| Human Molecular Genetics | Pages |
Isoleucylation properties of native human mitochondrial tRNAIle and tRNAIle transcripts. Implications for cardiomyopathy-related point mutations (4269, 4317) in the tRNAIle gene
Introduction
Results
Obtaining and characterizing the tRNA isoleucylation system from human placental mitochondria
Isoleucylation properties of in vitro tRNA transcripts
Discussion
Importance of modified nucleotides for isoleucylation by human mt IleRS
Towards understanding tRNAIle-related pathologies
Conclusion
Materials And Methods
Materials
Partial purification of the human mt aminoacyl-tRNA synthetases
ATP-PPi exchange activity
Purification of native human mt tRNAIle
Cloning and in vitro transcription
In vitro aminoacylation
Acknowledgements
References
Isoleucylation properties of native human mitochondrial tRNAIle and tRNAIle transcripts. Implications for cardiomyopathy-related point mutations (4269, 4317) in the tRNAIle gene
A growing number of mutated mitochondrial tRNA genes have been found associated with severe human diseases. To investigate the potential interference of such mutations with the primordial function of tRNAs, i.e. their aminoacylation by cognate aminoacyl-tRNA synthetases, a human mitochondrial in vitro aminoacylation system specific for isoleucine has been established. Both native tRNAIle and isoleucyl-tRNA synthetase activity have been recovered from human placental mitochondria and the kinetic parameters of tRNA aminoacylation determined. The effect of pathological point mutations present in the mitochondrial gene encoding tRNAIle has been tackled by investigating the isoleucylation properties of wild-type and mutated in vitro transcripts. Data show that: (i) modified nucleotides contribute to efficient isoleucylation; (ii) point mutation A4269G in the gene (A[rarr]G at nt 7 in the tRNA), associated with a cardiomyopathy, does not affect aminoacylation significantly; (iii) point mutation A4317G (A[rarr]G at nt 59 in the tRNA), reported in a case of fatal infantile cardiomyopathy, induces a small but significant decrease in isoleucylation. The potential implications of these findings on the understanding of the molecular mechanisms involved in the expression of pathology are discussed.
INTRODUCTION
In the past 7 years, an increasing number of point mutations in human mitochondrial (mt) DNA have been associated with pathologies (1-3). These point mutations are located either in the encoding genes for respiratory chain subunits, in rRNA genes or, in a higher proportion, in tRNA genes (4). Point mutations occurring in tRNA genes may be expected to result in a deficiency in translation and, indeed, investigations on mt protein synthesis in mt DNA-less cells repopulated with mutant mitochondria (cybrid cells) showed a decrease in the rate of mt protein synthesis and/or in steady-state levels of the translation products for six mutations (5-11). The molecular mechanisms leading to such decreases are, however, not well understood. Since mutations are spread over any parts of tRNA genes (1-3), several possible mechanisms have to be considered, including impaired maturation of the polycistronic transcripts or, if the tRNA is properly processed, production of tRNA with altered structures and hence functions. So, mutations may interfere with correct folding and/or post-transcriptional modification or hinder efficient recognition of tRNAs by partners in the protein synthesis machinery. The best yet documented molecular mechanism of pathogenesis concerns the MERRF A8344G transition in the tRNALys gene. In this case the mutated tRNA was shown to be less stable in vivo than the wild-type counterpart and, moreover, its steady-state aminoacylation level was reduced by 30%. These two effects cause reduced protein synthesis(10).
Since specific aminoacylation of tRNAs by their cognate aminoacyl-tRNA synthetases (aaRSs) is the most important prerequisite for faithful translation of the genetic information, we believe that investigation of the aminoacylation properties of mutated mt tRNAs versus the corresponding wild-type deserves more attention. In line with this view, in vitro aminoacylation systems involving human mt aaRSs have been set up in order to compare the aminoacylation properties of wild-type/mutant couples of in vitro transcribed human mt tRNAs (Brul et al., in preparation). Here we report the aminoacylation properties of wild-type and mutant in vitro transcripts of the mt tRNAIle. Although in vitro transcribed tRNAs have not undergone post-transcriptional modifications, a typical event for tRNAs, they proved to be efficient substrates for aminoacyl-tRNA synthetases in a large variety of systems from different organisms, such as Escherichia coli, yeast, mammals, and from organelles, such as mitochondria (12-14). Only a few exceptions have been described (12-14). The great technical difficulties of purifying mutated tRNA species from patients' mitochondria was a further argument in favor of using the in vitro transcription approach for preparation of human mt tRNAs.
