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Genes to Cells (2004) 9, 243-252. doi:10.1111/j.1356-9597.2004.00718.x
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The pathogenic A4269G mutation in human mitochondrial tRNAIle alters the T-stem structure and decreases the binding affinity for elongation factor Tu

Narumi Hino1, Tsutomu Suzuki1, Takehiro Yasukawa1, Kohji Seio2, Kimitsuna Watanabe1 and Takuya Ueda1,*

1 Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8562, Japan
2 Frontier Collaborative Research Centre, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama, Japan


   Abstract
Top
 Abstract
Introduction
 Discussion
 Experimental procedures
 References
 
The A4269G mutation in the human mitochondrial (mt) tRNAIle gene is associated with fatal cardiomyopathy. This mutation completely inhibits protein synthesis in mitochondria, thereby significantly reducing their respiratory activity. The steady-state amount of tRNAIle in cells bearing the A4269G mutation is almost half that of control cells. We previously reported that this mutation causes tRNAIle to be unstable both in vivo and in vitro. To investigate whether the instability of the mutant tRNAIle is due to structural alterations, a nuclease-probing experiment was performed with a mitochondrial enzymatic extract, which showed that the A4269G mutation destabilizes the T-stem of the mutant tRNAIle. In addition, measurements of the binding affinity of the aminoacylated mutant tRNAIle for mt elongation factor Tu (EF-Tu) showed that the mutant tRNAIle binds mt EF-Tu less efficiently than the wild-type does. This observation provides insight into the molecular pathology associated with tRNA dysfunction caused by this pathogenic point mutation.


   Introduction
Top
 Abstract
 Introduction
Discussion
 Experimental procedures
 References
 
Mitochondrial (mt) DNA mutations cause disorders of various tissues that rely heavily on oxidative phosphorylation for their energy supply. These tissues include the central nervous system, skeletal muscle, heart muscle and the liver. More than 100 different pathogenic point mutations or rearrangements in mt DNA have been described so far (Kogelnik et al. 1998). A number of these point mutations reside in tRNA genes.

It has been reported that pathogenic point mutations in mt tRNAs impair various aspects of tRNA maturation or function (Florentz 2002). For example, certain point mutations in mt tRNALeu(UUR) and mt tRNALys cause defects in post-transcriptional modification (Suzuki et al. 2002; Yasukawa et al. 2001, 2000b,c). A mt tRNALys with the A8344G mutation lacks the wobble modification and was found to be incapable of translating cognate codons due to the complete loss of codon–anti-codon pairing on the ribosome (Yasukawa et al. 2001). We recently also found that mt tRNAIle bearing the A4317G mutation, which is associated with fatal infantile cardiomyopathy, and mt tRNAGly bearing the A10044G mutation, which is associated with sudden infant death syndrome, exhibit decreased CCA-addition (Tomari et al. 2003). In addition, it has been reported that some pathogenic point mutations in mt tRNAIle reduce the efficiency of 3' end processing in vitro (Levinger et al. 2003) and that others reduce the efficiency of aminoacylation (Kelley et al. 2000, 2001).

Cardiomyopathy and ophthalmoplegia patients have been found to harbour 10 different point mutations of the mt tRNAIle gene. The A4269G mutation, which is associated with fatal cardiomyopathy (Taniike et al. 1992), was characterized in cybrid cells, which are constructed by the intercellular transfer of patient mt DNA to human cells lacking mt DNA ({rho}0 cells)(Hayashi et al. 1994). This study revealed that this point mutation completely inhibits protein synthesis in mitochondria, thereby significantly reducing their respiratory activity. The steady-state amount of the mt tRNAIle molecule bearing the A4269G mutation was also substantially lower (~50%) in cybrid cells than that of the wild-type tRNAIle in control cybrid cells (Hayashi et al. 1994). Moreover, we previously demonstrated by in vivo and in vitro experiments that the mutant tRNAIle is degraded more rapidly than wild-type tRNAIle (Yasukawa et al. 2000a). The observation that the mutant tRNAIle is partially denatured even at a physiological temperature (Yasukawa et al. 2000a) prompted us to speculate that it is more sensitive to mitochondrial nucleases than its wild-type equivalent. However, considering the steady state level (~50%) of the mutant tRNAIle, its rapid degradation cannot fully explain the complete loss of mitochondrial translation. In this study, we investigated the molecular mechanism by which the A4269G point mutation affects the translational activity of the mutant tRNAIle. We hypothesized that since it is located at the end of the acceptor stem, where the elongation factor Tu (EF-Tu) binds (Nissen et al. 1995), it modulates the binding of EF-Tu and thereby inhibits translation. To address this possibility, we estimated the binding affinity of mutant tRNAIle for mt EF-Tu.

