| Nucleic Acids Research | Pages |
Pyrophosphate mediates the effect of certain tRNA mutations on aminoacylation of yeast tRNAPhe
Introduction
Materials And Methods
Materials
In vitro transcription
Aminoacylation assay
Results
Selective enhancement of tRNAPhe mutants aminoacylation in the presence of PPase
PPase-dependent activation correlates with distortion of tRNA tertiary structure
Enhancement of tRNA aminoacylation by PPAse is not related to the amino acid activation step of the reaction
Discussion
Acknowledgements
References
Pyrophosphate mediates the effect of certain tRNA mutations on aminoacylation of yeast tRNAPhe
Received July 23, 1999; Revised and Accepted September 27, 1999
ABSTRACT The influence of pyrophosphate hydrolysis by inorganic pyrophosphatase on homologous aminoacylation of different yeast tRNAPhe mutants was studied. The addition of pyrophosphatase significantly improved the aminoacylation efficiency of tRNAPhe structural mutants as well as the mutant with substitution at position 20, while having no effect on the charge of wild-type tRNAPhe. Aminoacylation of tRNAPhe anticodon and discriminator base (N73) mutants was not affected by pyrophosphatase. Activation of wild-type tRNAPhe transcript aminoacylation by inorganic pyrophosphatase was observed only at low Mg2+ concentrations due to distortion of the tRNAPhe structure under these conditions. Our results demonstrate that pyrophosphate dissociation becomes a rate-limiting step of the reaction in yeast phenylalanyl-tRNA synthetase catalyzed aminoacylation of tRNAPhe variants with altered tertiary structure. A possible mechanism of pyrophosphate-mediated inhibition of tRNA mutants aminoacylation is discussed.
INTRODUCTION
Specificity and efficiency of tRNA aminoacylation by cognate aminoacyl-tRNA synthetase (aaRS) relies on the correct presentation of a certain number of nucleotides in the tRNA molecule to the enzyme. Any substitution at these key positions (called identity elements) generally results in a dramatic decrease of the aminoacylation efficiency of such mutant tRNAs (1,2).
The formation of aminoacyl-tRNA by aminoacyl-tRNA synthetases is generally described as a two-step reaction (1,3). Firstly, the amino acid (AA) is activated by forming enzyme-bound aminoacyladenylate (AA-AMP) with the release of pyrophosphate (PPi).
| E + AA + ATP -> E·AA-AMP + PPi |
In the second step, the activated amino acid is transferred to the terminal ribose of tRNA, followed by dissociation of AMP and aminoacylated tRNA.
| E·AA-AMP + tRNA -> AA-tRNA + AMP + E |
The process of tRNA aminoacylation consists of multiple elementary steps, such as binding of substrates and dissociation of products, conformational changes of both tRNA and aminoacyl-tRNA synthetase, and chemical reactions occurring in the enzyme active site (1-3). The observed overall reaction rate of tRNA aminoacylation is determined by the slowest elementary step of the reaction. The point mutations in tRNA may, as earlier predicted (4), differentially affect the rate of specific elementary steps in the aminoacylation reaction (for example by influencing the binding efficiency or by impending the conformational changes in the enzyme-substrate complex). Although the effects of numerous tRNA mutations on kinetic parameters of aminoacylation have been described, surprisingly little is known about the correlation between a given mutation and the reaction step affected.
It is logical to expect that mutations in tRNA would affect transfer of the amino acid to tRNA. Indeed, substitutions of discriminator base (N73) of tRNAAla directly affected kcat of the amino acid transfer reaction (5), probably through interference with the proper orientation of the tRNA CCA terminus in the active site (6,7). However, substitutions of tRNAGln identity elements affect aminoacylation by an entirely different mechanism, through interference with aminoacyladenylate formation by decreasing the enzyme affinity for Gln (8,9).
These examples demonstrate the limits of our current knowledge of the biochemical functions of tRNA identity elements. In this paper we present evidence that PPi dissociation is selectively affected by certain tRNA mutations and becomes the limiting step in tRNA aminoacylation catalyzed by yeast phenylalanyl-tRNA synthetase (PheRS).