Figure
In human pathology mutations in the mitochondrial gene encoding tRNAIle were first reported for two patients with fatal cardiopathies (15,16). These mutations, occurring at positions 4317 and 4269 respectively, correspond to A[rarr]G transitions. More recently five additional point mutations in the same gene were reported as associated with cardiopathies [mutation A4300G (17), mutation C4320T (18) and mutation A4295G (19)] or external ophthalmoplegia [mutation T4285C (20) and mutation T4274C (21)]. Biochemical data, available for mutations A4317G and A4269G, allow us to tackle the molecular mechanisms underlying pathogenic expression. Thus a decrease in both complex I and complex IV activities was reported in heart muscle of the patient carrying the A4317G mutation (15) and reduced complex IV activity was shown to occur in skeletal muscle and brain of the patient harboring the A4269G mutation (16,22). Characterization of cybrid cells containing predominant mt DNA carrying mutation A4269G (>95%) allowed the conclusion that accumulation of the mutation leads to a significant decrease in overall mt DNA-encoded polypeptide synthesis (9). Furthermore, the content of tRNAIle in these cells was substantially lower (~50%) than in control cybrid cells (9). In an attempt to understand the molecular mechanisms of the mt dysfunction associated with these two mutations, the in vitro isoleucylation properties of transcripts carrying mutation A4317G (referred to in what follows as [Ile(A59G)]) or mutation A4269G (referred to as [Ile(A7G)]) were established and compared with those of the corresponding wild-type transcript [IleWT]. These molecules are presented in Figure 1. The double nomenclature recalls both the position of the mutation within the mitochondrial genome (4317 or 4269) and within the transcribed tRNA (59 and 7 respectively). Characterization of human mt isoleucyl-tRNA synthetase activity (IleRS) towards its natural substrate, mt tRNAIle, is presented as well as a comparison of kinetic parameters for aminoacylation of pure native tRNAIle, wild-type and mutated tRNAIle transcripts.
RESULTS
Obtaining and characterizing the tRNA isoleucylation system from human placental mitochondria
In order to obtain large amounts of crude enzymatic extract and tRNA fractions the unique human tissue available without limitation, placenta was chosen as the source. Mitochondria were prepared by differential centrifugation and their purity checked with enzymatic markers. To avoid contamination by cytosolic proteins the external membranes of the mitochondria were removed by digitonin treatment, yielding mitoplasts.
Mitochondria purification. The quality of purification of human placental mitochondria was followed by lactate dehydrogenase activity (LDH), a cytosolic marker, and citrate synthase activity (CS), a mitochondrial matrix enzyme (Table 1). Contamination of the mitoplast extract by cytosolic enzymes was quantified by measurement of residual LDH activity. Only 0.13% of the total LDH activity detected in the homogenate fraction was found in the mitoplast fraction, indicating that most of the cytosolic proteins were removed during the purification process. The yield of mitochondrial purification was estimated as 61% as measured by CS activity in the mitoplasts and homogenate fractions (Table 1).Preparation of crude enzymatic mitochondrial extract. Preparation of the crude enzymatic extract was performed by consecutive lysis of mitoplasts, ultracentrifugation, dialysis and chromatography on a DEAE-cellulose column. This last step eliminates contaminating tRNA. Starting from one placenta, 75 mg protein containing IleRS activity were obtained. IleRS activity was monitored by its specific ATP-PPi exchange reaction and found to be 15 nmol ATP/min/mg protein. This activity, dependent on the presence of the specific amino acid, has the advantage of not requiring the cognate tRNA, a molecule very difficult to access in human mitochondria.
