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Characterization and tRNA Recognition of Mammalian Mitochondrial Seryl-tRNA Synthetase

2000; Elsevier BV; Volume: 275; Issue: 26 Linguagem: Inglês

10.1074/jbc.m908473199

1083-351X

Takashi Yokogawa, Nobukazu Shimada, Nono Takeuchi, L.A. Benkowski, Tsutomu Suzuki, Akira Omori, Takuya Ueda, Kazuya Nishikawa, Linda Spremulli, Kimitsuna Watanabe,

Mitochondrial Function and Pathology

Animal mitochondrial protein synthesis systems contain two serine tRNAs (tRNAsSer) corresponding to the codons AGY and UCN, each possessing an unusual secondary structure; the former lacks the entire D arm, and the latter has a slightly different cloverleaf structure. To elucidate whether these two tRNAsSer can be recognized by the single animal mitochondrial seryl-tRNA synthetase (mt SerRS), we purified mt SerRS from bovine liver 2400-fold and showed that it can aminoacylate both of them. Specific interaction between mt SerRS and either of the tRNAsSer was also observed in a gel retardation assay. cDNA cloning of bovine mt SerRS revealed that the deduced amino acid sequence of the enzyme contains 518 amino acid residues. The cDNAs of human and mouse mt SerRS were obtained by reverse transcription-polymerase chain reaction and expressed sequence tag data base searches. Elaborate inspection of primary sequences of mammalian mt SerRSs revealed diversity in the N-terminal domain responsible for tRNA recognition, indicating that the recognition mechanism of mammalian mt SerRS differs considerably from that of its prokaryotic counterpart. In addition, the human mt SerRS gene was found to be located on chromosome 19q13.1, to which the autosomal deafness locus DFNA4 is mapped. Animal mitochondrial protein synthesis systems contain two serine tRNAs (tRNAsSer) corresponding to the codons AGY and UCN, each possessing an unusual secondary structure; the former lacks the entire D arm, and the latter has a slightly different cloverleaf structure. To elucidate whether these two tRNAsSer can be recognized by the single animal mitochondrial seryl-tRNA synthetase (mt SerRS), we purified mt SerRS from bovine liver 2400-fold and showed that it can aminoacylate both of them. Specific interaction between mt SerRS and either of the tRNAsSer was also observed in a gel retardation assay. cDNA cloning of bovine mt SerRS revealed that the deduced amino acid sequence of the enzyme contains 518 amino acid residues. The cDNAs of human and mouse mt SerRS were obtained by reverse transcription-polymerase chain reaction and expressed sequence tag data base searches. Elaborate inspection of primary sequences of mammalian mt SerRSs revealed diversity in the N-terminal domain responsible for tRNA recognition, indicating that the recognition mechanism of mammalian mt SerRS differs considerably from that of its prokaryotic counterpart. In addition, the human mt SerRS gene was found to be located on chromosome 19q13.1, to which the autosomal deafness locus DFNA4 is mapped. aminoacyl-tRNA synthetase seryl-tRNA synthetase mitochondrial serine-specific tRNA corresponding to the anticodon GCU serine-specific tRNA corresponding to the anticodon UGA phenylalanine-specific tRNA corresponding to the anticodon GAA phenylmethylsulfonyl fluoride polyacrylamide gel electrophoresis nucleotide position(s) base pair(s) untranslated region reverse transcription polymerase chain reaction expressed sequence tag fast protein liquid chromatography nuclear respiratory factor The fidelity of protein synthesis relies on the specific attachment of amino acids to their cognate tRNA species. This process is catalyzed by aminoacyl-tRNA synthetase (ARS),1 which discriminates with high selectivity among many structurally similar tRNAs and amino acids (1.Normanly J. Abelson J. Annu. Rev. Biochem. 1989; 58: 1029-1049Crossref PubMed Scopus (223) Google Scholar, 2.Schulman L.H. Prog. Nucleic Acids Res. Mol. Biol. 1991; 41: 23-87Crossref PubMed Scopus (162) Google Scholar). To avoid misacylation of tRNAs from any of the 19 noncognate groups within each tRNA sequence, tRNAs possess identity elements that are unambiguously recognized only by the cognate synthetase. These recognition elements are most commonly located in the tRNA anticodon, the acceptor stem and the “discriminator” base at position 73 (2–5). However, in the Escherichia coli system, several biochemical approaches have revealed that identity elements of the tRNAAla and tRNASer isoacceptors are not located in the anticodon and discriminator (4.Shimizu M. Asahara H. Tamura K. Hasegawa T. Himeno H. J. Mol. Evol. 1992; 35: 436-443PubMed Google Scholar, 6.Himeno H. Hasegawa T. Ueda T. Watanabe K. Shimizu M. Nucleic Acids Res. 1990; 18: 6815-6819Crossref PubMed Scopus (130) Google Scholar, 7.Sampson J.R. Saks M.M. Nucleic Acids Res. 1993; 21: 4467-4475Crossref PubMed Scopus (77) Google Scholar, 8.Hou Y.M. Schimmel P. Nature. 