Recognition of yeast tRNAPhe (GAA) is determined by five major identity elements (nucleotides G20, G34, A35, A36 and A73) located in three separate regions of the tRNA molecule (the D-loop, the anticodon and the acceptor stem) (10). Previously we have shown that dissociation of the pyrophosphate is the rate-limiting step in the case of heterologous aminoacylation of Escherichia coli tRNAPhe by yeast PheRS (11). This slow PPi dissociation correlates with the single difference in the identity element sets between yeast and E.coli tRNAPhe (substitution G->U at position 20). In this paper, we have screened the set of tRNAPhe variants to identify mutations resulting in pyrophosphate-dependent inhibition of aminoacylation.
MATERIALS AND METHODS
Materials
L-[3H]phenylalanine (21 Ci/mmol) was from Amersham, [alpha]- and [[gamma]-32P]ATP were from NEN, PEI-cellulose plates were from Selecto Scientific, yeast PheRS was purified as described previously (12), purified T7 RNA polymerase was a kind gift of Dr R. Aphasizhev (Institute of Molecular Biology, Moscow, Russia). Restriction enzyme BstNI was from New England Biolabs. Plasmids containing yeast tRNAPhe variants (pYF0, pYF1, pYF2, pYF3, pYF4, pYF66 and pYF91) were kindly provided by Prof. O. Uhlenbeck (University of Colorado, Boulder, CO) and described previously (10,13). Inorganic pyrophosphatase (PPase) was from Sigma.
In vitro transcription
All tRNA transcripts used in this study were obtained by in vitro transcription of the corresponding synthetic genes placed under T7 promoter (14). Transcription was performed in the reaction mixture containing 40 mM Tris-HCl pH 8.1 (at 37°C), 22 mM MgCl2, 5 mM dithiothreitol, 0.01% Triton X-100, 1 mM spermidine, 1 mM each nucleotide triphosphate, 5 mM GMP, 0.1 mg/ml linearized plasmid and 2.5 U/ml T7 RNA polymerase. Incubation was performed for 3 h at 37°C and the reaction terminated by phenol/chloroform extraction. Full-length tRNA transcripts were purified by single-nucleotide resolution electrophoresis on 12% polyacrylamide denaturing gels and recovered by electroelution. Concentration of tRNA transcripts was measured by optical density at 260 nm. Functional integrity of tRNA transcripts was verified by their aminoacylation at high PheRS concentration (200 nM). Aminoacylation plateau values of all the transcripts used in this study generally exceed 90% (81% for the YF2 mutant).
Aminoacylation assay
Aminoacylation reactions were performed in the reaction buffer containing 100 mM Tris-HCl pH 7.5, 15 mM MgCl2 (unless indicated), 3 mM ATP, 30 mM KCl, 5 µM [3H]phenyl-alanine, between 50 nM and 4 µM of tRNA transcript and 0.5-20 nM PheRS, depending on the nature of the transcript and the observed reaction rate. When indicated, PPase was added to the reaction mixtures to a final concentration of 20 U/ml. Renaturation of all tRNA transcripts was performed by heating in the presence of 1 mM MgCl2 for 90 s at 65°C followed by slow cooling to room temperature. Incubation was done at 37°C and aminoacylated tRNA samples were precipitated by 5% trichloroacetic acid, collected on GF/C filters (Whatman) and radioactivity was counted. The kinetic constants were calculated from Lineweaver-Burk plots and non-linear regression of the data. All values represent an average of the results obtained in at least two independent experiments. The relative efficiency of aminoacylation for different variants as compared to the wild-type tRNAPhe transcript was estimated using apparent kinetic parameters (in Michaelis-Menten approximation) and in particular by comparison of the specificity constant kcat/Km.
