alterations. A standard aminoacylation reaction mixture with 5 D 260 units per mL of the purified tmRNA transcript and 5 n M of alanyl-tRNA synthetase was transferred from 30 °Cto80°C and backward during measure- ments of the amount of [ 3 H]Ala$tmRNA synthesized. 468 V. G. Stepanov and J. Nyborg (Eur. J. Biochem. 270) Ó FEBS 2003 E. coli tRNA Ala at 37 °C, or with the k cat value of 0.71 s )1 calculated from the specific activity of T. thermophilus alanyl-tRNA synthetase at 60 °C with unfractionated tRNA as a substrate [13]. In order to characterize the interaction of EF-Tu with Ala$tmRNA, we attempted to study EF-Tu protection of Ala$tmRNA against spontaneous base-promoted hydro- lysis. By analogy with E. coli tmRNA [5], we expected to observe an increase of Ala$tmRNA yield in the amino- acylation reaction and a decrease of Ala$tmRNA deacy- lation rate in the nonenzymatic hydrolytic reaction in the presence of EF-Tu and 5¢-Guanylylimidodiphosphate (GDPNP), a stable analog of GTP. Surprisingly, tmRNA charging with alanine was found to be strongly inhibited by the elongation factor (Fig. 8). Moreover, the influence of EF-Tu on the Ala$tmRNA deacylation rate revealed a deviation from the mechanism of aminoacyl ester bond protection upon formation of the canonical ternary complex between EF-Tu, nucleotide cofactor and aminoacyl-tRNA. While the velocity of Ala$tRNA Ala decay decreased monotonously with the increase of EF-Tu*GDPNP con- centration in the reaction mixture, the apparent rate of Ala$tmRNA hydrolysis first decreased to a certain level and then started to increase again (Fig. 9). The simplest kinetic model that can describe this phenomenon implies an existence of two interacting binding sites for EF-Tu*GDPNP on tmRNA: E þ P ÀÀ* )ÀÀ k 1 k À1 AP ð7Þ E þ P ÀÀ* )ÀÀ k 2 k À2 BP ð8Þ E þ AP ÀÀ* )ÀÀ k 3 k À3 ABP ð9Þ E þ BP ÀÀ* )ÀÀ k 4 k À4 ABP ð10Þ P À! k b S ð11Þ BP À! k b BS ð12Þ where E, P and S correspond to EF-Tu*GDPNP, Ala$tmRNA and deacylated tmRNA, respectively, AP represents a complex in which EF-Tu is bound to the acceptor stem of Ala$tmRNA in the same way as in the Fig. 7. Kinetics of aminoacylation of the tmRNA transcript (A) and tRNA Ala (B) at different concentrations of alanyl-tRNA synthetase at 50 °C. The aminoacylation reaction mixtures contained all compo- nents at standard concentrations except ATP whose concentration was decreased to 20 l M . The drawing represents an output of the DYNAFIT program, where lines correspond to the best fit of the experimental points to the proposed reaction mechanism. (A) Concentration of alanyl-tRNA synthetase was 0.30 (circles), 0.60 (squares), 1.50 (trian- gles), 3.00 (reverse triangles) and 4.50 (diamonds) l M . (B) Concen- tration of alanyl-tRNA synthetase was 0.034 (circles), 0.068 (squares), 0.102 (triangles), 0.171 (reverse triangles), 0.342 (diamonds) l M . Table 2. Kinetic parametes of tmRNA and tRNA Ala aminoacylation with T. thermophilus alanyl-tRNA synthetase at 50 °C. tmRNA tRNA Ala Constants Value Standard error Value Standard error k f ,m M )1 s )1 12.9 ± 3.8 19.8 ± 7.3 k b ,10 )3 s )1 4.57 ± 2.12 26.1 ± 7.9 k cat ,10 )3 s )1 0.0956 ± 0.0079 2.23 ± 0.56 k rev ,m M )1 s )1 0.313 ± 0.081 0.874 ± 0.182 K d ¼ k b /k f , l M 0.354 1.319 K m ¼ (k b + k cat )/k f , l M 0.361 1.432 k cat /K m , M )1 s )1 265 1560 k h ,10 )3 s )1 2.21 ± 0.19 2.59 ± 0.16 S 0 , l M 1.59 ± 0.11 0.125 Number of datapoints 60 59 Ó FEBS 2003 tmRNA from Thermus thermophilus (Eur. J. Biochem. 