Results tRNAIle matures normally in cybrid cells bearing the A4269G mutation

It has been reported that some pathogenic mutations affect steps in tRNA maturation, including-3' end processing and the addition of CCA (Levinger et al. 2003; Tomari et al. 2003). We first analysed the steady-state level of the mutant tRNAIle (A4269G) and its precursor RNA in cybrid cells using Northern blotting. We employed mt tRNAMet as a positive control, since the mt tRNAMet gene is located downstream of the mt tRNAIle gene and is transcribed into the same precursor RNA (Fig. 1A). As shown in Fig. 1B, the levels of mutant tRNAIle in the mutant cybrid are reduced (< 50% of that in the control cybrid), as expected, while both cells have equivalent levels of tRNAMet (data not shown). We then assessed the effect of the mutation on 3' end processing. If this step is impaired by the mutation, precursor tRNAIle molecules bearing the 3' trailer sequence should be detectable (Fig. 1A). However, no band other than that corresponding to the processed tRNAIle could be observed in mutant cybrid cells as well as in control cybrid cells (Fig. 1B), even after the gels were subjected to longer exposure (data not shown). Thus, the processing step of the maturation of the tRNAIle molecule proceeds normally in cybrid cells bearing the A4269G mutation.



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  		Figure 1  Effects of pathogenic point mutations on tRNA maturation. (A) Organization of the tRNAIle locus in the mt genome. The arrowheads indicate processing sites and the bold line indicates the normally processed tRNAIle transcript (69 nucleotides). The position of the Ile oligonucleotide probe used in Northern blot analysis is shown as a grey line. (B) Northern blot analysis of tRNAIle levels in cybrid cells with or without the A4269G mutation. RNA samples were prepared from cybrid cells with (A4269G) or without (control) the A4269G mutation. (C) Effect of pathogenic point mutations on CCA-addition. The ability of the human mt CCA-adding enzyme to repair wild-type, A4269G-bearing and A4317G-bearing tRNAIle transcripts that lack the 3'-terminal CCA sequence was assessed in vitro in the presence of CTP and [{alpha}-32P]-ATP.

 
We recently reported that the A4317G mutation in mt tRNAIle seriously hinders the CCA-adding process (Tomari et al. 2003). Thus, we examined the CCA-accepting activity of the A4269G tRNAIle and found that it is almost the same as the wild-type in this respect, while the A4317G tRNAIle shows little activity, as reported previously (Fig. 1C) (Tomari et al. 2003). These results are also supported by the fact that an extra band representing the mutant tRNAIle with a shortened CCA-terminus is not observed on Northern blots of total RNA prepared from cybrid cells (Fig. 1B). Thus, the CCA-adding process in the mutant cybrid does not appear to be affected by the A4269G mutation.