Rate of ATP-PPi exchange was measured in reaction buffer containing either 5 or 15 mM MgCl2, 1 mM PPi, 2 mM [[gamma]-32P]ATP (~2000 c.p.m./pmol) and 1 nM PheRS. Reaction progress was measured by the rate of ATP consumption by spotting 1 µl aliquots of the reaction mixture on PEI-cellulose plates. Plates were developed in 0.8 M LiCl and quantified using a phosphor-imager.
Rate of AMP production was measured in the standard reaction buffer containing either 5 or 15 mM MgCl2, 2 µM tRNA and 30 µM [[alpha]-32P]ATP at 100 mCi/mmol. PheRS concentration was 2 nM for YF0 aminoacylation at 15 mM MgCl2, 10 nM at 5 mM MgCl2 and 100 nM for aminoacylation of YF4. The reaction was monitored by TLC as described above.
PPi inhibition was measured by addition of PPi into standard reaction mixtures containing either 15 or 5 mM MgCl2, 1 µM YF0 tRNA and 0.5 or 2 nM PheRS, respectively. PPi concentration was varied between 1 and 100 µM. KI was deduced either from apparent Km of YF0 aminoacylation with and without 10 µM PPi, or from Dixon plots, using tRNA concentrations between 0.5 and 2 µM and PPi concentrations between 1 and 50 µM.
RESULTS
Selective enhancement of tRNAPhe mutants aminoacylation in the presence of PPase
In order to identify mutations leading to pyrophosphate-dependent reduction of the aminoacylation rate, we screened a set of yeast tRNAPhe transcripts with mutations in all three major identity regions (10): two mutants with the disrupted G19-C56 tertiary base pair, which occur between the D- and T-loops, and the compensatory mutant containing the inverted G19-C56 base pair (Fig. 1). The aminoacylation of these tRNA transcripts was tested both in the absence and in the presence of yeast inorganic pyrophosphatase in the reaction mixture. In agreement with our previous results (14), the addition of the PPase had almost no effect on the aminoacylation of wild-type tRNAPhe transcript (YF0) under the test conditions (15 mM Mg2+), while the charging of the YF4 mutant (G20U substitution) was considerably activated (Fig. 2 and Table 1). However, aminoacylation of the mutants with the A35U substitution in the anticodon (YF91) and the A73U substitution at the discriminator base position (YF66) were almost insensitive to the presence of PPase in the reaction mixture (Table 1)-only 2-fold activation was observed. Both tRNAPhe mutants with disrupted 3D structure (YF1 and YF4) appeared to be rather poor substrates for PheRS in our experimental conditions, but their aminoacylation was significantly improved in the presence of PPase (Fig. 2 and Table 1). Aminoacylation of the YF2 mutant with the compensatory mutations (G19C and C56G), which by itself is a much better substrate than both structural variants, was also improved by PPi cleavage but to a lesser extent (Table 1). It is worth mentioning that the influence of the mutations on the kinetic parameters of aminoacylation measured in this study is qualitatively the same but quantitatively stronger, than pre-viously reported (10,12). Most probably, this may be attributed to the differences in the conditions used for renaturation and amino-acylation of tRNA transcripts.
Figure 1. Cloverleaf structures of yeast tRNAPhe mutants used in this study. tRNAPhe identity elements are shaded. Mutations are shown by arrows. Corresponding mutants names are shown in parentheses.
Figure 2. Effect of PPase on aminoacylation of tRNAPhe variants. Closed symbols indicate no PPase, open symbols indicate presence of PPase. (A) YF1, circles; YF2, inverted triangles; YF3, squares; insert displays YF1 aminoacylation on a different scale. (B) YF0, inverted triangles; YF4, circles. Concentration of tRNAs was 1 µM. PheRS concentration was varied between 2 and 20 nM depending on the nature of the transcript and the observed reaction rate.
