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• W2084325654 abstract "The mammalian mitochondrial methionyl-tRNA transformylase (MTFmt) was partially purified 2,200-fold from bovine liver mitochondria using column chromatography. The polypeptide responsible for MTFmt activity was excised from a sodium dodecyl sulfate-polyacrylamide gel and the amino acid sequences of several peptides were determined. The cDNA encoding bovine MTFmt was obtained and its nucleotide sequence was determined. The deduced amino acid sequence of the mature form of MTFmt consists of 357 amino acid residues. This sequence is about 30% identical to the corresponding Escherichia coli and yeast mitochondrial MTFs. Kinetic parameters governing the formylation of various tRNAs were obtained. Bovine MTFmtformylates its homologous mitochondrial methionyl-tRNA and the E. coli initiator methionyl-tRNA (Met-tRNAfMet) with essentially equal efficiency. The E. coli elongator methionyl-tRNA (Met-tRNAmMet) was also formylated although with somewhat less favorable kinetics. These results suggest that the substrate specificity of MTFmt is not as rigid as that of the E. coli MTF which clearly discriminates between the bacterial initiator and elongator Met-tRNAs. These observations are discussed in terms of the presence of a single tRNAMet gene in mammalian mitochondria. The mammalian mitochondrial methionyl-tRNA transformylase (MTFmt) was partially purified 2,200-fold from bovine liver mitochondria using column chromatography. The polypeptide responsible for MTFmt activity was excised from a sodium dodecyl sulfate-polyacrylamide gel and the amino acid sequences of several peptides were determined. The cDNA encoding bovine MTFmt was obtained and its nucleotide sequence was determined. The deduced amino acid sequence of the mature form of MTFmt consists of 357 amino acid residues. This sequence is about 30% identical to the corresponding Escherichia coli and yeast mitochondrial MTFs. Kinetic parameters governing the formylation of various tRNAs were obtained. Bovine MTFmtformylates its homologous mitochondrial methionyl-tRNA and the E. coli initiator methionyl-tRNA (Met-tRNAfMet) with essentially equal efficiency. The E. coli elongator methionyl-tRNA (Met-tRNAmMet) was also formylated although with somewhat less favorable kinetics. These results suggest that the substrate specificity of MTFmt is not as rigid as that of the E. coli MTF which clearly discriminates between the bacterial initiator and elongator Met-tRNAs. These observations are discussed in terms of the presence of a single tRNAMet gene in mammalian mitochondria. During the initiation of protein biosynthesis the initiator methionyl-tRNA is bound to the ribosomal P-site. In prokaryotes, this step is facilitated by initiation factor 2 (IF-2) 1The abbreviations used are: IF-2, initiation factor-2; PAGE, polyacrylamide gel electrophoresis; MTF, methionyl-tRNA transformylase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; bp, base pair; THF, tetrahydrofuran. while in the eukaryotic cytoplasm this step is mediated by eIF-2. In contrast, all other aminoacyl-tRNAs function as elongator tRNAs and enter the A-site of the ribosome in a complex with elongation factor Tu (EF-Tu) in prokaryotes or eEF-1 in the eukaryotic cell cytoplasm (1Kozak M. Microbiol. Rev. 1983; 47: 1-45Crossref PubMed Google Scholar). In most organisms, the initiator tRNA has distinct features that ensure its selection during the initiation process and its exclusion from the steps of polypeptide chain elongation. In prokaryotes and eukaryotic organelles, such as mitochondria and chloroplasts, the methionine attached to the initiator tRNA undergoes formylation at its amino group through the action of the enzyme methionyl-tRNA transformylase (MTF) (2Marcker K. Sanger F. J. Mol. Biol. 1964; 8: 835-840Crossref PubMed Scopus (154) Google Scholar, 3Mahler H. Crit. Rev. Biochem. 