Nuclease-probing of mutant tRNAIle by mitochondrial enzymatic extract

We previously reported that the A4269G tRNAIle molecule is degraded much more rapidly than its wild-type counterpart in cybrid cells (Yasukawa et al. 2000a). Moreover, when the A4269G tRNAIle was obtained from cybrid cells and treated with a mitochondrial enzymatic extract, it was degraded more quickly than the wild-type tRNAIle. In addition, we observed that the digestion pattern of the mutant tRNA was different from that of the wild-type (Yasukawa et al. 2000a), indicating that the A4269G mutation alters the conformation of the tRNAIle molecule. Thus, we next investigated the structure of the mutant tRNAIle by a nuclease-probing experiment. Before we did this experiment, we examined the degradation rate of in vitro-transcribed tRNAIle with or without the A4269G mutation by the mitochondrial enzymatic extract and found that the transcribed mutant tRNAIle is more rapidly degraded than the wild-type tRNA (Fig. 2A), which nicely conforms to our previous result of the native tRNAs (Yasukawa et al. 2000a). Moreover, the in vitro-transcribed wild-type and mutant tRNAIle molecules had similar isoleucine-accepting activities with partially purified mitochondrial isoleucyl-tRNA synthetase (data not shown). This result indicates that the in vitro-transcribed tRNAIle with or without the mutation adopts a largely correct tertiary structure that is sufficient for aminoacylation. Hence, we employed in vitro-transcribed tRNA molecules in the following experiments.



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  		Figure 2  Nuclease-probing of mutant and wild-type tRNAIle molecules. (A) In vitro degradation of in vitro-transcribed wild-type ({circ}) and mutant ({blacksquare}) tRNAIle molecules. 32P-labelled tRNAIle molecules were incubated with a mitochondrial enzymatic extract, and intact tRNAIle molecules were measured as described (Yasukawa et al. 2000a). The average values at 0 h were arbitrarily defined as 100%. Bars indicate the standard deviation for 4 measurements. (B) An enlarged view of nuclease cleavage patterns corresponding to the area connecting the acceptor stem and the D-arm of wild-type and mutant tRNAIle molecules. The abbreviations –E, Al, T1 and U2 indicate no treatment, alkaline digestion, RNase T1 digestion (specific for G) and RNase U2 digestion (specific for A > G), respectively. The S100 lanes contain tRNAs that were partially digested with increasing amounts of the mitochondrial enzymatic extract under non-denaturing conditions. (C) An enlarged view of nuclease cleavage patterns corresponding to the T-arm region. (D) The secondary structure of the tRNAIle molecule and the unique cleavage sites in the mutant molecule. The black arrowheads indicate positions where the mutant is more nuclease-sensitive than the wild-type. The uridine-specific cleavage sites, whose products bear 5' hydroxyl group, and the nonspecific cleavage sites, whose products bear 5' phosphates, that are common to both the wild-type and mutant tRNAsIle are shown by the open and grey arrowheads, respectively. The size of each arrowhead indicates the cleaving efficiency.

 
Mutant tRNAsIle were subjected to a nuclease-probing experiment using the mitochondrial enzymatic extract. The patterns of cleavage (Fig. 2) reveal that the mitochondrial extract contains a uridine-specific RNase whose products probably bear a 5' hydroxyl group, as well as nonspecific nucleases whose products probably bear a 5' phosphate at the end of the cleaved fragments, judging from their migration patterns compared with alkaline-digested ladders of molecules bearing 5' hydroxyl groups. As expected, mitochondrial nucleases preferentially cleaved bases in the loops (Fig. 2D). Since many cleavage sites are common to both the mutant and wild-type tRNAIle molecules, it appears that the overall structure of the mutant tRNAIle is similar to that of the wild-type tRNAIle. However, some differences were observed. First, a site that is 3' adjacent site (U8-G9) to the mutation site (A7G) is cleaved, whereas the same position in the wild-type tRNAIle is not (Fig. 2B). Second, the T-stem (C61-U64) of the mutant tRNAIle is also somewhat more sensitive to mitochondrial nucleases than the wild-type (Fig. 2C), which suggests that the A4269G mutation destabilizes the T-stem structure. These differences in digestion profile demonstrate that the point mutation alters the structure of the mutant tRNAIle and renders it susceptible to mitochondrial nucleases.