Table 1. Influence of inorganic pyrophosphatase on the kinetic parametersa of aminoacylation of various tRNAPhe mutants
| Name | Mutation | PPase | Plateaub | Plateau ratio | Km | kcat | kcat ratio | kcat/Km | kcat/Km ratio |
| % | (+/-) | (nM) | (1/min) | (+/-) | 1/nM.min | (+/-) | |||
| YF0 | - | 94 | 1.06 | 500 | 6.01 | 0.9 | 1.2 × 10-2 | ||
| + | 100 | 370 | 5.40 | 1.4 × 10-2 | 1.2 | ||||
| YF1 | C56G | - | 0.8 | 25 | 2300 | 0.03 | 16.6 | 1.3 × 10-5 | |
| + | 19.1 | 2200 | 0.50 | 2.2 × 10-4 | 16.9 | ||||
| YF3 | G19C | - | 0.7 | 30 | 3600 | 0.76 | 6.3 | 2.1 × 10-4 | |
| + | 21.3 | 700 | 4.82 | 6.8 × 10-3 | 32.4 | ||||
| YF2 | G19C | - | 9.1 | 6 | 2000 | 2.62 | 1.9 | 1.3 × 10-3 | |
| C56G | + | 54.1 | 340 | 5.14 | 1.5 × 10-2 | 11.5 | |||
| YF4 | G20U | - | 1.1 | 13 | 3200 | 0.12 | 14.8 | 3.7 × 10-5 | |
| + | 13.5 | 2900 | 1.77 | 6.1 × 10-4 | 16.5 | ||||
| YF91 | A35U | - | 6.2 | 1.2 | 1800 | 0.31 | 1.5 | 1.7 × 10-4 | |
| + | 8.4 | 1200 | 0.46 | 3.8 × 10-4 | 2.2 | ||||
| YF66 | A73U | - | 4.5 | 1.3 | 750 | 1.80 | 1.8 | 2.4 × 10-3 | |
| + | 6.1 | 1000 | 3.40 | 3.4 × 10-3 | 1.4 |
bPlateau values were determined at the same concentration of enzyme used for the kinetic constant measurements (see Materials and Methods).
Kinetic constants for aminoacylation of mutant tRNA transcripts are summarized in Table 1. Pyrophosphate hydrolysis generally results in the increase of kcat and decrease of Km values, although the extent of their variations differs considerably from one mutant to another. Taking into account the rather complex nature of the observed (apparent) Km and kcat parameters, we mostly use the specificity factor (kcat/Km) value as a measure of the overall catalytic efficiency, rather than consider the variations on these kinetic constants independently.
PPase-dependent activation correlates with distortion of tRNA tertiary structure
If inhibition of the aminoacylation reaction by PPi is related to the compromised tRNA tertiary structure, it should be observed even for wild-type tRNAPhe transcript aminoacylation in conditions where its tertiary structure is disrupted. Since distortion of the correct 3D structure of tRNAPhe transcript occurs at low Mg2+ concentration (14,15), we have studied the effect of PPase on the aminoacylation of wild-type and YF4 (G20U) tRNAPhe transcripts at different Mg2+ concentrations (Fig. 3). At Mg2+ concentrations below 5 mM, addition of PPase considerably activated the aminoacylation of wild-type transcript, while it had no effect at higher Mg2+ concentrations. PPase-dependent activation of wild-type tRNAPhe aminoacylation at low Mg2+ concentration is mostly due to the ~10-fold decrease of Km value in the presence of PPase (Table 2). The influence of PPi cleavage on the aminoacylation of YF4 mutant (G20U), tested at low Mg2+ concentration, was much more profound even than at 15 mM (Fig. 3).
Figure 3. Mg2+ dependence of PPase activation effect on YF0 (circles) and YF4 (squares) aminoacylation. Initial rate of aminoacylation was measured at different MgCl2 concentrations. PheRS concentration was 0.5 nM for YF0 and 5 nM for YF4 aminoacylation. Concentration of transcripts was 1 µM. Activation is calculated as a ratio of initial rate of aminoacylation in the presence of PPase to the rate in its absence at a given MgCl2 concentration.