1988; 333: 140-145Crossref PubMed Scopus (516) Google Scholar, 9.McClain W.H. Foss K. Science. 1988; 240: 793-796Crossref PubMed Scopus (292) Google Scholar). In the case of tRNAAla, the G3-U70 base pair in the acceptor stem is a major determinant of tRNAAla identity (8.Hou Y.M. Schimmel P. Nature. 1988; 333: 140-145Crossref PubMed Scopus (516) Google Scholar, 9.McClain W.H. Foss K. Science. 1988; 240: 793-796Crossref PubMed Scopus (292) Google Scholar). tRNAs can be divided into two groups according to the length of the extra arm: those with a short extra arm of 4–5 nucleotides (type 1) and those with a long extra arm of at least 11 nucleotides (type 2) (10.Lenhard B. Orellana O. Ibba M. Weygand-Durasevic I. Nucleic Acids Res. 1999; 27: 721-729Crossref PubMed Scopus (63) Google Scholar). tRNAs that belong to the latter type are restricted to only three species in prokaryotes: tRNAsTyr, tRNAsLeu, and tRNAsSer, and two species in eukaryotes: tRNAsLeu and tRNAsSer (Fig.1). Biological experiments have shown that the long extra arm of E. coli tRNASercontributes the most to the specificity of serylation (6.Himeno H. Hasegawa T. Ueda T. Watanabe K. Shimizu M. Nucleic Acids Res. 1990; 18: 6815-6819Crossref PubMed Scopus (130) Google Scholar, 7.Sampson J.R. Saks M.M. Nucleic Acids Res. 1993; 21: 4467-4475Crossref PubMed Scopus (77) Google Scholar, 11.Asahara, H., Himeno, H., and Shimizu, M. (1991) Chem. Lett. 363–366Google Scholar, 12.Schatz D. Leberman R. Eckstein F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6132-6136Crossref PubMed Scopus (98) Google Scholar, 13.Asahara H. Himeno H. Tamura K. Nameki N. Hasegawa T. Shimizu M. J. Mol. Biol. 1994; 236: 738-748Crossref PubMed Scopus (74) Google Scholar). Moreover, Himeno et al. (6.Himeno H. Hasegawa T. Ueda T. Watanabe K. Shimizu M. Nucleic Acids Res. 1990; 18: 6815-6819Crossref PubMed Scopus (130) Google Scholar) reported that the different orientations of the long extra arms in these three species are a key element for discrimination by E. coli seryl-tRNA synthetase (SerRS), which is a plausible reason why neither the length nor the sequence of the extra arm is conserved among tRNASerisoacceptors (14.Sprinzl M. Hartmann T. Weber J. Blank J. Zeidler R. Nucleic Acids Res. 1989; 17: 1-172Crossref PubMed Scopus (354) Google Scholar). These results are consistent with the crystallographic structures of SerRS-tRNASer complexes from E. coli andThermus thermophilus (15.Price S. Cusack S. Borel F. Berthet-Colominas C. Leberman R. FEBS Lett. 1993; 324: 167-170Crossref PubMed Scopus (35) Google Scholar, 16.Biou V. Yaremchuk A. Tukalo M. Cusack S. Science. 1994; 263: 1404-1410Crossref PubMed Scopus (428) Google Scholar, 17.Cusack S. Yaremchuk A. Tukalo M. EMBO J. 1996; 15: 2834-2842Crossref PubMed Scopus (157) Google Scholar). tRNASer binds across both subunits of the dimer. The terminal part of the acceptor end contacts the active site of one subunit, whereas the rest of the tRNASer is bound to the other subunit, in which is located the N-terminal long helical arm-like domain that is important for recognition of the long extra arm and TΨC loop of tRNASer. In eukaryotic systems, cytoplasmic tRNASer also has a long extra arm (Fig. 1), and several biochemical studies on Saccharomyces cerevisiae and human tRNAsSer have indicated that the major identity element of tRNASer is located in this arm (18.Achsel T. Gross H.J. EMBO J. 1993; 12: 3333-3338Crossref PubMed Scopus (78) Google Scholar, 19.Dock-Bregeon A.C. Garcia A. Giege R. Moras D. Eur. J. Biochem. 1990; 188: 283-290Crossref PubMed Scopus (40) Google Scholar, 20.Wu X.-Q. Gross H.J. Nucleic Acids Res. 1993; 21: 5589-5594Crossref PubMed Scopus (87) Google Scholar, 21.Himeno H. Yoshida S. Soma A. Nishikawa K. J. Mol. Biol. 1997; 268: 704-711Crossref PubMed Scopus (46) Google Scholar). Thus, it can be concluded that the major identity element of both prokaryotic and eukaryotic cytoplasmic tRNAsSer for specific recognition by SerRS is located in the characteristic long extra arm. The recognition mechanism using the long extra arm appears evolutionarily conserved in the tRNASer-SerRS system. On the other hand, because all animal mitochondrial (mt) tRNAsSer possess a short extra arm (10.Lenhard B. Orellana O. Ibba M. Weygand-Durasevic I. Nucleic Acids Res. 1999; 27: 721-729Crossref PubMed Scopus (63) Google Scholar), the recognition mechanism described above would not be applicable in the mt system. Also, animal mt tRNASer isoacceptors differ structurally from those of other mt tRNAs; the tRNASer specific for codons AGY (Y = C or U; tRNAGCUSer) lacks the entire D arm (22.Steinberg S. Gautheret D. Cedergren R. J. Mol. Biol. 1994; 236: 982-989Crossref PubMed Scopus (68) Google Scholar), whereas the isoacceptor for codons UCN (N = A, G, C, or U; tRNAUGASer) lacks the invariant U8 between the acceptor and D stems and has a small D loop and an extended anticodon stem consisting of 6 base pairs (23.Yokogawa