Isoleucylation properties of human native tRNAIle. The activity of the IleRS preparation was characterized by charging assays of native tRNAIle purified from human placenta. A low amount of pure tRNA was specifically extracted by hybridization to a biotinylated oligonucleotide. At the optimal enzyme concentration native tRNAIle was isoleucylated to 27% (Fig. 2a). To further characterize the isoleucylation properties of the tRNA kinetic parameters were established. A Lineweaver-Burk plot analysis gave an apparent Km for tRNA of 0.127 ± 0.02 µM and a Vmax of 392 ± 51 pmol/min/mg protein (Fig. 2b).
Table
Figure
Optimal isoleucylation levels of three in vitro transcripts derived from tRNAIle were measured with an enzyme concentration allowing maximal isoleucylation of the natural substrate. The wild-type transcript was isoleucylated to 17.8 ± 4.1%, mutant [Ile(A7G)] to 12.4 ± 1.9% and variant [Ile(A59G)] only to 5.3 ± 0.59% (Fig. 3a). Initial rates of isoleucylation were determined with increasing concentrations of transcript (0.6-30 µM) and the kinetic parameters for isoleucylation of the transcripts were derived from a standard Lineweaver-Burk plot (Fig. 3b). The apparent Km of the two mutants as compared with the wild-type transcript are similar Km[IleWT] = 2.92 ± 0.29 µM, Km[Ile(A7G)] = 3.08 ± 0.56 µM, Km[Ile(A59G)] = 3.01 ± 0.15 µM. The Vmax for [IleWT] was estimated as 187 ± 11 pmol/min/mg protein, whereas those of Vmax for [Ile(A7G)] and [Ile(A59G)] were 140 ± 28 and 47 ± 8 pmol/min/mg protein respectively. These data are summarized in Table 2.
Figure
Mitochondrial marker
Cytosolic marker
CS activity (U)
Recovery of mitochondria (%)
LDH activity (U)
Residual LDH activity (%)
Homogenate
302
100
12 040
100
Mitochondria
249
82
42
0.35
Mitoplasts
184
61
15.6
0.13
Isoleucylation properties of in vitro tRNA transcripts
DISCUSSION
Importance of modified nucleotides for isoleucylation by human mt IleRS
According to the endosymbiotic theory mitochondria evolved from sequestered bacteria (23). They share many molecular mechanisms and properties of their biomolecules with those of prokaryotic organisms. However, some of their aminoacylation systems diverge, since it has been shown that several E.coli synthetases are unable to aminoacylate mammalian mt tRNA (see for example 24). Thus, to investigate the in vitro aminoacylation properties of human mt tRNA, a homologous enzymatic activity has been prepared. The human mitochondrial crude enzymatic extract prepared from placenta and containing IleRS activity allowed us to isoleucylate native human tRNAIle as well as in vitro transcribed tRNA [IleWT] to appreciable levels (27 and 18% respectively). Although we cannot exclude the possibility that these moderate plateau levels reflect some special structural features relevant to preparation of the RNA molecules, they are consistent with those usually observed for mitochondrial aminoacylation systems. Typical examples concern native mitochondrial tRNAMet from Ascaris suum, which is aminoacylated to 20% (25), or transcripts from marsupial mitochondrial tRNAGly, aminoacylated to 10% (26).
Comparison of the kinetic parameters of aminoacylation of both native and wild-type in vitro transcribed tRNAIle (Table 2) reveals a marked difference in recognition by IleRS of the two substrates. The in vitro transcript is 48-fold less efficiently isoleucylated than the fully modified native molecule, as revealed by comparing the Vmax/Km ratios. This result shows an important contribution of modified nucleotides to isoleucylation. The large Km and the low Vmax of the transcript compared with the values for native tRNAIle strongly suggest that the modified residues in the native tRNA are involved in both the binding and catalytic steps of the aminoacylation reaction.
Modified nucleotides are generally not required to ensure aminoacylation of tRNAs by their cognate synthetases (12-14). However, some exceptions have been described, including E.coli tRNAGlu (27,28), tRNAIle (29,30) and tRNALys (31). The modification pattern of human mitochondrial tRNAIle has not yet been established, but should be similar to that of bovine mt tRNAIle, where three modifications were determined, namely m1G9, [psi]27 and t6A37 (32). Interestingly, in E.coli the absence in the isoacceptor tRNAIle1 of the modified base t6A37 leads to a strong kcat effect (30), whereas in the human mt system the absence of modified nucleotides leads to a mostly Km effect. Modified nucleotides in human tRNAIle may either contribute to isoleucylation as an identity element(s), i.e. elements responsible for specific aminoacylation, or in an indirect way through structural features (33).