Table 2. Influence of inorganic pyrophosphatase and PPi on the kinetic parametersa of wild-type tRNAPhe (YF0) aminoacylation at different Mg2+ concentrations
| [Mg2+] | PPase | Km | kcat, relative | kcat/Km | kcat/Km ratio | KI (PPi) | SI50 (PPi)b |
| (mM) | (nM) | (+/-) | (µM) | (µM) | |||
| 5 | - | 2000 | 0.12 | 0.03 | 2.1 | 4.5 | |
| 5 | + | 200 | 0.15 | 0.37 | 12.3 | ||
| 15 | - | 500 | 1 | 1 | 4.2 | 8.1 | |
| 15 | + | 370 | 0.9 | 1.35 | 1.2 |
bSI50 (PPi) was measured at 1 µM of YF0 transcript.
Enhancement of tRNA aminoacylation by PPAse is not related to the amino acid activation step of the reaction
Since it is possible that the rate of amino acid activation can be altered at 5 mM MgCl2, the rate of phenylalanyl-adenylate formation was measured at both 15 and 5 mM Mg2+. Activation rates, as measured by ATP-PPi exchange appeared to be the same (data not shown). Thus the lower rate of tRNA aminoacylation at 5 mM Mg2+ may be attributed to an alteration of tRNA structure.
PPase-dependent activation of aminoacylation at low Mg2+ presumes a stronger sensitivity of reaction to PPi inhibition in these conditions. Inhibition experiments showed that PPi is a potent inhibitor of aminoacylation even at standard conditions with IS50 of 8 µM (Table 2). However, a comparison of PPi inhibition of aminoacylation at low and high Mg2+ concentrations did not reveal substantial differences. In both cases PPi inhibition is competitive to tRNA with only a 2-fold decrease in KI and IS50 at 5 mM Mg2+.
One may expect that the observed PPase activation effect is due to the higher rate of PPi production (i.e. higher ATP hydrolyzed/Phe-tRNA formed ratio) upon aminoacylation of mutated tRNAs or wild-type transcript at low Mg2+ concentration. This possibility was tested by parallel measuring of tRNA aminoacylation and AMP production rates in the course of aminoacylation of the YF4 mutant and wild-type transcript at low MgCl2 concentration compared to YF0 aminoacylation in standard conditions. No significant enhancement of AMP production was found under our experimental conditions (data not shown).
DISCUSSION
By screening the set of tRNAPhe mutants we found that they fall into two groups according to sensitivity of their aminoacylation to PPi inhibition. Aminoacylation of tRNA mutants with single substitution of the discriminator base (N73) and in the anticodon is not activated in the presence of PPase. On the contrary, aminoacylation of the mutant with substitution at position 20 as well as of the structural mutants is significantly improved upon the cleavage of PPi. Since point mutations in the anticodon and at position 73 have no significant impact on the overall structure of the tRNA molecule (16), our results indicate that PPi dissociation becomes the limiting step of the reaction only when tRNA tertiary structure is disturbed. For both structural variants tested here, the introduced mutations disrupt the tertiary base pair stabilizing the interaction between D- and T-loops, thus changing significantly the overall structure of the molecule (16). Aminoacylation of these mutants is significantly improved in the presence of PPase, mainly due to the increase in the kcat. The double mutant with the reconstructed N19-N56 base pair, and thus mostly restored tertiary structure (16), still shows the increased Km value. It may reflect the presence of some residual structural distortions, probably due to the impaired stacking interaction involving G67, G18 and G19 (17), which are apparently sensed by PheRS. Hydrolysis of PPi in this case results in a several-fold decrease of Km.