270) 469 ternary complexes between EF-Tu, GTP and aminoacyl- tRNAs (therefore the alanylated tmRNA acceptor stem is further referred to as the canonical EF-Tu binding site), BP represents a complex in which EF-Tu is bound to a hypothetical alternative site, and ABP represents a complex in which EF-Tu molecules are bound simultaneously to both the canonical and alternative sites. Qualitatively, the observed dependence of the Ala$tmRNA deacylation rate on EF-Tu concentration may result from negative cooper- ativity upon EF-Tu binding to the canonical and alternative sites, if we assume that (a) the protein protects the aminoacyl ester bond only when it is bound to the canonical site, and (b) the canonical site has higher affinity towards EF-Tu*GDPNP than the alternative one. In order to quantify the interrelationships between the elementary reactions of the proposed kinetic model, we attempted to determine the corresponding kinetic constants or, at least, to estimate the limits of their admissible dispersion. The dynamics of the Ala$tmRNA/EF–Tu interaction can be represented by a nonlinear system of differential equations: d½E=dt ¼Àk 1 ½E½Pþk À1 ½APÀk 2 ½E½Pþk À2 ½BP À k 3 ½E½APþk À3 ½ABPÀk 4 ½E½BP þ k À4 ½ABPð13Þ d½P=dt ¼Àk 1 ½E½Pþk À1 ½APÀk 2 ½E½Pþk À2 ½BPÀk h ½P ð14Þ d½AP=dt ¼ k 1 ½E½PÀk À1 ½APÀk 3 ½E½APþk À3 ½ABP ð15Þ d½BP=dt ¼ k 2 ½E½PÀk À2 ½BPÀk 4 ½E½BPþk À4 ½ABP À k h ½BPð16Þ d½ABP=dt ¼ k 3 ½E½APÀk À3 ½ABPþk 4 ½E½BP À k À4 ½ABPð17Þ Fig. 8. Kinetics of aminoacylation of the tmRNA transcript with alanyl- tRNA synthetase in presence (black diamonds) or in absence (grey squares) of Th. aquaticus EF-Tu*GDPNP. A45-lL aliquot with 23 l M EF-Tu*GDPNP complex in the exchange buffer (25 m M Hepes/ NaOH (pH 7.9), 5 m M GDPNP, 0.2 M NH 4 Cl, 2 m M b-mercapto- ethanol) was added to 150 lL of the standard aminoacylation reaction mixture 20 s before the aminoacylation was started. In the case of the control reaction mixture, 45 lL of the exchange buffer was added to 150 lL of the standard reaction mixture. Fig. 9. Dependence of the apparent velocity of Ala$tmRNA (A) and Ala$tRNA Ala (B) hydrolysis on the concentration of the EF- Tu*GDPNP complex in the deacylation reaction mixture. The drawing represents an output of the DYNAFIT program, where lines correspond to the best fit of the experimental points to the proposed reaction mechanism. 470 V. G. Stepanov and J. Nyborg (Eur. J. Biochem. 270) Ó FEBS 2003 d½S=dt ¼ k h ½Pþk h ½BPð18Þ The experimental dataset contained the values of the initial Ala$tmRNA deacylation rates determined at seven differ- ent concentrations of EF-Tu*GDPNP in the reaction mixture. The numerical analysis of the model was accom- plished through an iterative procedure, which took advant- age of the fact that the elementary reactions of the kinetic model contribute differently to the initial rate of Ala$tmRNA decay. Briefly, on the basis of preliminary simulation tests of the model, the kinetic constants were divided into three groups according to their influence on the fitting quality characterized by the standard deviation of the theoretical curve from the experimental data. The first group consisted of parameters k 1 , k 3 and k h , whose influence on the fitting efficiency was determinative. In general, when k 1 , k 3 and k h were fixed, the fitting quality could not be significantly improved by compensatory adjustment of all the remaining parameters. The second group contained parameters k -2 , k -3 , k 4 , which could vary 5–6 orders of magnitude without serious effect on the deviation of the model from the experimental data. The third group included parameters k -1 , k 2 , k -4 , whose variability upon fitting was more moderate than in the previous case and depended on the current values of k 1 , k 3 and k h . At the first stage, the value of k h was estimated from the kinetics of Ala$tmRNA decay in the absence of EF-Tu. The search for k 1 and k 3 was performed by systematic sampling of the (k 1 , k 3 )-spacewhenalltheremaining constants (except k h )wereallowedtobeadjustedinorderto reach minimal standard deviation of the model from the experimental data for each given pair of k 1 , k 3 . At the point where the fitting quality was maximal, all the parameters of the first group (k 1 , k 3 and k h ) have been fixed. Then the limits of admissible dispersion for each rate constant of the third group were studied by systematic sampling of the (k -1 , k 2 , k -4 )-space, while other kinetic parameters were kept fixed at their currently