Towards understanding tRNAIle-related pathologies
The wild-type in vitro tRNAIle transcript, being a rather efficient substrate for IleRS, has been considered an effective reference for comparative studies of the effects of point mutations on aminoacylation. The isoleucylation properties of the two variants [Ile(A7G)] and [Ile(A59G)] revealed that neither mutation drastically affects aminoacylation. However, some significant differences appear. The charging level as well as the kinetic parameters of isoleucylation of variant [Ile(A7G)] (Table 2) are not significantly changed as compared with the wild-type. In contrast, variant [Ile(A59G)] has a low charging level and a slightly but significantly decreased aminoacylation efficiency.
Mutation A7G occurs at the root of the acceptor stem of the tRNA and replaces an A-U base pair by a G-U pair (Fig. 1). G-U pairs are often found in tRNAs (see tRNA sequence compilation in 32) and were shown to introduce both local structural perturbations (see for example 34) and particular access to chemical groups (35). Interestingly, base pair 7-65 is always of canonical type (A-U) in mammalian mt tRNAIle (32). According to our data the presence of a G-U pair instead of an A-U pair does not perturb recognition by human mt IleRS in vitro. This is in line with present knowledge on isoleucine identity elements. Indeed, in E.coli tRNAIle1 identity elements do not include this residue. The complete set of determinants is formed by most of the anticodon loop nucleotides, the so-called discriminator base (A73) and three base pairs (C29-G41, U12-A23 and C4-G69) and involves structural elements (30). In conclusion, our data suggest that the pathogenetic effect of mutation A7G is not related to direct impairment of isoleucylation properties.
Table 2.
| Level of charging (%) | Vmax (pmol/min/mg) | Km (µM) | Vmax/Km (pmol/min/mg)/µM | Loss (relative to native) | |
| Native tRNAIle | 27 ± 2.6 | 392 ± 51 | 0.127 ± 0.02 | 3087 ± 887 | 1 |
| [IleWT] | 17.8 ± 4.1 | 187 ± 11 | 2.92 ± 0.29 | 64 ± 10 | 48 |
| [Ile(A7G)] | 12.4 ± 1.9 | 140 ± 28 | 3.08 ± 0.56 | 45 ± 17 | 69 |
| [Ile(A59G)] | 5.3 ± 0.59 | 47 ± 8 | 3.01 ± 0.15 | 16 ± 3.5 | 193 |
In vivo data concerning the patient carrying mutation A4269G showed a marked decrease in cytochrome c oxidase (COX) activity but no deficiency in the other respiratory chain complex activities in the nearly homoplasmic mutant patient's skeletal muscle (22). An ex vivo study, based on the establishment and analysis of cybrid cells bearing mutation A4269G, led to the conclusion that overall mt protein synthesis is decreased (9). In this latter study a decrease in the steady-state level of tRNAIle transcripts was also demonstrated. Interestingly, a recent study showed that aminoacylation induces conformational changes in human mt tRNAs, potentially hindering recognition by proteins of the translation apparatus (36). In line with this, mutation A4269G would not affect isoleucylation of the tRNA per se, but interfere with subsequent conformational changes and thus lead to a defective interaction with partners in the translational machinery. Elongation factor may be a good candidate, since this protein was shown to recognize aminoacylated tRNAs at the level of the acceptor arm (37). Related to this view is the recent finding of an anti-determinant box within the acceptor arm of the specialized selenocysteine-inserting tRNASec from E.coli that prevents its recognition by elongation factor (38); interestingly, this box contains a G-U pair at a location near to that of the mutant G-U in the mt tRNAIle pathogenetic variant.
