A Disease-causing Point Mutation in Human Mitochondrial tRNAMet Results in tRNA Misfolding Leading to Defects in Translational Initiation and Elongation
2008; Elsevier BV; Volume: 283; Issue: 49 Linguagem: Inglês
10.1074/jbc.m806992200
ISSN1083-351X
AutoresChristie N. Jones, Christopher I. Jones, William D. Graham, Paul F. Agris, Linda Spremulli,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNAMet (hmtRNAMet) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNAMet with a C8. The hmtRNAMet serves both in translational initiation and elongation in human mitochondria making this tRNA of particular interest in mitochondrial protein synthesis. Here we show that the single 8U→C mutation leads to a failure of the tRNA to respond conformationally to Mg2+. This mutation results in a drastic disruption of the structure of the hmtRNAMet, which significantly reduces its aminoacylation. The small fraction of hmtRNAMet that can be aminoacylated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initiation, and it is unable to form a stable ternary complex with elongation factor EF-Tu preventing any participation in chain elongation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg2+, the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg2+, the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg2+-binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans. The mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNAMet (hmtRNAMet) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNAMet with a C8. The hmtRNAMet serves both in translational initiation and elongation in human mitochondria making this tRNA of particular interest in mitochondrial protein synthesis. Here we show that the single 8U→C mutation leads to a failure of the tRNA to respond conformationally to Mg2+. This mutation results in a drastic disruption of the structure of the hmtRNAMet, which significantly reduces its aminoacylation. The small fraction of hmtRNAMet that can be aminoacylated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initiation, and it is unable to form a stable ternary complex with elongation factor EF-Tu preventing any participation in chain elongation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg2+, the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg2+, the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg2+-binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans. Human mitochondria are subcellular organelles that produce more than 90% of the energy required by the cell. The mitochondrial genome encodes 13 proteins necessary for energy production, two rRNAs and all of the 22 tRNAs required for the synthesis of these proteins (1Attardi G. Int. Rev. Cytol. 1985; 93: 93-145Crossref PubMed Scopus (237) Google Scholar, 2Anderson S. de Brujin M. Coulson A. Eperon I. Sanger F. Young I. J. Mol. Biol. 1982; 156: 683-717Crossref PubMed Scopus (1188) Google Scholar). Mammalian mitochondrial tRNAs have several unusual features that distinguish them from canonical tRNAs. In many cases, they lack a number of the conserved or semi-conserved nucleotides that play important roles in creating the L-shaped tertiary structure of prokaryotic and eukaryotic cytoplasmic tRNAs (3Dirheimer G. Keith G. Dumas P. Westhof E. RajBhandary U. Soll D. tRNA: Structure, Biosynthesis and Function. American Society for Microbiology, Washington, DC1995: 93-126Google Scholar). There is little detailed structural information on these tRNAs. No data are currently available that examine the structure of mammalian mitochondrial tRNAs with single nucleotide resolution. However, chemical and enzymatic probing has lead to the idea that these tRNAs have retained the basic cloverleaf structure of canonical tRNAs but that they lack several conserved tertiary interactions leading to a weaker three-dimensional structure (4Watanabe Y.-I. Kawai G. Yokogawa T. Hayashi N. Kumazawa Y. Ueda T. Nishikawa K. Hirao I. Miura K.-I. Watanabe K. Nucleic Acids Res. 1994; 22: 5378-5384Crossref PubMed Scopus (56) Google Scholar, 5Yokogawa T. Watanabe Y.-I. Kumazawa Y. Ueda T. Hirao I. Miura K.-I. Watanabe K. Nucleic Acids Res. 1991; 19: 6101-6105Crossref PubMed Scopus (68) Google Scholar, 6Wakita K. Watanabe W. Yokogawa T. Kumazawa Y. Nakamura S. Ueda T. Watanabe K. Nishikawa K. Nucleic Acids Res. 1994; 22: 347-353Crossref PubMed Scopus (75) Google Scholar, 7Helm M. Giege R. Florentz C. Biochemistry. 