1973; 1: 625-637Google Scholar, 4Schwartz J.H. Meyer R. Eisenstadt J.M. Brawerman G. J. Mol. Biol. 1967; 25: 571-574Crossref PubMed Scopus (43) Google Scholar). In Escherichia coli , MTF discriminates strictly between the initiator tRNA and the tRNAs used for chain elongation by recognizing specific determinants in the initiator tRNA (5RajBhandary U.L. J. Bacteriol. 1994; 176: 547-552Crossref PubMed Google Scholar). Formylation of methionyl-tRNA is necessary for the interaction of the tRNA with IF-2. Formylation also eliminates any significant interaction with EF-Tu. In the yeasts and plants, the initiator tRNA is not formylated. However, Met-tRNAiMet is excluded from chain elongation by the presence of a 2′-O -ribosyl phosphate modification at position 64 of the initiator tRNA (6Åström S.U. Byström A.S. Cell. 1994; 79: 535-546Abstract Full Text PDF PubMed Scopus (65) Google Scholar). The translational system in animal mitochondria is thought to be more closely related to that of prokaryotes than to that of the eukaryotic cell cytoplasm (7Galper J.B. Darnell J.E. J. Mol. Biol. 1971; 57: 363-367Crossref PubMed Scopus (35) Google Scholar, 8Kumazawa Y. Schwartzbach C.J. Liao H.X. Mizumoto K. Kaziro Y. Miura K. Watanabe K. Spremulli L.L. Biochim. Biophys. Acta. 1991; 1090: 167-172Crossref PubMed Scopus (38) Google Scholar). This idea is based on the use of fMet-tRNA for initiation, on the antibiotic sensitivity of the ribosomes, and on the ability of the mammalian mitochondrial elongation factors to function on bacterial ribosomes. However, animal mitochondrial protein synthesis has a number of unusual features that distinguish it from other translational systems. In general, mitochondrial tRNAs are shorter than their prokaryotic or eukaryotic cytoplasmic counterparts (59–75 nucleotides in length). They display numerous primary structural differences from “normal” tRNAs. In some cases, they cannot be folded into the typical cloverleaf secondary structure and lack one or more of the invariant or semi-invariant residues found in other tRNAs (9Dirheimer G. Keith G. Dumas P. Westhof E. Süll D. RajBhandary U.L. tRNA. ASM Press, Washington, D. C.1995: 93-126Google Scholar). There are genes for 22 tRNAs in the mammalian mitochondrial genome (10Anderson S. de Bruijin M.H. Coulson A.R. Eperon I.C. Sanger F. Young I.G. J. Mol. Biol. 1982; 156: 683-717Crossref PubMed Scopus (1193) Google Scholar). This number is sufficient to read the altered genetic code found in this organelle. There is a single tRNA for each amino acid except for leucine and serine for which two tRNAs are required. A single gene for tRNAMet is present and no tRNAs appear to be imported into mammalian mitochondria (11Roe B.A. Wong J.F.H. Chen E.Y. Walton A.G. Proceedings of the Third Cleaveland Symposium on Macromolecules. Elsevier, Amsterdam1981: 167-176Google Scholar). It is unclear how a single tRNAMet species can play the dual roles of an initiator and an elongator tRNA. Translational initiation in mammal mitochondria requires fMet-tRNA for the IF-2mt-dependent binding to ribosomes. However, the unformylated form is required by EF-Tumt for chain elongation (12Liao H.X. Spremulli L.L. J. Biol. Chem. 1990; 265: 13618-13622Abstract Full Text PDF PubMed Google Scholar, 13Schwartzbach C.J. Spremulli L.L. J. Biol. Chem. 1989; 264: 19125-19131Abstract Full Text PDF PubMed Google Scholar). Thus, the single tRNAMet gene must give rise to two species of tRNA (fMet-tRNA and Met-tRNA). This process requires a mechanism to adjust the ratio of formylated to non-formylated Met-tRNAs to meet the needs of both initiation and elongation. As a first step toward the investigation of this process, we report here the purification, cloning, and characterization of bovine mitochondrial MTF (MTFmt). Folinic acid and CHAPS were purchased from Sigma. [35S]Methionine (37 TBq/mmol) and [14C]methionine (1.85 GBq/mmol) were obtained from Amersham. DEAE-Sepharose fast flow, Mono S (HR5/5), Hi Trap Blue, and Hi Trap Heparin columns were purchased from Pharmacia. An affinity column using an immobilized E. coli tRNA mixture