In vitro isoleucylation properties of variant [Ile(A59G)] are different from those of the wild-type to a low but significant extent. The relevance of these effects to in vivo aminoacylation properties of the mutated species remains uncertain. However, a moderate effect is expected to be sufficient to lead to pathological consequences in vivo. Indeed, it is not expected that mt protein synthesis is totally blocked in patient cells, a situation which would lead to cell death. The sole in vivo studies described so far (10) are in favor of this view. Indeed, a 50% decrease in lysylation of human mt tRNALys carrying the 8344 point mutation was observed in a cybrid system by studying the steady-state level of lysyl-tRNA using an acid gel method. This decrease in mutant tRNA lysylation leads to alteration of the elongating peptide chain by premature mt translation termination and to an overall reduction in protein synthesis in cybrids. If a decrease of 50% in lysylation alters mt protein synthesis, it is believable that the 70% decrease in isoleucylation level found for the A59G mutation in vitro could reduce mt protein synthesis in a dramatic way as well.
Mutation A59G occurs in the T loop of the tRNA, at a position where the sequence is highly conserved in isoleucine-specific tRNAs (with one exception; see below). Nucleotide 59 has not been found as an aminoacylation identity element in cytosolic tRNAs investigated so far (12-14). In the case of animal mt tRNASer(AGY), however, the T loop region was shown to be a main recognition site for homologous seryl-tRNA synthetase (39). Thus nt 59 might contribute to identity in a direct or indirect way.
Mutation A59G may have a structural impact. Indeed, the presence of G59 allows formation of an additional base pair with C54. This, in turn, may lead to complete rearrangement of the T stem and shortening of the T loop, as suggested by Tanaka et al. (15) (see inset in Fig. 1). The natural occurrence of G59 in a single tRNAIle species (that from mouse) where restructuring of the T stem is not possible is an argument in favor of a structural interference of A59G with aminoacylation in human mitochondria. However, the fact that Vmax rather than Km is affected and the fact that melting profiles of both wild-type and variant A59G are identical (unpublished data) is not in favor of this hypothesis.
CONCLUSION
Isoleucine is an amino acid of predominant importance in human mitochondria. Indeed, 8.5% of the 3789 codons found in human mt protein gene sequences (40) encode for isoleucine. This percentage ranks isoleucine in third place, after leucine (16.9% of codons) and threonine (9.2% of codons). Furthermore, an isoleucine codon instead of a methionine codon initiates ND2 synthesis (subunit 2 of NADH coenzyme Q reductase) (40). For these reasons even faint changes in the aminoacylation properties of tRNAIle (a loss of isoleucylation efficiency or acquisition of mischarging properties) or a faint change in the level of available charged tRNAIle are expected to lead to important impairment of protein synthesis. Our data strongly suggest that mutation A59G affects the aminoacylation level of tRNAIle. Moreover, they also show that modified bases contribute to efficient aminoacylation in vitro. Acquisition of such modifications in vivo may, however, depend on integrity of the nucleotide sequence (41). Thus both mutations A7G and A59G may also affect aminoacylation in an indirect way. Furthermore, the lack of modified bases may contribute to increased instability of the tRNA (33). Thus the involvement of modified bases in expression of the pathological effects induced by point mutations in the tRNAIle gene might be important.
In conclusion, studies on specific mechanistic aspects of the protein synthesis machinery of in vitro transcribed human mt tRNAs has shed new light on our understanding of human mt tRNA-related diseases. They designate modified nucleotides as major contributors to the mt isoleucylation process, in both a direct and an indirect way. Further investigations on the modification patterns of wild-type and mutated native tRNAIle extracted from human cells would help in understanding the molecular mechanisms of the related pathologies. Unfortunately, no cell lines are available for mutation A59G (Dr Tanaka, personal communication). Our data suggest that further studies of structural and functional properties of other tRNA variants prepared in vitro is likely to be productive. It is noteworthy that five other point mutations have recently been described in the tRNAIle gene in patients presenting with cardiomyopathy (mutation C4320T, located very close to mutation A4317G, mutation A4300G and mutation A4295G) or external ophthalmoplegia (mutation T4285C and mutation T4274C), making the mt tRNAIle gene a hot-spot for pathogenic mutations. In a general view, 34 different point mutations occurring in tRNA genes and associated with pathologies are presently described in the literature. We are investigating some of the corresponding tRNAs by the in vitro approach.