T. Watanabe Y. Kumazawa Y. Ueda T. Hirao I. Miura K. Watanabe K. Nucleic Acids Res. 1991; 19: 6101-6105Crossref PubMed Scopus (68) Google Scholar) (Fig.1). The primary and secondary structures of these two tRNAsSer are too different for a common region in these tRNAs to be identified. To date, it remains unclear whether the single ARS recognizes two cognate tRNAs with apparently different structures, like animal mt tRNAsSer. It thus is of interest to ascertain whether the single mitochondrial seryl-tRNA synthetase (mt SerRS) recognizes the two distinct tRNASer isoacceptors and, if so, what kind of tRNA recognition mechanism is needed for the system. To obtain information on the recognition mechanism of animal mt SerRS, we previously studied the recognition sites of bovine mt tRNAGCUSer (24.Ueda T. Yotsumoto Y. Ikeda K. Watanabe K. Nucleic Acids Res. 1992; 20: 2217-2222Crossref PubMed Scopus (50) Google Scholar). We have recently undertaken further biochemical investigations to elucidate the recognition mechanism of animal mt SerRS by purifying bovine mt SerRS from bovine liver, cloning its gene, and characterizing the native bovine mt SerRS. The results are presented here. Phenylmethylsulfonyl fluoride (PMSF) and DEAE-Sepharose were purchased from Sigma; hydroxyapatite and a protein assay kit were from Bio-Rad; Centriprep-10, Centricon-10, and Microcon-10 were from Amicon; [14C]l-serine (4.4 GBq/mmol) was from NEN Life Science Products; and Superdex 200 prep grade, HiTrap heparin (1 ml), Mono S (HR5/5), and Mono Q (HR5/5) were from Amersham Pharmacia Biotech. Other chemicals were from Wako Pure Chemicals. E. coli total tRNAs were from Roche Molecular Biochemicals. Native mt tRNAsSer and mt tRNAGAAPhe were purified from bovine mitochondria by the selective hybridization method using a solid phase DNA probe as described by Wakita et al. (25.Wakita K. Watanabe Y. Yokogawa T. Kumazawa Y. Nakamura S. Ueda T. Watanabe K. Nishikawa K. Nucleic Acids Res. 1994; 22: 347-353Crossref PubMed Scopus (75) Google Scholar). Procedures were generally performed at 4 °C; only the FPLC system (Amersham Pharmacia Biotech) was operated at room temperature. For For step 1, digitonin-treated bovine liver mitochondria, isolated mt pellets, and the mt S-30 fraction were prepared as described previously (26.Schwartzbach C.J. Farwel M. Liao H.X. Spremulli L.L. Methods Enzymol. 1996; 264: 248-261Crossref PubMed Google Scholar, 27.Schwartzbach C.J. Spremulli L.L. J. Biol. Chem. 1989; 264: 19125-19131Abstract Full Text PDF PubMed Google Scholar). For step 2, fresh S-30 (2800 mg) was applied onto a DEAE-Sepharose column (2.7 × 17.5 cm) equilibrated and washed with Buffer A (20 mm Tris-HCl (pH 7.6), 40 mm KCl, 1 mm MgCl2, 0.1 mm EDTA, 6 mm β-mercaptoethanol, 10% glycerol, and 100 μm PMSF), and developed with a linear gradient (1000 ml) from 40 to 500 mm KCl in Buffer A. Fractions (10 ml) were collected at a flow rate of 1.0 ml/min. Active fractions were precipitated with ammonium sulfate (60% saturation). For step 3, the above precipitate was dissolved and dialyzed extensively against Buffer B (10 mm potassium phosphate (pH 7.4), 6 mm β-mercaptoethanol, 10% glycerol, and 100 μm PMSF). The dialyzed sample (360 mg of proteins) was applied onto a hydroxyapatite column (1.5 × 11 cm) equilibrated with Buffer B and developed with a linear gradient (200 ml) from 10 to 200 mm potassium phosphate in Buffer B. Fractions (5 ml) were collected. Aliquots (200 μl) were taken from every second fraction and dialyzed against Buffer C (20 mm Hepes-KOH (pH 7.0), 40 mm KCl, 1 mm MgCl2, 0.1 mm EDTA, 6 mm β-mercaptoethanol, 10% glycerol, and 100 μm PMSF) with Microcon 10 to remove phosphate. These were used for the aminoacylation assays. The concentrated sample (5 ml, 55 mg of proteins) collected by Centriprep 10 from active fractions was immediately applied onto a Superdex 200 column (2.5 × 60 cm) equilibrated with Buffer C. For step 4, the column was developed with Buffer C. Fractions (5 ml) were collected at a flow rate of 0.5 ml/min. Active fractions were concentrated with Centriprep 10. This procedure was used in the subsequent steps. For step 5, the concentrated sample (5.6 mg of proteins) was diluted with Buffer D (20 mm Hepes-KOH (pH 7.0), 1 mmMgCl2, 0.1 mm EDTA, 2 mmdithiothreitol, and 10% glycerol) 4-fold and immediately applied onto a HiTrap heparin column (1 ml), which was developed with a 25-ml linear gradient from 0 to 500 mm KCl in Buffer D at a flow rate of 0.5 ml/min using a FPLC system. Fractions of 1 ml were collected. For step 6, the sample (0.36 mg) dialyzed against Buffer D with Centricon 10 was immediately applied onto a Mono S column (0.5 × 5 cm) and developed with a 20-ml linear gradient from 0 to 400 mm KCl in Buffer D at a flow rate of 0.5 ml/min by FPLC. Fractions of 1 ml were collected. For step 7, the sample (0.14 mg) dialyzed against Buffer D with Centricon 10 was immediately applied onto a Mono Q column (0.5 × 5 cm) and developed with a 25-ml linear gradient from 0 to 300 mm KCl in Buffer D at a flow rate of 0.5 ml/min by FPLC. Fractions of 1 ml were collected. To check their purity, active fractions were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) using the method of Laemmli (28.