was prepared as described (14Cuatrecasas P. J. Biol. Chem. 1970; 245: 3059-3065Abstract Full Text PDF PubMed Google Scholar). Buffer TG contains 20 mm Tris-HCl (pH 7.6), 1 mm MgCl2, 0.1 mm EDTA, 6 mm β-mercaptoethanol, 10% glycerol, 0.1 mmphenymethylsulfonyl fluoride. Buffer PG contains 20 mmpotassium phosphate (pH 6.8), 10% glycerol, 1 mmdithiothreitol, 0.1 mm phenymethylsulfonyl fluoride, 0.5% CHAPS. Protein concentrations were determined by the Bio-Rad protein assay kit using bovine serum albumin as a standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to Laemmli (15Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Mitochondrial Met-tRNA synthetase (MetRSmt) was partially purified from mitochondrial extracts by chromatography on DEAE-Sepharose and ceramic hydroxyapatite (Bio-Rad). E. coli MetRS was partially purified from E. coli extracts by chromatography on DEAE-Sepharose and ceramic hydroxyapatite (Bio-Rad). Mitochondrial tRNAMet was purified by a solid-phase hybridization method using DNA probes complementary to the 30 bases at the 3′-end of the tRNA (16Wakita 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). E. coli tRNAfMetand tRNAmMet were purified as described (17Nishimura S. Cantoni G.L. Davies D.R. Procedures in Nucleic Acid Research. 2. Harper & Row, New York1971Google Scholar, 18Yoshida M. Takeishi K. Ukita T. Biochim. Biophys. Acta. 1971; 228: 153-166Crossref PubMed Scopus (63) Google Scholar). Further purification was carried out on a 6% native polyacrylamide gel if necessary. Aminoacylation of mitochondrial tRNAMet was carried out in reaction mixtures (100 μl) containing 100 mm Tris-HCl (pH 8.5), 14 mm Mg(OAc)2, 20 mm KCl, 2 mm dithiothreitol, 4 mm ATP, 1 mmspermine, 20 μm [35S]methionine (200 GBq/mmol), 2–4 μm tRNAMet, and saturating amounts of partially purified MetRSmt. The tRNA was extracted using phenol equilibrated at pH 5.0 and the remaining ATP and methionine were removed on a Hi Trap desalting gel (Pharmacia). Aminoacylation of E. coli tRNA was carried out with [35S] or [14C]methionine as described (19Gite S. RajBhandary U.L. J. Biol. Chem. 1997; 272: 5305-5312Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and the Met-tRNAs were purified as described above. The bovine liver mitochondria were prepared as described (20Schwartzbach C.J. Farwell M. Liao H.X. Spremulli L.L. Methods Enzymol. 1996; 264: 248-261Crossref PubMed Google Scholar). About 60 g of mitochondria were resuspended in 240 ml of Buffer TG containing 0.005 m KCl (TG.005), and disrupted by sonication using five 20-s bursts at 100 watts followed by 40-s cooling periods. The homogenate was subjected to centrifugation at 100,000 × g for 180 min. The supernatant fraction (S100) was either processed immediately or frozen quickly and stored at −70 °C. CHAPS was added to a final concentration of 0.2% (w/v) to all the buffers indicated below. The S100 (5,700 mg) was applied to a 100-ml DEAE-Sepharose fast flow column (17.5 × 2.7 cm) equilibrated with Buffer TG.005, at a flow rate of about 2.0 ml/min. The column was washed by Buffer TG.005 until the absorbance at 280 nm became less than 0.1 and the proteins bound to the column were eluted by a 1.0-liter linear gradient of 5–400 mm KCl in Buffer TG. Fractions (10 ml) were collected at a flow rate of 2.0 ml/min. Fractions containing MTFmt activity were pooled and concentrated by ammonium sulfate precipitation (45–60% saturation). The pellet was then dissolved in Buffer PG and dialyzed against Buffer PG containing 0.15m KCl (PG.15) for 6 h with two changes of buffer. The sample (1,300 mg) was applied to a Mono S column (0.5 × 5 cm) equilibrated in Buffer PG.15 at a flow rate of 0.25 ml/min. The column was then washed with Buffer PG.15 and developed with a 10-ml linear gradient from 0.15 to 0.4 m KCl in Buffer PG. Fractions (0.25 ml) were collected at the flow rate of 0.25 ml/min. The fractions with MTFmtactivity were pooled and diluted with Buffer PG until the concentration of KCl was less than 0.25 m. The sample