MATERIALS AND METHODS
Materials
Human placentae were recovered from a local hospital within 1 h after delivery and transported to the laboratory on ice. [[gamma]-32P]ATP (3000 Ci/mmol) and L-[4,5-3H]isoleucine (94 Ci/mmol) were from Amersham (Les Ulis, France), [32P]pyrophosphate (Ppi) was from NEN Research Product (Les Ulis, France). Digitonin, saccharose, a bicinchoninic protein determination kit and phenol were from Sigma (St-Quentin-Fallavier, France), T4 polynucleotide kinase from Biolabs (Ozyme, Paris, France), RNasin and streptavidin-coated magnetic beads from Promega (Charbonnière, France). Oligonucleotides were purchased from Genset (Paris, France) and nylon membranes (Biotrans 0.2 µm) were from ICN (Orsay, France). DEAE-cellulose (Whatman DE52) was from OSI (Maurepas, France). All other chemicals were of the highest quality available.
Partial purification of the human mt aminoacyl-tRNA synthetases
All steps were carried at 4°C with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM [beta]-mercaptoethanol in all buffers. Human placental mitochondria were obtained by conventional differential centrifugation after homogenization of the tissue by grinding (42). The mitochondria were then subjected to digitonin treatment (12 mg/100 mg protein) to remove the external membrane (43). CS (a mitochondrial matrix enzyme) and LDH (a cytosolic enzyme) activities were assayed as previously described (44,45). Mitoplasts were then lysed and homogenized (15 strokes of a Potter homogenizer) in the presence of 0.1% Triton X-100, 100 mM NH4Cl, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 50 mM Tris-HCl, pH 7.5. The lysate was centrifuged at 100 000 g in a Beckman Ti60 rotor for 1 h at 4° C. After 2 h dialysis against buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol) the supernatant was loaded onto a DEAE-cellulose column equilibrated in buffer A in order to remove tRNAs. Elution was performed in one step with 250 mM potassium phosphate, pH 6.5, 10% glycerol. The corresponding fraction was then dialyzed against buffer A, then against bufferA containing 50% glycerol and stored at -20°C. Protein concentration determination was by the bicinchoninic acid method using bovine serum albumin as the standard (46). In a typical experiment, starting from one placenta, 30 ml crude enzymatic extract at a protein concentration of 2.5 mg/ml were obtained.
ATP-PPi exchange activity
The presence of IleRS activity in the enzymatic extract has been established by measurement of its ATP-PPi exchange activity (47), which accounts for isoleucyl-adenylate formation, the first step in the tRNAIle aminoacylation process. This specific assay, in the present case, does not require the specific tRNA, a rare species in human mitochondria (see below) and thus is a useful alternative to the tRNA aminoacylation assay for characterization of IleRS. Amino acid-dependent ATP-PPi exchange measures incorporation of [32P]PPi under conditions of equilibrium of the amino acid activation step. Rates of ATP-PPi exchange catalyzed by IleRS were measured at 37°C in 100 µl containing 100 mM HEPES, pH 7.2, 1 mM ATP, 1 mM dithiothreitol, 10 mM MgCl2, 10 mM KF, 1 mM [32P]PPi (2 c.p.m./pmol) and 10 µl of the enzymatic fraction under study. The reaction was initiated by addition of 1 mM isoleucine. Labeled ATP was adsorbed on charcoal, filtered and counted.
Purification of native human mt tRNAIle
Mitochondria were purified on a sucrose gradient (48) and frozen in liquid nitrogen. Mitochondrial tRNAs were then prepared by acid phenol extraction, followed by a DEAE-cellulose column as described previously (49). This method allows us to obtain tRNAs, but still contaminated with high molecular weight RNAs. tRNAs were further purified on a Qiagen tip 500 column. The RNA solution was adjusted to 50 mM MOPS, 15% ethanol, pH 7.0, prior to being loaded on the column and the elution step was carried out with the same buffer containing 0.8 M NaCl. tRNAs were precipitated with isopropanol. The pellets were dissolved in water and tRNAs analyzed on 12% acrylamide-7 M urea gels. The concentration of tRNA was determined by the A260 absorbance (1 OD unit = 40 µg/ml). From 500 g placenta, ~500 µg total mt tRNAs were obtained. tRNAIle was extracted from total mt tRNAs according to Mörl et al. (50) by hybridization to a complementary biotinylated oligonucleotide bound to streptavidin-coated particles and purification on a denaturing 8% polyacrylamide-7 M urea gel. The sequence of the oligonucleotide was 5[prime]-biotin-TGGTAGAAATAAGGGGGTTTAAGCTCCTATTAT-3[prime]. With this technique 5 µg purified tRNAIle were recovered from 750 µg total human mt tRNAs as quantified by OD260. The purity of this native tRNA has been checked by occurrence of a major band (>90%) on a denaturing 8% acrylamide-7 M urea gel after 5[prime]-end-labeling. Furthermore, the RNase T1 digestion pattern of the purified native tRNAIle was similar to the transcript T1 profile (data not shown).