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). The mt SerRS fraction was frozen quickly and stored at −70 °C. The tRNASer-SerRS complex was formed by incubation at 37 °C for 10 min in a 10-μl aliquot containing 50 mm Tris-HCl (pH 8.5), 15 mmMgCl2, 5 mm dithiothreitol, 1 mmspermine, about 0.02 A 260 unit of mt tRNA, and about 0.5 μg of mt SerRS fraction. Native PAGE was done as described by Hornung et al. (29.Hornung V. Hofmann H. Sprinzl M. Biochemistry. 1998; 37: 7260-7267Crossref PubMed Scopus (21) Google Scholar), and the gels were stained with both Coomassie Brilliant Blue and toluidine blue to analyze the components of the tRNASer-SerRS complex. The band of the complex was cut out and subjected to SDS-PAGE, and the gel was silver-stained. About 15 μg of the purified mt SerRS was digested with 1 μg of lysyl endopeptidase at 37 °C overnight in a 50-μl aliquot containing 100 mm Tris-HCl (pH 9) and 20 mm EDTA. The resultant product was loaded onto a C8 column (2.1 × 30 mm) in a high performance liquid chromatography system and separated at a flow rate of 0.2 ml/min with a 6-ml linear gradient from 0 to 35% acetonitrile containing 0.1% trifluoroacetate and then with a 3-ml linear gradient from 35 to 70% acetonitrile containing 0.1% trifluoroacetate. The amino acid sequence of each separated peptide was determined with an Applied Biosystems 477A/120A protein sequencer. In parallel, the sequences of peptides digested with endoproteinase V8 were obtained according to the method of Cleveland et al.(30.Cleveland D.W. Fisher S.G. Kirschner M.W. Laemmli U.K. J. Biol. Chem. 1977; 252: 1102-1106Abstract Full Text PDF PubMed Google Scholar) with the modifications indicated in Ref. 31.Omori A. Yoshida S. J. Protein Chem. 1994; 13: 471Google Scholar. The assays were carried out at 37 °C for 5 min with reaction mixtures (15 μl) containing 100 mm Tris-HCl (pH 8.5), 10 mmMgCl2, 60 mm KCl, 2 mm ATP, 10 mm dithiothreitol, 42 μm[14C]l-serine, 0.5A 260 unit of E. coli total tRNAs, and an appropriate amount of the enzyme fraction (32.Nishimura S. Novelli G.D. Biochim. Biophys. Acta. 1964; 80: 574-586PubMed Google Scholar). One unit of activity is defined as the amount of enzyme that catalyzes the formation of 1 pmol of seryl-tRNASer for 1 min. The protein concentration was determined with a Bio-Rad protein assay kit using bovine serum albumin as a standard. Aminoacylation reactions to determine the kinetic parameters of bovine mt SerRS were carried out at 37 °C in a buffer containing 50 mm Tris-HCl (pH 8.5), 15 mm MgCl2, 5 mm dithiothreitol, 1 mm spermine, 2 mm ATP, 60 mm KCl, 33 μm[14C]l-serine (5.59 GBq/mmol) purchased from Amersham Pharmacia Biotech, and 5.7 nm purified bovine mt SerRS. Although l-serine was used at the subsaturating concentration, it was slightly above K m (23 μm) of bovine mt SerRS according to Kumazawa et al. (33.Kumazawa Y. Studies on the Mitochondrial Protein Synthesis. Ph.D. Thesis. The University of Tokyo, Tokyo, Japan1988Google Scholar), and we made compromise between unreliable results because of the concentration around K m and low counting efficiency because of low specific activity of the labeledl-serine caused by dilution with nonlabeledl-serine (18.Achsel T. Gross H.J. EMBO J. 1993; 12: 3333-3338Crossref PubMed Scopus (78) Google Scholar). The initial rates of aminoacylation were determined by using six different concentrations of native tRNAsSer ranging from 0.04 to 1.5 μm (0.04, 0.10, 0.30, 0.70, 1.0, and 1.5 μm) for tRNAGCUSer or from 0.03 to 1.3 μm (0.03, 0.10, 0.25, 0.60, 0.90, and 1.3 μm) for tRNAUGASer at a fixed concentration of mt SerRS, which gave reasonable kinetics plots for determining the apparent K m andk cat values. Partial peptide sequences of bovine mt SerRS were subjected to a BLAST search of the DDBJ/EBI/GenBankTM nucleotide sequence data bases and a human EST clone (accession number T78174) was obtained. A sense primer (np 1207–1227; see Fig. 4) and an antisense primer (np 1453–1472) were designed from the partial region in the clone that was highly identical to the partial peptide sequences of bovine mt SerRS. To obtain the bovine cDNA clone, RT-PCR was performed using these two primers, 2 μg of laboratory stock bovine poly(A)-tailed mRNA, and a TaKaRa RNA PCR kit (AMV) version 2.1. cDNA screening, cloning, and sequencing of the plasmid DNA obtained were done according to Takeuchi et al. (34.Takeuchi N. Kawakami M. Omori A. Ueda T. Spremulli L.L. Watanabe K. J. Biol. Chem. 1998; 273: 15085-15090Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The 5′-region of the cDNA corresponding to the N-terminal region of the mature mt SerRS was obtained by RT-PCR. First strand cDNA synthesis and first and nested PCR were carried out according to Nakayama (35.Nakayama H. Baio Jikken Irasutoreiteddo. 