was immediately frozen and stored at −70 °C. The sample (1.6 mg) was applied at a flow rate of 0.25 ml/min to a Hi Trap Blue column (1 ml) equilibrated in Buffer PG containing 0.25 m KCl (PG.25). After washing with Buffer PG.25, bound proteins were eluted with a 10-ml linear gradient from 0.25 to 0.75 m KCl in Buffer PG. Fractions (0.25 ml) were collected at the flow rate of 0.25 ml/min. The fractions with MTFmt activity were pooled and diluted with Buffer PG to decrease the KCl concentration to less than 0.10 m. The sample was then frozen quickly and stored at −70 °C. E. coli tRNA was immobilized on CNBr-activated Sepharose 4B (Pharmacia). The partially purified sample (0.24 mg) containing MTFmt activity was applied to the column (0.5 × 2.1 cm) which had been equilibrated in Buffer PG containing 0.1 m KCl (PG.10). The column was developed with a linear gradient (0.10 to 0.60 m KCl in Buffer PG). Fractions (0.1 ml) were collected at the flow rate of 0.1 ml/min. The fractions showing MTFmt activity were diluted with Buffer PG to reduce the concentration of KCl to less than 0.25 mand stored at −70 °C. The sample (0.13 mg) was applied to a Hi Trap Heparin column (1 ml) equilibrated with Buffer PG.25. The column was washed with Buffer PG.25 and developed with a 10-ml linear gradient from 0.25 to 0.75 m KCl in Buffer PG. Fractions of 0.25 ml were collected at the flow rate of 0.25 ml/min. Fractions with MTFmt activity were pooled and dialyzed against buffer containing 20 mm Tris-HCl (pH 7.6), 10 mm KCl, 5 mm MgCl2, 10% glycerol, 1 mm dithiothreitol, and 0.5% CHAPS. The sample was divided into small aliquots, fast-frozen, and stored at −70 °C. The assay of the formylation activity was carried out according to Ref. 21Blanquet S. Dessen P. Kahn D. Methods Enzymol. 1984; 106: 141-153Crossref PubMed Scopus (32) Google Scholar with a slight modification as follows. Reaction mixtures (50 μl) contained 20 mm Tris-HCl (pH 7.6), 10 mmKCl, 5 mm MgCl2, 0.1 mg/ml bovine serum albumin, 0.5% (w/v) CHAPS, 1 mm dithiothreitol, 1 μm E. coli [14C]Met-tRNAfMet, 0.3 mm N 10-formyltetrahydrofolate, and the indicated amounts of MTFmt. The partially purified sample containing MTFmt was subjected to SDS-PAGE and blotted onto a siliconized glass-fiber membrane (22Eckerskron C. Mewes W. Goretzki H. Lottspeich F. Eur. J. Biochem. 1988; 176: 509-519Crossref PubMed Scopus (247) Google Scholar). The band believed to be MTFmt (based on the correlation between the intensity of this band and the activity of MTFmt) was excised. The amino-terminal sequence was obtained on an Applied Biosystems 477A/120A protein sequencer. The sequences of internal peptides were obtained according to Clevelandet al . (23Cleveland 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 modifications indicated in Ref. 24Omori A. Yoshida S. J. Protein Chem. 1994; 13: 471Google Scholar. Approximately 1 × 106 plaques from a bovine heart cDNA library (Uni-ZAPTM XR, Stratagene) were screened by hybridization with a putative human MTFmtcDNA probe labeled by random priming (25Gottesman M.M. Methods Enzymol. 1987; 151: 363-364Google Scholar). Hybridizations were carried out at 65 °C with 6 × SSC buffer containing 20 mm NaH2PO4 and 0.4% (w/v) SDS (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Positive plaques were isolated and the pBluescript SK(−) plasmid clones were excised in vivo according to the manufacturer's instructions (Stratagene). Plasmid DNA was subjected to autosequencing using a HITACHI SQ-5500 sequencer (27Biggin M.D. Gibson T.J. Hong G.