Binding affinity of mt EF-Tu for the chemically aminoacylated mutant tRNAIle

It has been reported by Hayashi et al. (1994) that A4269G cybrid cells exhibit complete loss of overall mtDNA-encoded protein synthesis, as well as a significant reduction in respiratory chain activity. In this study, we have found that the T-stem of the mutant tRNAIle is destabilized by the A4269G mutation. As this structure is a binding site for EF-Tu (Nissen et al. 1995), we speculated that the destabilized T-stem modulates the binding affinity of EF-Tu and thereby inhibits mitochondrial translation, which might be a molecular mechanism contributing to pathogenesis along with the reduced steady-state level of the mutant tRNAIle. This prompted us to measure the binding affinity of mt EF-Tu for the aminoacylated mutant tRNAIle.

To obtain sufficient amounts of aminoacylated tRNAsIle for this purpose, we synthesized chemically aminoacylated tRNAIle by ligating N-(4-pentenoyl)valyl-pdCpA (Fig. 3A), because valyl-tRNA is known to be resistant to deacylation (Schofield & Zamecnik 1968) and EF-Tu can also bind misacylated tRNA. The aminoacylated tRNAs were homogeneous due to the high ligation efficiency of T4 RNA ligase (Fig. 3B). Prior to the binding experiment, the 4-pentenoyl group (Fig. 3A) of the aminoacylated tRNA was removed by iodine deprotection according to the method of Lodder et al. (1997). The complete deprotection of the aminoacylated tRNAs was confirmed by nucleoside analysis using LC/MS (Fig. 3C). This allowed us to observe the presence of 4-pentenoyl-valylated adenosine in aminoacylated tRNAIle before deprotection. After deprotection, valylated adenosine appeared in the place of 4-pentenoyl-valylated adenosine.



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  		Figure 3  Preparation of aminoacylated tRNAIle using T4 RNA ligase. (A) The chemical structure of N-(4-pentenoyl)-(S)-valyl-pdCpA-tRNAIle. (B) Ligation efficiency, as estimated by denaturing polyacrylamide gel electrophoresis of the 5'-32P-labelled tRNAIle before and after ligation. (C) Mass spectrometric analysis of deprotection efficiency. Mass chromatograms for proton adducts of N-(4-pentenoyl)-(S)-valyl-adenosine (m/z 449.2) and valyl-adenosine (m/z 367.2) are shown in the upper and lower panels, respectively.

 
A fixed amount of EF-Tu:GDPNP and increasing amounts of the valyl-tRNAsIle (Val-tRNAsIle) with and without the mutation were coincubated and the resulting EF-Tu:GDPNP:Val-tRNAIle ternary complex (TC) was analysed by gel mobility shift analysis (Fig. 4). Upon complex formation, the increased negative charge of the bound Val-tRNAIle caused the TC to be highly mobile. As shown in Fig. 4A, bovine mt EF-Tu has a higher affinity for wild-type tRNAIle than for the mutant tRNAIle, although the TC appeared as smeared bands due to the high dissociation rate of mt EF-Tu from Val-tRNAsIle. The apparent Kd values for TC formation were calculated from the band intensities of free mt EF-Tu and the TC. By Scatchard plot, the apparent Kd values of the binding of Val-tRNAIle with and without the mutation to bovine mt EF-Tu were estimated to be 5.27 µM and 11.29 µM, respectively. To confirm this difference in binding affinity for EF-Tu, we performed the same experiment using Thermus thermophilus EF-Tu, which forms a more stable TC than bovine mt EF-Tu does. The T. thermophilus EF-Tu formed stable ternary complexes in the gel (Fig. 4B). The apparent Kd values for the binding of Val-tRNAIle with and without the mutation to T. thermophilus EF-Tu were determined to be 0.16 µM and 0.31 µM, respectively. The two-fold weaker affinity of the mutant tRNA for T. thermophilus EF-Tu is consistent with the result obtained with bovine mt EF-Tu. These results show that the A4269G mutation causes inefficient TC formation, which might affect the efficiency of mitochondrial translation.