Cloning and in vitro transcription
Synthetic genes corresponding to the T7 RNA polymerase promoter directly connected to the downstream human mt tRNAIle wild-type (40) and mutated (15,16) sequences, all ending at a BstNI recognition site, were constructed from four overlapping and complementary oligonucleotides. The oligonucleotides were hybridized and ligated into pTFMA, a pUC derivative, linearized at HindIII and BamHI sites. Amplification of the plasmids was with transformed E.coli TG2 cells. In vitro transcription was performed as previously described (51). Purification of transcripts was performed on short run preparative denaturing 10% polyacrylamide-7 M urea gels. Long runs of electrophoresis (18 h) did not allow detection of 3[prime]-end heterogeneity, as often occurs for in vitro transcripts. Transcripts were then excised from the gel, electroeluted and ethanol precipitated, desalted by gel filtration on Sephadex-G25 spin columns and quantitated by optical density measurement at 260 nm, assuming that 1 OD260 unit = 40 µg/ml. Based on nucleotide composition, 1 µg tRNAIle transcript corresponds to 43 pmol.
In vitro aminoacylation
Prior to being aminoacylated, the in vitro synthesized tRNAs were denatured for 5 min at 68°C in water and renatured from a completely melted molecule by cooling slowly to room temperature. Aminoacylation assays were performed at 30°C in mixtures containing 100 mM Tris-HCl, pH 8.5, 5 mM MgCl2, 2 mM ATP, 10 mM KCl, 10 µM L-[4,5-3H]isoleucine (4500-7500 c.p.m./pmol), 0.6 U/µl RNasin and 0.5 mM CTP. These optimal aminoacylation conditions were chosen after screening pH (7.0-9.0), different MgCl2/ATP ratios (1-3 with ATP at 2 mM) and temperature (37 and 30°C). For the natural substrate a preliminary deacylation step at 75°C for 5 min in 10 mM Tris-HCl, pH 8.5, did not significantly change the isoleucylation level, showing that the purified tRNAIle is no longer charged. In the same fashion, renaturation of transcripts with 10 mM MgCl2 did not enhance aminoacylation capacity.
For determination of the kinetic parameters of the transcripts two independent experiments were realized for each concentration of substrate. Various concentrations of enzyme were tested to ensure initial velocities. Aliquots were withdrawn at different times, spotted on 3MM Whatman papers and treated by the trichloroacetic acid method as usual. Radioactivity retained on papers was measured by liquid scintillation counting. To correct for the low counting efficiency of free [3H]Ile in comparison with that of [3H]Ile-tRNA, total charging assays with 3H- and 14C-labeled isoleucine were performed in parallel so that a conversion factor could be calculated.
ACKNOWLEDGEMENTS
This work benefited from support by the Association Française contre les Myopathies (AFM), by the Institut National de la Santé et de la Recherche Médicale (INSERM) and by the Centre National de la Recherche Scientifique (CNRS). H.B. was the recipient of a Poste d'Accueil INSERM and M.H. a grant from the European Communities. We thank G.Attardi for critical reading of the manuscript.
REFERENCES
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[Full Text]
S. O. Kelley, S. V. Steinberg, and P. Schimmel
Fragile T-stem in Disease-associated Human Mitochondrial tRNA Sensitizes Structure to Local and Distant Mutations
J. Biol. Chem.,
March 30, 2001;
276(14):
10607 - 10611.
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