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3963-3965Crossref PubMed Scopus (1412) Google Scholar). The initiation of protein synthesis in mitochondria requires the use of the formylated initiator tRNA (fMet-tRNA). Hence, this organelle must possess a factor equivalent to the bacterial methionyl-tRNA transformylase. When extracts of bovine mitochondria were tested for a factor that could carry out the formylation of E. coli Met-tRNAfMet, a small amount of activity could be detected. The partial purification of this activity (MTFmt) was carried out by successive column chromatography as described under “Experimental Procedures” (TableI).Table IPurification of mitochondrial MTFmt from bovine liverPurification stepProteinTotal unitsSpecific activity1-aSpecific activity; 1 unit of the enzyme is the capacity of formylating 1 pmol of E. coli Met-tRNAfMet in 1 min at 30 °C.Total recoveryPurification×10 3 mg×10 3unit/mg%-foldMitochondrial extract1-bFrom 63 g (wet weight) of bovine mitoplast.(S-100)5,7001,1000.191001DEAE-Sepharose1,3004200.32381.7Mono S1.627172.589Hi Trap Blue0.247.5310.068160Affinity column (tRNAE. colimix-Sepharose)0.137.0540.064280Hi Trap Heparin0.0104.24200.0382,2001-a Specific activity; 1 unit of the enzyme is the capacity of formylating 1 pmol of E. coli Met-tRNAfMet in 1 min at 30 °C.1-b From 63 g (wet weight) of bovine mitoplast. Open table in a new tab The purification scheme resulted in a 2,200-fold purification of MTFmt with an overall yield of about 0.04%. Throughout the purification scheme, the recovery of MTFmt was significantly improved by the addition of the detergent CHAPS to all of the buffers used. This observation suggests that there are hydrophobic patches on MTFmt that lead to the absorption of this factor on the matrices of various resins or that reduce its solubility resulting in substantial losses of activity. Analysis of the partially purified preparation of MTFmt on SDS-PAGE (Fig.1 B ) showed the presence of three major polypeptide bands. The band with a molecular mass of about 40,000 daltons was tentatively identified as MTFmt. The intensity of this band correlated with the amount of MTFmtactivity observed. In addition, the transformylase would be expected to be about this size. This polypeptide represented about 25% of the protein in the partially purified sample. About 2.5 μg of MTFmt were obtained from 2 kg of bovine liver. In order to obtain cDNA clones of MTFmt, partial peptide sequences were determined. MTFmt was first subjected to NH2-terminal Edman degradation. Second, for the determination of internal amino acid sequences, peptides resulting from digestion with endoproteinase V8 were purified by polyacrylamide gel electrophoresis and subjected to Edman degradation. Three peptide sequences were obtained (Table II).Table IISequences of peptide fragments derived from bovine liver MTFmtPeptidePosition1 = NH2-ASPPWED1–72 = EVVTVPSPSP53–623 = ETVPVPPKST162–171 Open table in a new tab These sequences were then used to search the data bases. The partial sequence of one human cDNA (GenBank number 108908) included the sequence EVVTVPSPSP found as an internal peptide in bovine MTFmt. The nucleotide sequence of this cDNA contained a 400-bp region which was homologous to E. coli MTF. This cDNA clone was, thus, predicted to encode a portion of human MTFmt. Probes prepared from this putative human MTFmt cDNA hybridized with the E. coli MTF gene (data not shown). A bovine heart Uni-ZAPTMXR cDNA library was screened using the human cDNA clone as a probe. Five positive plaques were isolated among 1 × 106 plaques, and the plasmids carrying the cDNA inserts of interest were excisedin vivo . The largest clone characterized carried a 1355-bp insert (Fig. 2). Sequence analysis indicated that this clone contained the entire coding region for the mature form of MTFmt (1071 bp) and a portion of a putative mitochondrial import signal (45 bp). The 3′-untranslated region was 220 bp in length and contained a conventional polyadenylation signal (AAUAAA) 17 nucleotides upstream of the poly(A) tail (Fig. 2) (28Wickens M. Stephenson P. Science. 1984; 226: 1045-1051Crossref PubMed Scopus (313) Google Scholar). The mature form of MTFmt is 357 amino acids in length and has a molecular weight of 40,017. This value is consistent with the molecular weight of the band identified as MFTmt on SDS-PAGE. The amino acid sequence of MTFmt is about 30% identical to the corresponding prokaryotic factors (Fig. 3 A ) (29Guillon J.M. Meinnel T. Mechulam Y. Lazennec C. Blanquet S. Fayat S. J. Mol. Biol. 1992; 244: 359-367Crossref Scopus (86) Google Scholar, 30Meinnel T. Blanquet S. J. Bacteriol. 