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  		Figure 4  Binding affinity of EF-Tu for chemically aminoacylated tRNAIle molecules. Gel mobility shift assays of the binding of bovine mt EF-Tu:GDPNP (A) or Thermus thermophilus EF-Tu:GDPNP (B) to Val-tRNAIle with or without the mutation. The free and bound Val-tRNAsIle were separated by electrophoresis on 5% polyacrylamide gels.

 

   Discussion
Top
 Abstract
 Introduction
 Discussion
Experimental procedures
 References
 
It has been reported that pathogenic point mutations in mt tRNAs impair various steps of their maturation (Florentz 2002). In the case of the A4269G mutation, the in vitro 3' end processing efficiency (Vmax/KM) of the mutant tRNAIle has been estimated to be reduced 12.5-fold lower than that of the wild-type transcript (Levinger et al. 2003). In this study, we tried to detect the precursor tRNAIle by Northern blot analysis but did not observe an extra band corresponding to this transcript. This result suggests that A4269G tRNAIle is transcribed and processed normally in cybrid cells, although it is possible that the precursor tRNAIle is degraded too rapidly to be detected in this manner.

We found that the A4269G mutation alters the tRNAIle tertiary structure, and that this destabilizes the T-stem, which is a binding site for bacterial EF-Tu (Nissen et al. 1995). Supporting this interpretation is the finding that the U50-G64 wobble base pair in the T-stem of the E. coli initiator tRNAMet acts as an anti-determinant for EF-Tu (Stortchevoi et al. 2003). In addition, in yeast and plant initiator tRNAs, anti-determination with respect to EF-Tu can be caused by a bulky ribose modification at position 64 in the T-stem (Forster et al. 1993; Kiesewetter et al. 1990). Since mt EF-Tu structurally resembles bacterial EF-Tu (Andersen et al. 2000), it is plausible that this conformational change in the T-stem reduces the binding affinity of mt EF-Tu. In this study, we examined the binding affinity of EF-Tu for the chemically aminoacylated mutant tRNAIle molecule by a gel mobility shift assay. We found that mt EF-Tu binds to the mutant tRNAIle less efficiently than to the wild-type tRNAIle. The Kd value of the binding affinity between the mutant tRNAIle and EF-Tu is about twice that of the wild-type molecule and EF-Tu. It has been reported that the Kd value for the bovine mt EF-Tu: GTP: E. coli Phe-tRNAPhe ternary complex is 18 ± 4 nM, as determined by an RNase A protection assay (Cai et al. 2000). In contrast, in our study, the Kd values using bovine mt EF-Tu were in the micromolar range, as revealed by gel mobility shift analysis. The large difference in Kd values is likely due to the dissociation of the TC during electrophoresis, since we detected TCs as smeared bands. The gel mobility shift assay was established to compare the relative binding affinities of the TC or other RNA-protein complexes (Ohndorf et al. 2001; Wagner et al. 1984; Wu & RajBhandary 1997). As the apparent Kd values for the affinity of bovine mt EF-Tu for Ala-mt tRNASer variants, as determined by this method (Hanada et al. 2001), are almost the same as that for the affinity of bovine mt EF-Tu for Val-mt tRNAsIle we report here, we conclude that the gel mobility shift experiments are reliable. When we employed T. thermophilus EF-Tu, which forms a stable TC as seen in the gel mobility shift assay, the dissociation of the TC in the gel was reduced (Fig. 4B), and the apparent Kd values became much lower than that for mt EF-Tu but still larger than the value determined by the RNase A protection method. In this study, aminoacylated tRNA was prepared by chemical acylation. To prevent self-ligation of tRNA, the 5' phosphate group of the tRNA was removed prior to ligation. According to the crystal structure of the TC, the 5' phosphate of yeast Phe-tRNAPhe forms a salt bridge with a conserved residue, Arg300, of T. aquaticus EF-Tu (Nissen et al. 1995), indicating that the absence of a 5' phosphate from Val-tRNAIle partly explains the large Kd values. In addition, in vitro transcribed tRNA without any modifications might reduce the binding affinity of EF-Tu. In both experiments using bovine mitochondrial or T. thermophilus EF-Tu, the apparent Kd value of EF-Tu for the mutant tRNA is about twice that of of EF-Tu for the wild-type tRNA. We consider this difference in binding affinity to be reliable.