best values. The kinetic constants were characterized either by an optimized value within a 95% confidence interval, or by the upper or lower limit that was defined as the point where the stable increase of the standard deviation reaches 1% of its minimal value at the current conditions. Then the rate contstants of the second group were estimated in the same way. The full set of the rate constants was then refined by repeating an optimization procedure, which assumed an improvement of the fitting quality through the adjustment of the kinetic parameters belonging to one group, while the rate constants from the two other groups remained fixed. After two cycles of this refinement, further adjustment of the kinetic parameters could not decrease the difference between the model and the experimental data anymore. For comparison, the kinetic parameters of Ala$tRNA Ala protection by EF-Tu*GDPNP were calculated using the same approach. The Ala$tRNA Ala decay in the presence of the elongation factor was described by the Eqns (7) and (11), where P and S represented alanylated and deacylated tRNA Ala , respectively. The calculated rate constants are listed in Table 3. The affinity of the elongation factor towards the alanylated tRNA-like module of tmRNA (the canonical binding site) does not seem to be much different from its affinity towards Ala$tRNA Ala when the alternative EF-Tu binding site on tmRNA is empty. However, when it is occupied, EF-Tu binding to the canonical site deteriorates dramatically, the K d value being increased at least 10 5 times. On the other hand, the first EF-Tu molecule should bind to Ala$tmRNA predominantly at the canonical site, because the corresponding association rate (k 1 ) is 13 times higher than that for the alternative site (k 2 ). Therefore, the first event in a major sequence of elementary interactions of EF-Tu with Ala$tmRNA should be the formation of a complex between EF-Tu*GDPNP and the alanylated acceptor stem of tmRNA, in which the aminoacyl residue is protected against hydrolysis. Then, the second EF-Tu molecule binds to the alternative site on tmRNA. This causes a quick ejection of the first EF-Tu molecule from the canonical binding site, which is expressed by the drastic increase of the corresponding dissociation rate constant (k -4 is approximately 5 orders of magnitude higher than k -1 ). As a result, the alanyl ester bond loses the protection and becomes susceptible again to the nucleophilic attack of hydroxyl anions (Fig. 10). To test experimentally our suggestion that Ala$tmRNA possesses a second EF-Tu binding site besides its alanylated tRNA-like module, we checked whether EF-Tu*GDPNP can form a complex with uncharged tmRNA. By analogy with tRNA, we assumed that efficient EF-Tu binding to the tRNA-like module of tmRNA is only possible when tmRNA is aminoacylated. EF-Tu*GDPNP and tmRNA were mixed and incubated for 10 min at the same conditions as those used in the studies on Ala$tmRNA protection with EF-Tu. Electrophoretic separation of these mixtures revealed a change of tmRNA mobility in the presence of the elongation factor (Fig. 11). Thus, even being uncharged, tmRNA still retains an ability to bind EF-Tu*GDPNP. Discussion In the present study we investigated the interaction of the T. thermophilus tmRNA transcript with thermophilic Table 3. Kinetic parameters for Ala$tmRNA and Ala$tRNA Ala pro- tection against alkaline hydrolysis at 40 °CbyTh. aquaticus EF-Tu in complex with GDPNP. Constants Ala$tmRNA Ala$tRNA Ala Value Standard error Value Standard error k 1 , l M )1 s )1 1.03 0.08 0.368 0.027 k -1 ,s )1 0.0602 0.0371 0.010 upper limit k 2 , l M )1 s )1 0.0805 0.0251 k -2 ,s )1 0.01 a upper limit k 3 , l M )1 s )1 0.149 0.006 k -3 ,s )1 10 a upper limit k 4 , l M )1 s )1 0.9 upper limit k -4 ,s )1 5000 lower limit k h ,10 )3 s )1 0.90 b 0.04 0.89 b 0.04 a Throughout the fitting procedure, strong covariation of k -2 and k -3 was observed, k -3 being equal approximately to 1000 k -2 . b The value has been evaluated from the kinetics of alanyl ester bond hydrolysis in the absence of EF-Tu, and was fixed upon fitting the model to the main massif of the experimental data. Ó FEBS 2003 tmRNA from Thermus thermophilus (Eur. J. Biochem. 