1994; 176: 7387-7390Crossref PubMed Google Scholar, 31Fleischmann R.D. et al.Science. 1995; 269: 496-512Crossref PubMed Scopus (4702) Google Scholar, 32Fraser C.M. et al.J. Mol. Biol. 1997; 266: 939-949Crossref PubMed Scopus (53) Google Scholar, 33Kunst F. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3134) Google Scholar). It is interesting to note that the sequence of bovine MFTmt is also only 28% identical to that of yeast MTFmt (34Skala J. Van Dyck L. Purnelle B. Goffeau A. Yeast. 1992; 8: 777-785Crossref PubMed Scopus (13) Google Scholar) (Table III).Table IIIIdentity of the known MTF amino acid sequences from various organismsE. coliT. thermophilusH. influenzaeM. genitaliumB. subtilisYeast (mt)Bovine (mt)%%%%%%%E. coli100T. thermophilus44100H. influenzae6540100M. genitalium262329100B. subtilis43394032100Yeast (mt)2929292427100Bovine (mt)292829242828100Sequence comparisons were done with the Maximum Matching program in GENETYX version 7.0. The designations used are as follows: E. coli, Thermus thermophilus (T. thermophilus), Haemophilus influenzae (H. influenzae), Mycoplasma genitalium (M. genitalium), Bacillus subtilis (B. subtilis), Saccharomyces cerevisiae mitochondria (Yeast (mt)), and bovine liver mitochondria (Bovine (mt)). Open table in a new tab Sequence comparisons were done with the Maximum Matching program in GENETYX version 7.0. The designations used are as follows: E. coli, Thermus thermophilus (T. thermophilus), Haemophilus influenzae (H. influenzae), Mycoplasma genitalium (M. genitalium), Bacillus subtilis (B. subtilis), Saccharomyces cerevisiae mitochondria (Yeast (mt)), and bovine liver mitochondria (Bovine (mt)). It should be noted that, while the NH2-terminal amino acid sequence obtained by peptide sequencing was ASPGWED, the cDNA sequence obtained gave the sequence ASPPWED when translated into the amino acid sequence. This apparent discrepancy could arise if there is more than one copy of MTFmt gene in the bovine nuclear genome with slightly different sequences. The crystal structure of E. coli MTF has recently been determined (35Schmitt E. Blanquet S. Mechulam Y. EMBO J. 1996; 15: 4749-4758Crossref PubMed Scopus (60) Google Scholar). Analysis of this structure indicates that MTF contains two domains, NH2-terminal and COOH-terminal domains. NH2-terminal domain carries the tetrahydrofolate (THF)-binding site in which the THF binding motif (SLLP motif) is contained, as well as a Rossman fold (35Schmitt E. Blanquet S. Mechulam Y. EMBO J. 1996; 15: 4749-4758Crossref PubMed Scopus (60) Google Scholar). The longest stretch of conserved residues among the MTFs from various sources is located in the THF-binding site proposed for E. coli MTF (35Schmitt E. Blanquet S. Mechulam Y. EMBO J. 1996; 15: 4749-4758Crossref PubMed Scopus (60) Google Scholar) (Fig. 3). The COOH-terminal domain provides a positively charged surface oriented toward the active center of the enzyme and is, presumably, involved in positioning the Met-tRNA substrate for the formylation reaction. The THF binding motif is present as SCLP in bovine MTFmt, and the Phe at position 14, which is assumed to be responsible for the interaction of E. coli MTF with the 3′-end of the Met-tRNA (35Schmitt E. Blanquet S. Mechulam Y. EMBO J. 1996; 15: 4749-4758Crossref PubMed Scopus (60) Google Scholar), is also conserved in the bovine mitochondrial counterpart (Phe-29) (Fig. 3). The COOH-terminal domain of E. coli MTF is characterized by an oligonucleotide binding fold termed the OB fold. The OB fold is formed by two orthogonal sheets consisting of five antiparallel strands folded into a β-barrel surrounded by α-helices (35Schmitt E. Blanquet S. Mechulam Y. EMBO J. 1996; 15: 4749-4758Crossref PubMed Scopus (60) Google Scholar). Secondary structure predictions using the Chou-Fasman or Robson methods (36Chou P.Y. Fasman G.D. Adv. Enzymol. 