The G20U substitution, unlike the other point mutations of identity elements, displayed the same kinetic phenotype as the structural mutations. Mutations introduced at this position do not change dramatically the overall tRNA structure, as was shown by Pb2+ cleavage test (16). Crystallographic data also indicates that G20 is not involved in any interactions with other nucleotides in tRNAPhe (18).Structural studies of yeast tRNAPhe indicate that the base of G20 is implicated in the formation of the Mg2+ binding site (19). Thus the substitutions introduced at this position may considerably affect magnesium binding through altering a local conformation of tRNAPhe at the `angle' region. This suggestion is in good agreement with the observed higher sensitivity to PPi inhibition of the G20U transcript aminoacylation at low Mg2+ concentrations. The other possibility is that G20 becomes involved either in tertiary interactions within the tRNA molecule or interacts with the protein upon the transitional conformational change of the tRNA-synthetase complex. The former possibility is well illustrated by the structural rearrangement of the conserved base G20b in tRNASer upon interaction with synthetase (20). Insertion of G20b from the D-loop into the tRNA core determines the correct orientation of the long variable loop of tRNA in the complex and directs the acceptor stem of the tRNA into the active site.
Correlation between tRNA structure and the phenomena of PPi inhibition is further confirmed by the PPase effect on Mg2+ dependence of aminoacylation. PPi dissociation becomes limiting at low Mg2+ concentration, where tRNA conformation is altered (14,15). Aminoacylation of wild-type transcript is not affected by PPase when Mg2+ concentration is sufficient to maintain native tRNA structure.
Thus the observed PPi-dependent inhibition of tRNAPhe mutants aminoacylation correlates with the distortions in the tertiary structure of the tRNA substrate. At the same, time point mutations in other major identity determinants such as anticodon and discriminator base clearly inhibit tRNA aminoacylation by another mechanism.
What could be a mechanism of PPi inhibition or, put another way, how could the distorted tertiary structure of the tRNA molecule be responsible for the observed change in the reaction mechanism? One possible explanation is that PPi dissociation is required for the enzyme to adopt the catalytically active conformation and the correctly folded tRNA molecule serves as a trigger of this process. Philosophically, from the aminoacyl-tRNA synthetase point of view, tRNA is just a rather complex device for the correct positioning of the terminal adenosine in the enzyme active site. For the vast majority of tRNAs, with identity elements located far away from the acceptor stem, this positioning may be achieved through the conformational changes of both the enzyme and tRNA (1-3). Evidence that both tRNA and aminoacyl-tRNA synthetase undergo significant mutual conformational changes upon the interaction comes both from biochemical experiments (1-3,21) and crystallographic data (20,22). Of special interest is the comparison of mutually exclusive adenylate- (A-) and tRNA-bound (T-) conformations of seryl-tRNA synthetase (23). Upon tRNA binding, a number of motif 2 active site residues previously found interacting with ATP or adenylate switch to tRNA binding. The switch from A- to T-conformation is most likely triggered by successful completion of the serine activation step. Pyrophosphate release may free Arg271 to interact with C74 and thus to stabilize correct tRNA 3[prime] end positioning in the active site to permit the aminoacylation step. Vice versa, tRNA binding may facilitate PPi release from the active site.
Our results can be described by a simplified scheme (Fig. 4). The proposed hypothetical mechanism is based on two assumptions: first, that PPi dissociation from enzyme-adenylate complex (complex I) is slower than the rate of its generation from ATP, which makes this complex a predominant adenylate-containing form of enzyme; and secondly that PPi-containing complex with tRNA (complex II) is catalytically inactive or marginally active. The second assumption is supported by the fact that complete inhibition by PPi can be achieved. According to the scheme, the major reaction pathway is through complex I to complex IV via complex II. tRNA binding to complex I triggers PPi dissociation with formation of the active complex IV. Apparently, certain mutant tRNA molecules cannot displace PPi and the reaction is blocked by formation of the inactive complex II. Since aminoacylation of neither anticodon nor N73 position substitutions is rescued by the PPi cleavage, it seems logical to suggest that interactions outside the identity elements are required to induce PPi dissociation. Besides identity elements, tRNA forms multiple backbone contacts with the enzyme (1,2) and it seems likely that this type of enzyme-tRNA interaction is responsible for the triggering of PPi dissociation. Distortion of the backbone interactions in structurally altered tRNA molecules apparently may slow down this process. Another question is why PPi dissociation is required for the productive interaction between tRNA and synthetase. As mentioned above, it is possible that the enzyme adopts a catalytically active conformation only after PPi dissociation. PPase may activate mutant aminoacylation by facilitating PPi dissociation from complex I and bypassing inactive complex II by rerouting the reaction through complex III. This scheme presumes that the reaction mechanism may be different in the presence or absence of PPase. Such PPase-induced changes of the aminoacylation mechanism have been observed for several aminoacyl-tRNA synthetases (3,24,25). Further experiments are required to confirm this hypothetical mechanism.