1996; 3: 85-100Google Scholar) with some modifications. A degenerate sense primer (np 103–122) was designed from the N-terminal peptide sequence. Antisense primers (np 1099–1118 and 1123–1143) were designed from the cDNA sequence obtained by cDNA screening and respectively used for first and nested PCR. The predominant PCR product was purified by agarose gel electrophoresis and cloned into a pCR®2.1-TOPO vector (Invitrogen). A MarathonTM cDNA amplification kit (CLONTECH) was used to further determine the 5′-region of the bovine mt SerRS cDNA. Antisense primers (np 151–168 and 126–144) were designed from the 5′-region sequence of the mature mt SerRS and used, respectively, for first and nested PCR. Sequencing was done using a Dye Terminator Cycle sequencing kit (Perkin-Elmer) and an ABI PRISMTM310 genetic analyzer. The major part of the putative human mt SerRS cDNA sequence and the whole putative cDNA sequence of mouse mt SerRS were obtained by connecting several EST clones whose peptide sequences are very homologous to that of bovine mt SerRS. The few unknown regions in the human mt SerRS cDNA were determined by RT-PCR using RT-PCR high (Toyobo). Primers were designed from the determined sequences on both sides. To elucidate whether the single animal mt SerRS recognizes the two tRNASer isoacceptors, which differ considerably in their secondary structures, we purified mt SerRS to homogeneity from bovine liver mitochondria by successive column chromatographies as described under “Experimental Procedures.” Only one peak fraction exhibiting serylation activity was observed in each step. The purification scheme resulted in 2400-fold purification of mt SerRS with 2.8% recovery (Table I). In the final step, the serylation activity completely coincided with the Mono Q column absorbance profile (Fig.2 A). The molecular mass of mt SerRS was estimated to be about 53,000 Da by SDS-PAGE (Fig.2 B). On the other hand, mt SerRS was eluted in the region of a molecular mass exceeding 100,000 Da on Superdex 200 column chromatography (data not shown). Because all the SerRSs known so far have an α2 subunit structure, bovine mt SerRS is thought to be a dimer.Table IPurification of mitochondrial seryl-tRNA synthetase from bovine liverPurification stepProteinTotal unitsSpecific activityTotal recoveryPurificationmgunitsunits/mg%foldMitochondrial extract (S-30)280080002.81001DEAE-Sepharose460680015855.360% Ammonium sulfate precipitation360560016705.6Hydroxyapatite553200644023Superdex 2005.6320058040210Heparin0.3678022009.8790Mono S0.1442030005.31100Mono Q0.03222068002.82400 Open table in a new tab To ascertain whether the single bovine mt SerRS recognizes the two mt tRNAsSer, we carried out gel retardation assays and aminoacylation reaction experiments. Fig.3 A shows that the main protein band was shifted as a consequence of adding mt tRNAGCUSer and mt tRNAUGASer to mt SerRS, whereas no such shift was observed when mt tRNAGAAPhewas used. Furthermore, the shifted band was found to contain both a 53,000-Da protein and the mt tRNASer on the SDS-containing gel (Fig. 3 B). It was thus demonstrated that the single mt SerRS recognizes and binds to the two tRNASer isoacceptors with different structures. The kinetic parameters of aminoacylation by the purified bovine mt SerRS are shown in Table II. The bovine mt SerRS is seen to aminoacylate the two tRNAsSer almost equally. The K m values determined in the present study are rather different from those reported previously using partially purified bovine mt SerRS (36.Yokogawa T. Kumazawa Y. Miura K. Watanabe K. Nucleic Acids Res. 1989; 17: 2623-2638Crossref PubMed Scopus (48) Google Scholar). The present data appear more reasonable because the K m values for each cognate tRNASer in the previous data differ considerably.Table IIKinetic parameters in aminoacylation of bovine mitochondrial serine tRNAsSubstrateK mk catk cat/K mμm1/s1/(μm × s)tRNAGCUSer0.37 ± 0.100.35 ± 0.100.95tRNAUGASer0.22 ± 0.030.63 ± 0.062.86Experimental conditions for aminoacylation are described under “Experimental Procedures.” Open table in a new tab Experimental conditions for aminoacylation are described under “Experimental Procedures.” To obtain cDNA clones of mt SerRS, partial peptide sequences were determined. N-terminal sequencing revealed that mt SerRS has two heterologous termini: NH2-ATERQDRNLLYEHAR and NH2-ERQDRNLLYEHAR (Fig. 4). Subsequently, five internal peptides were sequenced (Fig. 4) that were subjected to a BLAST search through the human EST data base. The search revealed one EST clone (accession number T78174) containing portions of the two peptide sequences at the C-terminal region of bovine mt SerRS (Fig. 4). For cDNA screening, a cDNA clone was obtained by RT-PCR using bovine