In mammalian mitochondria, the concentration of EF-Tu and total tRNA are estimated to be 0.3 µM and 0.9 µM, respectively (Cai et al. 2000; King & Attardi 1993; Schwartzbach & Spremulli 1989). Considering the Kd value (18 ± 4 nM) of the ternary complex, most EF-Tu molecules appear to participate in TC formation (Cai et al. 2000). Assuming that there are equal amounts of the 22 species of tRNA in the mitochondrion, the tRNAIle concentration is approximately 40 nM. The concentration of A4269G tRNAIle is estimated to be roughly 20 nM, according to the steady-state amount of the mutant tRNAIle (Hayashi et al. 1994). The concentration of the mutant tRNA is close to the Kd value of the TC, and under such conditions, the twofold increased Kd value would significantly affect TC formation in vivo.

Furthermore, the binding of EF-Tu to aminoacylated tRNAs is believed to protect them from degradation in vivo (Pingoud et al. 1977), suggesting that the high degradation rate of the mutant tRNAIle might partly be explained by a reduced efficiency of TC formation. If this is the case, it may be possible to protect the mutant tRNA from degradation by providing a large amount of mt EF-Tu. We examined the degradation rate of Val-tRNAIle by mitochondrial enzymatic extracts in the presence of EF-Tu and found that the mutant tRNAIle molecules became resistant to mitochondrial nucleases with increasing concentrations of mt EF-Tu (data not shown). This result indicates that high mt EF-Tu levels protect the mutant tRNAIle from rapid degradation in the cell. In support of this finding, it has been reported that pathogenic point mutations in yeast mt tRNA can be rescued by over-expressing mt EF-Tu (Feuermann et al. 2003). Further investigation will be required to clarify whether the mitochondrial dysfunction associated with the A4269G mutation can be compensated for by the over-expression of mt EF-Tu.

We summarize the molecular pathogenesis associated with the A4269G mutation as follows. The mutant tRNAIle matures normally in cybrid cells. The aminoacylation efficiency of the mutant tRNAIle is only slightly decreased (Degoul et al. 1998; Yasukawa et al. 2000a). The mutant tRNAIle is intrinsically unstable in vivo as well as in vitro (Yasukawa et al. 2000a). Moreover, the mutation destabilizes the T-stem of tRNA and decreases the affinity of binding of EF-Tu. The weak interaction with EF-Tu might affect mitochondrial translation, and it does not protect the mutant tRNAIle from degradation. The A4269G mutation thus appears to impair multiple processes regulating tRNAIle stability and function, and the translational defect is likely to be a ‘sum total’ of all of them.


   Experimental procedures
Top
 Abstract
 Introduction
 Discussion
 Experimental procedures
References
 
Northern blot analysis

Cybrid cell lines, which were constructed by the intercellular transfer of patient mtDNA to {rho}° HeLa cells (EB8), were kindly provided by Dr J. Hayashi and have been described (Hayashi et al. 1994). Total RNA was extracted from semiconfluent cultured cybrid cells by ISOGEN (Nippon Gene) according to the manufacturer's instructions. 5 µg of total RNA was subjected to Northern blot analysis using a 5'32P-labelled oligonucleotide probe specific for tRNAIle (5'-TAGAAATAAGGGGGTTTAAGC-3'). The hybridized membrane was exposed to an imaging plate, followed by analysis using a BAS5000 bio-imaging analyser (Fuji Photo Systems).

Assay with the CCA-adding enzyme

Preparations of tRNAIle transcripts without the 3'-terminal CCA sequence and of the human mitochondrial CCA-adding enzyme, and the assay conditions, have been previously described (Tomari et al. 2003).