1999; 38: 13338-13346Crossref PubMed Scopus (174) Google Scholar, 8Helm M. Brule H. Friede D. Giege R. Putz D. Florentz C. RNA (N. Y.). 2000; 6: 1356-1379Crossref PubMed Scopus (242) Google Scholar). In particular, a number of the long range interactions between the D- and T-arms of the tRNAs appear to be missing.All 22 tRNAs that function in mammalian mitochondria are encoded in the mitochondrial DNA. Considerable interest in mitochondrial tRNAs centers on the occurrence of diseases arising from mutations in their genes that lead to maternally inherited genetic disorders (9Wittenhagen L.M. Kelley S.O. Trends Biochem. Sci. 2003; 28: 605-611Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10King M. Koga Y. Davidson M. Schon E. Mol. Cell. Biol. 1992; 12: 480-490Crossref PubMed Scopus (404) Google Scholar, 11Sternberg D. Chatzoglou E. Laforet P. Fayet G. Jardel C. Blondy P. Fardeau M. Amselem S. Eymard B. Lombes A. Brain. 2001; 124: 984-994Crossref PubMed Scopus (79) Google Scholar, 12Enriquez J. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (257) Google Scholar). The diseases associated with mitochondrial tRNA mutations may arise from failure in the processing of the tRNA (13Levinger L. Jacobs O. James M. Nucleic Acids Res. 2001; 29: 4334-4340Crossref PubMed Scopus (51) Google Scholar), from reduced stability of the tRNA (14Hao H. Moraes C.T. Mol. Cell. Biol. 1997; 17: 6831-6837Crossref PubMed Scopus (57) Google Scholar, 15Kelley S.O. Steinberg S.V. Schimmel P. Nat. Struct. Biol. 2000; 7: 862-865Crossref PubMed Scopus (54) Google Scholar), from a reduction in aminoacylation (12Enriquez J. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (257) Google Scholar, 16Ling J. Roy H. Qin D. Rubio M.A. Alfonzo J.D. Fredrick K. Ibba M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 15299-15304Crossref PubMed Scopus (38) Google Scholar, 17Cenatiempo Y. Deville F. Dondon J. Grunberg-Manago M. Sacerdot C. Hershey J.W. Hansen H.F. Petersen H.U. Clark B.F. Kjeldgaard M. la Cour T.F.M. Mortensen K.K. Nyborg J. Biochemistry. 1987; 26: 5070-5076Crossref PubMed Scopus (49) Google Scholar), from a reduced ability of the mutated aminoacyl-tRNA to interact with mitochondrial elongation factor Tu (EF-Tumt) 3The abbreviations used are: EF-Tumt, mitochondrial elongation factor Tu; hmtRNAMet, human mitochondrial tRNAMet; MetRS, methionyl-tRNA synthetase; hmMetRS, human mitochondrial methionyl-tRNA synthetase; MTF, methionyl-tRNA transformylase; SHAPE, selective 2′-hydroxyl acylation analyzed by primer extension; PMSF, phenylmethylsulfonyl fluoride; ;ME, ;-mercaptoethanol; 1M7, 1-methyl-7-nitroisatoic anhydride. 3The abbreviations used are: EF-Tumt, mitochondrial elongation factor Tu; hmtRNAMet, human mitochondrial tRNAMet; MetRS, methionyl-tRNA synthetase; hmMetRS, human mitochondrial methionyl-tRNA synthetase; MTF, methionyl-tRNA transformylase; SHAPE, selective 2′-hydroxyl acylation analyzed by primer extension; PMSF, phenylmethylsulfonyl fluoride; ;ME, ;-mercaptoethanol; 1M7, 1-methyl-7-nitroisatoic anhydride. (the corresponding prokaryotic factor is also designated EF1A) (16Ling J. Roy H. Qin D. Rubio M.A. Alfonzo J.D. Fredrick K. Ibba M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 15299-15304Crossref PubMed Scopus (38) Google Scholar), and from the failure of the tRNA to be correctly modified leading to translational defects (18Kirino Y. Yasukawa T. Ohta S. Akira S. Ishihara K. Watanabe K. Suzuki T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15070-15075Crossref PubMed Scopus (205) Google Scholar).Normally, protein biosynthetic systems have two tRNAMet species. One is used solely for initiation, and the other functions in polypeptide chain elongation. Animal mitochondria are quite unusual in that they contain a single gene for tRNAMet, which functions in both polypeptide chain initiation and chain elongation. As a result of this dual role, mitochondrial Met-tRNAMet must be recognized by the mitochondrial Met-tRNA transformylase (MTFmt) and be brought as fMet-tRNAMet to the ribosome for translational initiation (19Spencer A.C. Spremulli L.L. Nucleic Acids Res. 2004; 32: 5464-5470Crossref PubMed Scopus (50) Google Scholar). In addition, Met-tRNAMet must interact with elongation factor EF-Tumt and bind to the A-site of the ribosome during translational elongation. Thus, this tRNAMet is of central importance in mitochondrial translation.Human