270) 471 alanyl-tRNA synthetase and elongation factor Tu. Despite the lack of post-transcriptional modifications, the tmRNA transcript possessed a remarkable thermostability, which may to a certain extent be explained by the large number of GC base pairs in double-stranded regions. tmRNA melting profile indicated structural constancy of this molecule in the temperature range 18–70 °C. This made us sure that the conformational state of the tmRNA transcript remains essentially the same under different thermal conditions of activity assays. The observed structural constancy makes T. thermophilus tmRNA a good target for structural studies. The thermal optima of tmRNA and tRNA Ala amino- acylation with T. thermophilus alanyl-tRNA synthetase were found to be lower than the optimum of tRNA Ala charging reported by Lechler et al.[13](% 50 °Cvs. % 60 °C, respectively). However, the real discrepancy may be smaller, taking into account that in both studies the initial aminoacylation velocity was measured in 10 °Csteps, and could be ascribed to the different composition of the reaction mixtures. Also, in our experiments no irreversible denaturation of alanyl-tRNA synthetase (or any other component of the aminoacylation reaction mixture) at 80 °C was observed, in contrast to the above-cited paper where irreversible thermoinduced precipitation of the enzyme from 65 °C and upward was described. This apparent disagreement is presumably due to a substrate protection effect, which may occur in our case because of the alanyl-tRNA synthetase stabilization in the presence of tmRNA, alanine and ATP. It is noteworthy that the in vitro determined thermal optimum of alanyl-tRNA synthetase activity is significantly lower than the characteristic temperatures of T. thermophi- lus growth (T opt 72–75 °C, T max 85 °C). This may be due to the intense spontaneous deacylation of Ala$tRNA Ala or Ala$tmRNA, which quickly becomes comparable with the enzyme-promoted aminoacylation reaction upon increase of temperature. The finding raises a question about the possible compensatory mechanisms, which allow an effi- cient production of Ala$tRNA Ala or Ala$tmRNA at 70– 80 °Ctooccurin vivo. Among those could be the protection Fig. 10. Proposed mechanism of EF–Tu*GDPNP interaction with Ala$tmRNA. The size of the arrows reflects the relative magnitude of the corresponding first-order or pseudo first-order kinetic constants at micromolar concentrations of EF-Tu. Fig. 11. Gel mobility shift study of the interaction between EF-Tu*GDPNP and uncharged tmRNA. Two equivalent gels were run at identical conditions and stained with Coomassie Blue R250 (A) and with pyronin Y enhanced by silver treatment (B). Mixtures containing 1.0 D 260 units per mL of tmRNA transcript and 0 (lanes 2), 3.8 (lanes 3), 7.5 (lanes 4) and 11.3 l M (lanes 5) EF-Tu*GDPNP in 80 m M Hepes/NaOH (pH 7.6), 8 m M MgCl 2 ,0.5m M Spermine*4HCl, 10% (v/v) glycerol were incubated for 10 min at 30 °C, then for 10 min in ice-cold water bath, and then separated in nondenaturing 8% polyacrylamide gel at room temperature, 120 V, for 2.5 h. The separation pattern of the mixture containing EF-Tu*GDPNP alone is shown on lanes 1. Lanes 6 and 7 represent two different amounts of EF-Tu*GDP loaded. 472 V. G. Stepanov and J. Nyborg (Eur. J. Biochem. 270) Ó FEBS 2003 [...]