Chemical synthesis of N-(4-pentenoyl)-Val-pdCpA

4-pentenoic acid (1001.2 mg, 10 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (1917 mg, 10 mmol) were dissolved in 50 mL dimethylformamide and stirred for 1 h at room temperature. L-valine methyl ester hydrochloride (1677 mg, 10 mmol) and triethylamine (1.4 ml, 10 mmol) were added to the solution and stirred for 2 h. The 4-pentenoyl-valine methyl ester was extracted from the mixture using ethyl acetate (200 ml). After evaporation, the product was dissolved in aqueous THF (50%) containing 0.25 N LiOH to hydrolyse the methyl ester. 4-pentenoyl-valine (99.6 mg, 0.5 mmol) was dissolved in triethylamine (139 µl, 1 mmol). Chloroacetonitrile (94.4 µl, 1.5 mmol) was added to the mixture and stirred for 24 h at room temperature. The 4-pentenoyl-valine cyanomethyl ester was extracted with dichloromethane and 1 M NaHSO4. pdCA tetrabutylammonium (2 µmol) and the 4-pentenoyl-valine cyanomethyl ester (50 µmol) were dissolved in 50 µl dimethylformamide and allowed to stand at room temperature for 5 h. The product was purified by reverse-phase column chromatography (Inertsil ODS-3, 10 x 250 mm; GL Sciences) using a Lachrom HPLC system (HITACHI inc). The solvent system consisted of 0.1 M NH4OAc (pH 4.5) (Solvent A) and 0.1 M NH4OAc (pH 4.5) in 80% methanol (Solvent B), and the chromatography was developed at a flow rate of 2 ml/min employing the following gradient program: 0–50% B in 0–10 min and 50–100% B in 10–35 min. The peak fraction containing N-(4-pentenoyl)-(S)-Val-pdCpA was collected and evaporated under reduced pressure.

Synthesis of chemically aminoacylated tRNAIle

In vitro transcription was performed as described by Yasukawa et al. (2000a) with slight modifications. The in vitro transcribed tRNAIle lacking the 3'-terminal-CA sequence was dephosphorylated by bacterial alkaline phosphatase (TaKaRa) to prevent self-ligation of the tRNA. N-(4-Pentenoyl)-Val-pdCpA was ligated to the 3'-truncated tRNAIle transcript by T4 RNA ligase at 10 °C for 3 h in a reaction mixture containing 5 A260/ml of the 3'-truncated tRNAIle transcript, N-(4-pentenoyl)-(S)-Val-pdCpA (a 10-fold molar excess over the tRNA), 50 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 3.5 mM dithiothreitol, 15 µg/mL BSA, 20% PEG6000, 0.5 mM ATP, 10% dimethylsulfoxide and 1.37 µg/µl T4 RNA ligase. N-(4-Pentenoyl)-(S)-Val-tRNAIle was extracted from the reaction mixture by ISOGEN (Nippon Gene) and deprotected in aqueous THF containing 25 mM iodine at room temperature for 15 min (Lodder et al. 1997). Val-tRNAIle was further purified by NAP5 gel chromatography (Amersham Biosciences).

Before and after ligation, a portion of the tRNAIle molecules was labelled at the 5'-termini with [{gamma}-32P]-ATP (110 TBq/mmol; Amersham Biosciences) and T4 polynucleotide kinase (Toyobo) and electrophoresed in a long denaturing polyacrylamide gel, followed by exposure to an X-ray film. The ligation efficiency was confirmed to be almost 100%.

Mass spectrometric analysis of aminoacylated nucleosides

10 µg of N-(4-pentenoyl)-Val-tRNAIle and the deprotected Val-tRNAIle were digested in 10 µl of a reaction mixture containing 20 mM HEPES-KOH (pH 8.0), 10 µg/ml nuclease P1 (Seikagaku Kogyo, Tokyo), and 9 units/ml bacterial alkaline phosphatase (TaKaRa). The hydrolysates were analysed by an LCQ ion-trap (IT) mass spectrometer (ThermoFinnigan) equipped with an electrospray ionization (ESI) source and a MAGIC 2002 liquid chromatography system (Michrom BioResources) as previously described (Kaneko et al. 2003).