Figure 4. Hypothetical kinetic scheme of PPase-dependent activation of tRNA aminoacylation (detailed in text).
Details of the PPase-dependent activation mechanism are still unclear. The most obvious explanation would be that activation occurs due to the removal of product inhibition. But activation takes place at low levels of tRNA aminoacylation where the concentration of PPi formed (<1 µM) is well below its SI50 (Table 2) and thus cannot have any strong inhibitive effect unless its production, upon aminoacylation, of `wrong' tRNAs is tremendously enhanced. However, such enhancement is not observed under our experimental conditions. Thus PPase activation occurs at very low PPi concentration and the simple explanation of uplifting of the product inhibition displayed by external PPi in inhibition experiments may not necessarily be the right one. Similar observations were made earlier when PPase-induced changes in the kinetic mechanism of tRNA aminoacylation by several aminoacyl-tRNA synthetases were found (24,25). Thus a more complicated mechanism may exist. It does not seem unreasonable to hypothesize that prior to dissociation PPi forms a very tight complex with the enzyme and its dissociation may become a limiting step in the reaction. Formation of such a complex by yeast PheRS has been demonstrated (12). Hydrolysis of PPi may either facilitate dissociation of this complex or PPi in this complex may be directly accessible to PPase and its cleavage may result in the observed activation.
Is the PPi inhibition phenomena unique for aminoacylation catalyzed by yeast PheRS? We do have reason to believe that it may be of general importance. First of all high sensitivity of tRNA aminoacylation (especially in heterologous systems) to PPi inhibition was noticed a long time ago (12,26-30). Moreover, covalent binding of PPi to various aminoacyl-tRNA synthetases (aaRSes) has been demonstrated, suggesting the existence of a covalent intermediate between certain aaRSes and PPi (31,32). Finally, results very similar to those described above for PheRS were also obtained for tRNA aminoacylation by yeast aspartyl-tRNA synthetase (A.M.Khvorova, A.D.Wolfson and R.Giege, manuscript in preparation).
Our data suggest that PPi dissociation is an important, active step of the aminoacylation reaction. It may be associated with substantial conformational transitions within the tRNA-synthetase complex. Only binding of correctly folded tRNA molecules to aminoacyl-tRNA synthetase efficiently triggers PPi dissociation, which becomes a rate-limiting step in aminoacylation of tRNAs with altered tertiary structure. Further studies, probably using a modification interference approach, may allow the determination of the exact locations of nucleotides responsible for the induction of PPi dissociation and thus directly involved in the conformational rearrangement of the complex.
ACKNOWLEDGEMENTS
We are grateful to Drs L. L Kisselev, R. Giege and O. Uhlenbeck for helpful discussion and to Dr K. Polach for critical reading of the manuscript. This study was supported by grants from International Science Foundation (grant MRT300) and RFFI (Russian Fundamental Science Foundation) grant 95-04-12591. This paper is dedicated to the memory of Dr K. L. Gladilin, our teacher and friend.
REFERENCES
*To whom correspondence should be addressed at present address: Department of Biochemistry, University of Colorado, Campus Box 215, Boulder, CO 80309, USA. Tel: +1 303 492 6088; Fax: +1 303 492 3586; Email: wolfsona{at}spot.colorado.edu Present addresses: Anastasia Khvorova, Department of Molecular and Cellular Biology, University of Colorado, Boulder, CO 80309, USA Yuri Motorin, Maturation des ARN et Enzymologie Moléculaire (MAEM), UMR 7567, CNRS-UHP, Faculté des Sciences, Vandoeuvre-les-Nancy, France
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