... parameters for tmRNA binding to alanyl-tRNA synthetase and elongation factor Tu from Escherichia coli Biochemistry 39, 2652–2658 6 Rudinger-Thirion, J., Giege, R & Felden, B (1999) Aminoacylated tmRNA from Escherichia coli interacts with prokaryotic elongation factor Tu RNA 5, 989–992 7 Wower, I.K., Zwieb, C.W., Guven, S.A & Wower, J (2000) Binding and cross-linking of tmRNA to ribosomal protein S1, on and off... the interaction of a E coli tmRNA transcript with wild-type E coli EF -Tu in the GDP or GTP form Their crosslinking and footprinting experiments revealed uncharged tmRNA to have a binding site, which can accommodate EF -Tu* GTP or EF -Tu* GDP Nucleotides located in helix 2 and pseudoknot pK4 were found to be in contact with the elongation factor upon complex formation The affinities of EF -Tu* GTP and EF -Tu* GDP... tmRNA from Thermus thermophilus (Eur J Biochem 270) 473 of the unstable aminoacyl ester bond in a complex with elongation factor Tu, increase of the intracellular concentration of alanyl-tRNA synthetase, or improvement of the substrate properties of tRNAAla and tmRNA in presence of cofactors (proteins, polyamines, metal ions) Also, compartmentalization of the components of the translational apparatus... EF Tu, the interaction of either alanyltRNA synthetase or EF -Tu with the tRNA-like module of tmRNA is strongly deteriorated The ability of deacylated tmRNA to form a complex with EF -Tu* GDPNP provides another evidence in favour of the existence on tmRNA of a EF -Tu binding site distinct from the tRNA-like module 474 V G Stepanov and J Nyborg (Eur J Biochem 270) Ó FEBS 2003 Our conclusions correlate with. .. given conditions, both Ala $tmRNA and Ala$tRNAAla associate with alanyltRNA synthetase much slower, than uncharged tmRNA and tRNAAla This reveals insignificant product inhibition in the aminoacylation reaction catalysed by T thermophilus alanyl-tRNA synthetase The difference between krev values for tmRNA and tRNAAla correlates with that between the corresponding kf values and allow us to suggest a similar... C-terminus of the cloned elongation factor The His-tag may block EF -Tu binding to the alternative site on tmRNA The role of the alternative EF -Tu binding site on tmRNA is unclear When it is occupied, T thermophilus tmRNA is practically inactivated Both aminoacylation of tmRNA and EF -Tu protection of the preformed aminoacyl ester bond become inefficient Consequently, at elevated temperatures and supramicromolar... rate of the tmRNA transcript from the complex with the enzyme (kb) was 5.7 times slower than that of tRNAAla It could be interpreted in the way that initial binding of alanyl-tRNA synthetase to both tRNAAla and tmRNA proceeds through the interaction with similar structural patterns (the alanylated acceptor stem with the specifically recognizable GU base pair, and the T loop) On the other hand, in an... components besides alanyl-tRNA synthetase and elongation factor Tu Acknowledgements This work has been supported by the Danish Natural Science Research Council through its Programme for Biotechnological Research References 1 Muto, A., Ushida, C & Himeno, H (1998) A bacterial RNA that functions as both a tRNA and an mRNA Trends Biochem Sci 23, 25–29 Ó FEBS 2003 tmRNA from Thermus thermophilus (Eur J... established complex with the enzyme, tmRNA may form more contacts (not necessary specific ones) with alanyl-tRNA synthetase than tRNAAla This should result in higher activation energy of the dissociation of the alanyl-tRNA synthetase *tmRNA complex and, consequently, in a smaller kb It is interesting to compare kf and krev values, which characterize the enzyme binding to uncharged and alanylated RNA,... made that the efficiency of T thermophilus tmRNA aminoacylation at elevated temperatures is cramped by the intense spontaneous hydrolytic decay of synthesized Ala $tmRNA At the same time, the elongation factor Tu by itself does not provide Ala $tmRNA with sufficient protection against deacylation, whereas it efficiently stabilizes Ala$tRNAAla The observed activity of EF -Tu on tmRNA can be described as regulatory . tmRNA from Thermus thermophilus Interaction with alanyl-tRNA synthetase and elongation factor Tu Victor G. Stepanov and Jens Nyborg Institute of. Molecular and Structural Biology, University of Aarhus, Denmark The interaction of a Thermus thermophilus tmRNA tran- script with alanyl-tRNA synthetase and elongation