1978; : 45-148PubMed Google Scholar, 37Garnier J. Osguthorpe D.J. Robson B. J. Mol. Biol. 1978; 120: 97-120Crossref PubMed Scopus (3423) Google Scholar) have not able been to clearly identify a similar structure in MTFmt. The Lys at position 207 in the linker region between the two domains of E. coli MTF is thought to be involved in the interaction with the 3′-end of the tRNA (19Gite S. RajBhandary U.L. J. Biol. Chem. 1997; 272: 5305-5312Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). This residue (Lys-214) has been conserved in the spacer region between the domains of MTFmt (Fig. 3). The initiation of translation in most prokaryotic organisms requires the formylation of the initiator Met-tRNA by MTF. In E. coli , the formyl group is a positive determinant for the specific interaction of IF-2 with the initiator tRNA. It also serves as a negative determinant that nearly eliminates the binding of EF-Tu to the initiator tRNA (5RajBhandary U.L. J. Bacteriol. 1994; 176: 547-552Crossref PubMed Google Scholar). Bacterial MTF will not formylate the Met-tRNAmMet species used for chain elongation. The strict substrate specificity of E. coli MTF is essential to ensure the accuracy and the efficiency of the initiation process. In contrast to all other systems, animal mitochondria do not contain two distinct methionyl-tRNA species that are used exclusively for the initiation or elongation phases of protein synthesis. Mammalian mitochondria have a single tRNAMetgene which is encoded in the organelle genome (10Anderson S. de Bruijin M.H. Coulson A.R. Eperon I.C. Sanger F. Young I.G. J. Mol. Biol. 1982; 156: 683-717Crossref PubMed Scopus (1193) Google Scholar). There is no evidence that cytoplasmic tRNAs are imported into animal mitochondria (11Roe B.A. Wong J.F.H. Chen E.Y. Walton A.G. Proceedings of the Third Cleaveland Symposium on Macromolecules. Elsevier, Amsterdam1981: 167-176Google Scholar). Thus, the single tRNAMet gene must, in some unknown manner, give rise to both an initiator tRNA (fMet-tRNA) and an elongator tRNA (Met-tRNA) (35Schmitt E. Blanquet S. Mechulam Y. EMBO J. 1996; 15: 4749-4758Crossref PubMed Scopus (60) Google Scholar, 19Gite S. RajBhandary U.L. J. Biol. Chem. 1997; 272: 5305-5312Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The unique presence of a single Met-tRNA species in mammalian mitochondria made it of considerable interest to address the substrate specificity of MTFmt. The kinetic parameters governing the formylation of three native tRNA molecules, bovine mitochondrial Met-tRNA, E. coli Met-tRNAfMet, and E. coli Met-tRNAmMet, were measured (TableIV). The results of these experiments indicated that MTFmt is clearly able to use E. coli Met-tRNAfMet with a V maxthat is about 3-fold higher than that observed with the mitochondrial Met-tRNAMet. The K m observed with theE. coli initiator tRNA is a little over 3-fold higher than with the mitochondrial tRNAMet. The net result is that the relative V max/K m for these two tRNAs are essentially the same. Surprisingly, MTFmt was also able to formylate the E. coli elongator Met-tRNAmMet. This tRNA is never a substrate for formylation by the homologous E. coli MTF (38Dickerman H.W. Steers Jr., E. Redfield B.G. Weissbach H. J. Biol. Chem. 1967; 242: 1522-1525Abstract Full Text PDF PubMed Google Scholar). TheK m value observed with Met-tRNAmMet is essentially the same as that observed for E. coli Met-tRNAfMet while the V max is about 3-fold lower than that observed with the mitochondrial Met-tRNA. MTFmt clearly does not discriminate between the bacterial initiator and elongator Met-tRNAs. This observation is compatible with the fact that there is a single Met-tRNA species in mammalian mitochondria.Table IVKinetic parameters in formylation of various Met-tRNAsMet-tRNAV max (×10−2μM/min)K mRelativeV max/K m4-aRelative V max/K mis the ratio of Vmax/Km of mitochondorial Met-tRNA toV max/K m of each Met-tRNA.