Cleavage-site mapping tRNA transcripts with or without the mutation were labelled at the 3'-terminus with [5'-32P]-(pCp) (111 TBq/mmol; NEN Life Science Products) and T4 RNA ligase. The mitochondrial enzymatic extract was prepared as described (Nagaike et al. 2001). The 3'-labelled tRNAIle molecules were partially digested by the mitochondrial enzymatic extract and the reaction was stopped by phenol extraction. The digested tRNAs were loaded on to 15% polyacrylamide-7 M urea gels containing 10% glycerol. An alkaline-treated tRNA ladder and the partial RNase T1 (Amersham Biosciences) and U2 (Seikagaku Kogyo) digests were also electrophoresed. The gel was exposed to an imaging plate, followed by analysis with a BAS 1000 bio-imaging analyser (Fuji Photo Film).

Degradation of the transcribed tRNAIle in mitochondrial extract

The tRNA transcript with or without the mutation was labelled at the 5'-terminus with [{gamma}-32P]-ATP and incubated at 37 °C with the mitochondrial extract (S100). The reaction was stopped immediately by phenol extraction. The extracted RNA fractions were then electrophoresed in a denaturing polyacrylamide gel and the gel was exposed to an imaging plate. The remaining intact tRNAIle was quantified with a BAS 1000 bioimaging analyser (Fuji Photo film). The details of this assay have been described (Yasukawa et al. 2000a).

Gel mobility shift analysis of the EF-Tu:GDPNP:Val-tRNAIle ternary complex

Bovine mitochondrial EF-Tu and Thermus thermophilus EF-Tu were expressed in E. coli as His-tagged proteins and purified by Ni-columns (Woriax et al. 1995). EF-Tu-bound GDP was digested by alkaline phosphatase in the presence of GDPNP to produce EF-Tu:GDPNP (Nissen et al. 1994). The various concentrations of Val-tRNAIle (0–400 pmol) were incubated at 37 °C for 15 min in a reaction mixture (10 µl) containing 50 mM Tris-HCl (pH 7.5), 65 mM NH4 acetate, 10 mM Mg acetate, 10 µM GDPNP and 10 µM EF-Tu. The samples were left on ice for 5 min and then subjected to electrophoresis on a 5% polyacrylamide gel. The gel was run in a buffer containing 50 mM Tris-HCl (pH 6.8 or 7.5), 65 mM NH4 acetate, 10 mM Mgacetate, 5 mM EDTA, 1 mM dithiothreitol and 10 µM GDPNP or GTP. The gel was stained with Coomassie Brilliant Blue (R250). The amounts of free EF-Tu and bound Val-tRNAIle were determined using a Fluor-S MultiImager with Quantity One software (BIO-RAD). The apparent dissociation constant for the ternary complex was calculated by means of a Scatchard plot.


   Acknowledgements
 
We thank Dr J. Hayashi (Tsukuba University) for kindly donating the cybrid cell lines. We would also like to express our appreciation to Dr S. Ohta (Nippon Medical School) for valuable discussions. We thank Dr T. Hanada, Dr Y. Shimizu, Mr T. Nagaike and Mr S. Chimnaronk (University of Tokyo) for their kind gifts of materials and Dr Linda L. Spremulli (University of North Carolina) for providing a plasmid of the mt EF-Tu gene. The authors also thank Drs Charlotte Knudsen and Gregers Rom Andersen (Aarhus University Denmark) for technical assistance with the gel mobility shift assay.


   Footnotes
 
Communicated by: Yoshikazu Nakamura

* Correspondence: E-mail: ueda{at}k.u-tokyo.ac.jp


   References
Top
 Abstract
 Introduction
 Discussion
 Experimental procedures
 References
 
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Received: 29 August 2003
Accepted: 17 December 2003




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L. Levinger, M. Morl, and C. Florentz
Mitochondrial tRNA 3' end metabolism and human disease
Nucleic Acids Res., October 11, 2004; 32(18): 5430 - 5441.
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