mRNA with primers designed from the sequences of this clone (Fig. 4). The cDNA screening gave one cDNA clone 998 base pairs (bp) in length that corresponded to the C-terminal region (np 892–1889) of mt SerRS (Fig. 4). Subsequently, the N-terminal region was amplified by RT-PCR using a degenerate primer based on the N-terminal peptide sequence, and one cDNA clone 1118 bp in length (np 103–1220) was obtained. Through 5′-rapid amplifiation of cDNA ends, four clones with identical sequences but different lengths were obtained. The longest cDNA fragment, 210 bp in length, contained one ATG codon. Assuming this to be the initiation codon, the 5′-untranslated region (UTR) consists of only 12 bases. It is possible that another ATG codon further upstream in the 5′-region functions as the initiation codon. However, human mt SerRS has a TAA codon at position −48 in frame that strongly suggests that the relevant ATG codon functions as the initiation codon. Additionally, the initiation context found in both sequences, possessing A at position −3 and G at position 4, conforms to the consensus feature for eukaryotic genes (37.Kozak M. EMBO J. 1997; 16: 2482-2492Crossref PubMed Scopus (414) Google Scholar) (Fig. 4). These facts strongly suggest that the sole ATG codon found in the cDNA sequence of bovine mt SerRS is the actual initiation codon. It is now clear that the bovine mt SerRS cDNA is composed of at least 12 bp of 5′-UTR, a 1557-bp coding sequence, and 331 bp of 3′-UTR. All the sequences of the five peptide fragments derived from bovine mt SerRS were identified within its complete amino acid sequence (Fig. 4). Based on the amino acid sequence of the 1557-bp coding sequence, analysis of the mature bovine mt SerRS revealed that the N-terminal 34-amino acid sequence of the precursor protein functions as the targeting peptide (Fig. 4). However, as noted above, two different N-terminal peptide fragments (i.e. two different precursor cleavage sites) were observed. This leaves the possibility of alternative cleavage of the mt SerRS precursor by the matrix processing protease (38.Gavel Y. von Heijne G. Protein Eng. 1990; 4: 33-37Crossref PubMed Scopus (276) Google Scholar). We next confirmed the C-terminal peptide of bovine mt SerRS. After digesting the mt SerRS with trypsin, peptide fragments were analyzed by liquid chromatography/mass spectrometry using electrospray ionization/iontrap mass spectrometry. The C-terminal peptide, LPGQPASS, was identified as a slightly charged ion with anm/z of 756.4 Da (data not shown). Peptide fragments generated from digestion by trypsin in H218O were similarly analyzed. No change in the molecular mass of the relevant fragment was observed, showing that the actual termination is executed at the putative termination codon expected from the bovine mt SerRS cDNA sequence. Thus, it was concluded that the cDNA sequence determined in this work is actually derived from the mature mt SerRS. The putative human mt SerRS cDNA was obtained by connecting human EST clones and RT-PCR (Fig.5 A); it is composed of at least 160 bp of 5′-UTR, a 1557-bp coding sequence, and 337 bp of 3′-UTR. The putative mouse mt SerRS cDNA was acquired only by connecting mouse EST clones (Fig. 5 A); it consists of at least 20 bp of 5′-UTR, a 1557-bp coding sequence, and 281 bp of 3′-UTR. Information on the position of the human mt SerRS gene on the genome was obtained by subjecting its cDNA sequence to a BLAST search. One of the acquired clones contained the complete human mt ribosomal protein S12 (MRPS12) gene (accession number AF058761). Only the first exon of the human mt SerRS gene was found in the sequence of the above-mentioned EST clone (Fig. 5 B). It is of interest that one of the putative binding sites of nuclear respiratory factor-1 (NRF-1), one of the transcription factors, is located in the coding sequence of human mt SerRS in the opposite direction (39.Johnson D.F. Hamon M. Fischel-Ghodsian N. Genomics. 1998; 52: 363-368Crossref PubMed Scopus (20) Google Scholar). According to the coding sequence of bovine mt SerRS, the predicted translation product has 518 amino acids. Mammalian mt SerRS has a long C-terminal sequence, but it is different from a basic C-terminal lysine-rich extension found in all eukaryotic cytoplasmic SerRSs that may be important for both stability and optimal substrate recognition (40.Weygand-Durasevic I. Lenhard B. Filipic S. Söll D. J. Biol. Chem. 1996; 271: 2455-2461Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Though it displays only 28–34% homology with both prokaryotic and eukaryotic cytoplasmic counterparts and even with yeast mt SerRS, relatively high homology is observed in the C-terminal region among all the sequences (Fig.6). Analyses of the crystal structures of E. coli and T. thermophilus SerRSs revealed that prokaryotic SerRS discriminates tRNASer from other noncognate tRNAs by means of the long helical arm located in the