μME. coli (f)2.40.0910.83E. coli (m)0.280.0950.091Bovine mitochondria0.80.02514-a Relative V max/K mis the ratio of Vmax/Km of mitochondorial Met-tRNA toV max/K m of each Met-tRNA. Open table in a new tab E. coli MTF is known to have some affinity for many different tRNAs that is mediated through an interaction between the 3′-end of the acceptor stem of the tRNA and regions surrounding Lys-207 in MTF (19Gite S. RajBhandary U.L. J. Biol. Chem. 1997; 272: 5305-5312Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) (Fig. 4). However, only Met-tRNAfMet is a substrate for the actual formylation process. It has been speculated that the formylation reaction is triggered by the melting of the acceptor stem of Met-tRNAfMet which is facilitated in the initiator tRNA by the unstable acceptor stem found in this tRNA (19Gite S. RajBhandary U.L. J. Biol. Chem. 1997; 272: 5305-5312Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 39Wallis N.G. Dardel F. Blanquet S. Biochemistry. 1995; 34: 7668-7677Crossref PubMed Scopus (29) Google Scholar). Several residues in MTF are thought to be involved in this melting process, such as basic residues in the loop I, and Phe-14 (35Schmitt E. Blanquet S. Mechulam Y. EMBO J. 1996; 15: 4749-4758Crossref PubMed Scopus (60) Google Scholar, 40Ramesh V. Gite S. Li Y. RajBhandary U.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13524-13529Crossref PubMed Scopus (26) Google Scholar) (Fig. 4). Phe-14 corresponds to Phe-29 in MTFmt, but Arg-42 in the loop I could not be identified clearly in MTFmt. A comparison of the sequences of prokaryotic MTF and yeast MTFmt with the sequence of bovine MTFmt does not provide a clear rationale for the structural basis for the Met-tRNA specificities of these enzymes. However, it should be noted that the region termed the “insertion sequence” in the Rossman fold, which is usually variable in length in class I aminoacyl-tRNA synthetases (41Arnez J.G. Moras D. Nagai K. Mattaj I.W. RNA-Protein Interactions. Oxford University Press, Oxford1994: 52-81Google Scholar), is shorter in the case of bovine MTFmt than in prokaryotic MTF (Figs. 3 and 4). The difference in this distance would be expected to place a number of residues in somewhat different positions in MTFmt. This difference might permit the mitochondrial factor to form the transition state with the elongator Met-tRNA easily. Hence it might be able to formylate the elongator Met-tRNA without the necessity for melting the acceptor stem. Further experiments will be designed to evaluate this idea. How the single tRNAMet gene gives rise to both an initiator and an elongator tRNAMet species still remains unknown at this time. One possibility is that the formylation of Met-tRNA converts it from the elongator to the initiator tRNA since formylation increases its affinity for IF-2mt and diminishes its ability to form a complex with EF-Tumt. The ratio of formylated to the non-formylated Met-tRNA might be regulated by a competition between MTFmt and EF-Tumt for Met-RNA. The purification and characterization of MTFmt as described here will allow us to investigate this issue by using purified IF-2mt and EF-Tumt. Alternatively, the activity of MTFmtmight be regulated by an unknown mechanism in vivo in conjunction with the initiation of mitochondrial protein synthesis. An investigation of this possibility must await the development of a better understanding of the initiation of mitochondrial protein synthesis. We are indebted to our previous co-workers, Dr. T. Yokogawa (Gifu University), Dr. C. Takemoto (University of Tokyo), T. Koike (National Institute of Genetics), and Dr. L. Benkowski (University of North Carolina), for their experimental supports and critical discussions. We thank S. Yoshida, Mitsubishikasei Institute of Life Science, for technical assistance in peptide sequence determination." @default.
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• W2084325654 title "Mammalian Mitochondrial Methionyl-tRNA Transformylase from Bovine Liver" @default.
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