N-terminal region and interacts with serine and ATP by the residues mainly located in the C-terminal region, in particular in motifs 2 and 3, which are highly conserved active sites among class II ARSs (Fig. 6). The high homology in the C-terminal region between prokaryotic SerRS and mammalian mt SerRS indicates that the C-terminal region also functions as the catalytic core in the latter, whereas the low homology in the N-terminal region accords well with the lack of the long extra arm in most animal mt tRNAsSer from the perspective of the co-evolution of ARS and its cognate tRNA. Our work has shown that the two distinct mitochondrial tRNASer isoforms are recognized by a single bovine mt SerRS, a 54,635-Da polypeptide. The low homology in the N-terminal region between mammalian mt SerRS and other SerRSs is consistent with the recognition mechanism of mammalian mt SerRS differing from that of prokaryotic SerRSs so far elucidated. On the other hand, the high homology in the C-terminal region is indicative of the conservation of the catalytic core in mammalian mt SerRSs, except for some residues involved in the interaction with the acceptor stem of tRNA. This local difference seems to be in agreement with the unique recognition mechanism of mammalian mt SerRS. Relevant details of our inspection of the C-terminal region of bovine mt SerRS are as follows. In the crystal structure of T. thermophilus SerRS, ATP is bound to the active site through interactions with Arg256, Glu258, Arg271, Phe275, Glu345, Glu348, and Arg386 (16.Biou V. Yaremchuk A. Tukalo M. Cusack S. Science. 1994; 263: 1404-1410Crossref PubMed Scopus (428) Google Scholar, 17.Cusack S. Yaremchuk A. Tukalo M. EMBO J. 1996; 15: 2834-2842Crossref PubMed Scopus (157) Google Scholar,41.Härtlein M. Cusack S. J. Mol. Evol. 1995; 40: 519-530Crossref PubMed Scopus (42) Google Scholar). (Fig. 6) Furthermore, serine specificity is ensured by the interaction of the hydroxyl group in the side chain of serine with Tyr380 in motif 3 (41.Härtlein M. Cusack S. J. Mol. Evol. 1995; 40: 519-530Crossref PubMed Scopus (42) Google Scholar). In particular, Glu281in yeast cytoplasmic SerRS, equivalent to Glu258 inT. thermophilus SerRS, is reported to be important for the binding of ATP and to contribute to the stabilization of the motif 2 loop (42.Lenhard B. Filipic S. Landeka I. Skrtic I. Söll D. Weygand-Durasevic I. J. Biol. Chem. 1997; 272: 1136-1141Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). All of these residues are also conserved in mammalian mt SerRS (Fig. 6). As reported by Cusack et al. (17.Cusack S. Yaremchuk A. Tukalo M. EMBO J. 1996; 15: 2834-2842Crossref PubMed Scopus (157) Google Scholar), the motif 2 loop of T. thermophilus SerRS can take either of two quite different conformations: one in the presence of tRNA (the T-conformation) and the other in the absence of tRNA but in the presence of ATP (the A-conformation). These two ordered conformations are each stabilized by different sets of interactions, often involving the same residues. The side chains of Glu258 and Arg271, key residues in the conformation switch, alter the conformation and bind to either ATP or tRNA in each conformation. These two residues are conserved in the mammalian mt SerRSs. On the other hand, Ser261, Phe262, and Arg267, which are involved in interactions with several bases in the acceptor stem in the T-conformation (17.Cusack S. Yaremchuk A. Tukalo M. EMBO J. 1996; 15: 2834-2842Crossref PubMed Scopus (157) Google Scholar), are scarcely conserved in mammalian mt SerRS. Cusack et al. (17.Cusack S. Yaremchuk A. Tukalo M. EMBO J. 1996; 15: 2834-2842Crossref PubMed Scopus (157) Google Scholar) speculate that the occurrence of two glycines in the motif 2 loop (Gly260 and Gly263) surrounded by small residues (Ala, Thr, or Val) in positions 259 and 266 may provide the flexibility necessary to facilitate the conformational switch. However, Gly260 and Val266 in T. thermophilus SerRS are not conserved in mammalian mt SerRSs. The conservation of Glu258 and Arg271(according to the T. thermophilus numbering) in the motif 2 loop of mammalian mt SerRSs also suggests the existence of the conformational switch from the serine activation step to the aminoacylation step in these enzymes. However, the lack of two out of the several residues necessary for providing flexibility to the motif 2 loop may reduce the flexibility of mammalian mt SerRS. Because the motif 2 loop of SerRS is the longest among other class II synthetases (17.Cusack S. Yaremchuk A. Tukalo M. EMBO J. 1996; 15: 2834-2842Crossref PubMed Scopus (157) Google Scholar), residues of the long motif 2 loop are able to extend down to the fifth base pair of the acceptor stem of T. thermophilustRNASer. The apparently lower flexibility of the motif 2 loop and the low level of conservation of Ser261 and Arg267 (T. thermophilus SerRS numbering) in mammalian mt SerRS (Fig. 6), raise the possibility that mammalian mt SerRS does not interact with the bases of the acceptor stem. This is fully consistent with our previous finding that substitution of A-U base pairs in the acceptor stem of bovine mt tRNAGCUSer with C-G pairs did not severely impair the charging activity of tRNAGCUSer by bovine mt SerRS (24.Ueda T. Yotsumoto Y. Ikeda K. Watanabe K. Nucleic Acids Res. 1992; 20: 2217-2222Crossref PubMed Scopus (50) Google Scholar). We previously demonstrated the significance of U54 and A58 of the T-loop in the recognition of bovine mt tRNAGCUSer by bovine mt SerRS (24.Ueda T. Yotsumoto Y. Ikeda K. Watanabe K. Nucleic Acids Res. 1992; 20: 2217-2222Crossref PubMed Scopus (50) Google Scholar). The corresponding residues are also found in another isoacceptor, tRNAUGASer, as U54 and m1A, respectively. Because the present work has shown that the single mt SerRS can aminoacylate the two structurally distinct tRNAsSer, it is reasonable to assume that both tRNAGCUSer and tRNAUGASer have the same recognition elements. Because tertiary U54-A58 pairing is widely conserved among nonmitochondrial tRNAs and is considered to play a general role in maintaining the L-shape of the tRNA molecule (43.Rich A. Kim S.H. Sci. Am. 1978; 238: 52-62Crossref PubMed Scopus (53) Google Scholar), it seems unlikely that this pairing is critical for enzyme recognition. Further study is necessary to determine the recognition elements common to both bovine mt tRNAsSer. Kumazawa et al. (44.Kumazawa Y. Himeno H. Miura K. Watanabe K. J. Biochem. (Tokyo). 1991; 109: 421-427Crossref PubMed Scopus (57) Google Scholar) showed that bovine mt SerRS not only charges cognate E. colitRNASer species but also extensively misacylates several noncognate E. coli tRNA species, whereas E. coliSerRS is unable to aminoacylate bovine mt tRNAsSer. This unilateral aminoacylation mechanism between bovine mitochondria andE. coli will be also elucidated through further research. A human EST data base search revealed that the human mt SerRS gene is located at a position 5′ adjacent to the RPMS12 gene on chromosome 19q13.1 (Fig. 5 B) (39.Johnson D.F. Hamon M. Fischel-Ghodsian N. Genomics. 1998; 52: 363-368Crossref PubMed Scopus (20) Google Scholar, 45.Shah Z.H. Migliosi V. Miller S.C.M. Wang A. Friedman T.B. Jacobs H.T. Genomics. 1998; 48: 384-388Crossref PubMed Scopus (17) Google Scholar). Recently, the autosomal dominant deafness locus DFNA4 was also mapped to 19q13.1 (46.Chen A.H. Ni L. Fukushima K. Marietta J. O'Neill M. Coucke P. Wiliems P. Smith R.J.H. Hum. Mol. Genet. 1995; 4: 1073-1076Crossref PubMed Scopus (66) Google Scholar). Because the ribosomal protein S12 is known to act as a core component of the highly conserved accuracy center in the ribosome, it is supposed that mutations in the S12 gene result in inaccurate mt translation (47.Alskne L.E. Anthony R.A. Liebman S.W. Warner J.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9538-9541Crossref PubMed Scopus (91) Google Scholar). A genetic study of the fruit fly indicates that a single point mutation in the mt ribosomal protein S12 causes a bang-senseless mutant called tko (48.Shah Z.H. O'Dell K.M.C. Miller S.C.M. An X. Jacobs H.T. Gene (Amst .). 1997; 204: 55-62Crossref PubMed Scopus (31) Google Scholar), the phenotype of which resembles a sensorineural hearing loss related to mt dysfunction (49.Prezant T.R. Agapian J.V. Bohlman M.C. Bu X. Ötzas S. Qiu W.-Q. Arnos K.S. Cortopassi G.A. Jaber L. Rotter J.I. Shohat M. Fischel-Ghodsian N. Nat. Genet. 1993; 4: 289-294Crossref PubMed Scopus (1005) Google Scholar). Although human RPMS12 has been suggested to be responsible for DFNA4 hearing loss (39.Johnson D.F. Hamon M. Fischel-Ghodsian N. Genomics. 1998; 52: 363-368Crossref PubMed Scopus (20) Google Scholar, 45.Shah Z.H. Migliosi V. Miller S.C.M. Wang A. Friedman T.B. Jacobs H.T. Genomics. 1998; 48: 384-388Crossref PubMed Scopus (17) Google Scholar), the human mt SerRS gene may also be a possible candidate, because mt SerRS contributes to the maintenance of translational fidelity in the mt protein synthesis reaction. Although many biochemical experiments on recognition elements in tRNAs, especially those of prokaryotes, have been reported, there has been no study in which the recognition mechanism of structurally different tRNAs by a single synthetase was elucidated. We have discussed the recognition mechanism of bovine mt SerRS in the light of the information revealed in the present study. Further experimental investigation will certainly reveal the essential recognition mechanism between SerRS and tRNAsSer and thereby deepen our understanding of the animal mitochondrial translation system. We thank Dr. Yoichi Watanabe (Tokyo University) for helpful discussions, Dr. Chie Takemoto (Gakushuin University) for kind advice concerning mt SerRS purification, and Takeo Suzuki (Tokyo University) for excellent technical assistance with mitochondrial tRNAs purification.