tRNAMet has a number of interesting features (Fig. 1A). The D-loop is somewhat small and lacks the G residues at positions 18 and 19 that facilitate interactions with the T-loop in the tertiary structure. The first position of the anticodon contains the rare modified base 5-formylcytidine. This modification may play a role in the unusual codon recognition requirements of this tRNA, which must recognize both AUG and AUA codons. The minor loop is short lacking the usual G47, and the T-stem has two adjacent pyrimidine:pyrimidine pairs (U-U and U-:). Furthermore, the T-loop lacks the T:C sequence and contains only six nucleotides instead of the normal seven. These unusual structural features suggest that human mitochondrial tRNAMet may have an intrinsically weak tertiary structure.Three interesting point mutations (T4409C, A4435G, and G4450A) occur in the gene for human tRNAMet (hmtRNAMet). The T4409C mutation (Fig. 1A) results in a U8 to C change at the corner of the acceptor stem and D-stem of hmtRNAMet. This mutation leads to mitochondrial myopathy resulting in dystrophic muscles and exercise intolerance (20Vissing J. Salamon M.B. Arlien-Soborg P. Norby S. Manta P. DiMauro S. Schmalbruch H. Neurology. 1998; 50: 1875-1878Crossref PubMed Scopus (59) Google Scholar). The A4435G mutation leads to the change of A37 to G37 in the anticodon loop of the tRNA (21Qu J. Li R. Zhou X. Tong Y. Lu F. Qian Y. Hu Y. Mo J.Q. West C.E. Guan M.X. Investig. Ophthalmol. Vis. Sci. 2006; 47: 475-483Crossref PubMed Scopus (119) Google Scholar). This mutation acts as a modulator of Leber's Hereditary Optic Neuropathy increasing the severity of this condition when it arises because of other mutations in the mitochondrial DNA. The G4450A mutation leads to loss of the final base pair in the T-stem (Fig. 1A). This mutation presents as splenic lymphoma, is largely confined to lymphocyte cells, and results in severely abnormal mitochondria leading to serious defects in energy production (22Lombes A. Bories D. Girodon E. Franchon P. Ngo M. Breton-Gorious J. Tulliez M. Goossens M. Hum. Mutat. 1998; 1: S175-S183Crossref PubMed Scopus (20) Google Scholar). A systematic examination of the structural and biochemical consequences of these mutations is lacking. Here we examine the structure of human mitochondrial tRNAMet and probe the effects of the 8U→C mutation on the structure and function of this tRNA.EXPERIMENTAL PROCEDURESRNA Synthesis—Human mitochondrial tRNAMet transcripts for aminoacylation experiments were prepared by in vitro transcription using the hammerhead ribozyme construct described previously (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar). The hmtRNAMet was purified by denaturing (10%) PAGE (29:1 acrylamide:bisacrylamide prepared with 7 m urea, 90 mm Tris borate, 2 mm EDTA), visualized by UV shadowing, excised from the gel, and recovered by passive elution in water followed by ethanol precipitation. hmtRNAMet transcripts for selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) experiments were prepared in the context of the structure cassette as described (24Wilkinson K.A. Merino E.J. Weeks K.M. Nat. Protoc. 2006; 1: 1610-1616Crossref PubMed Scopus (546) Google Scholar). D- and T-half-molecules were chemically synthesized (Dharmacon), purified, and analyzed as described previously (25Jones C. Spencer A.C. Hsu J. Spremulli L.L. Martinis S.A. DeRider M. Agris P.F. J. Mol. Biol. 2006; 362: 771-786Crossref PubMed Scopus (24) Google Scholar).Purification of E. coli Methionyl-tRNA Synthetase (MetRS) and Human Mitochondrial MetRS (hmMetRS)—A saturated overnight culture of JM109 cells carrying the pQE60-Escherichia coli MetRS plasmid construct (kindly provided by Uttam RajBhandary, Massachusetts Institute of Technology) was grown at 37 °C in 2× YT media (20 ml) supplemented with 50 ;g/ml ampicillin and used to inoculate 2 liters of 2×YT media (50 ;g/ml ampicillin). The cells were grown at 37 °C for 4 h (A600 = 0.6), induced with 50 ;m isopropyl ;-d-thiogalactopyranoside and then grown at 37 °C for 4 h post-induction. The cells were harvested by centrifugation at 4,000 rpm for 30 min. The cell pellet was resuspended in 500 ml of 10 mm Tris-HCl, pH 7.6, and then re-collected by low speed centrifugation. The cell pellet was fast frozen and stored at -80 °C until use.The cell pellet (7 g) was resuspended in 100 ml of lysis buffer (50 mm Tris-HCl, pH 7.6, 50 mm KCl, 10 mm MgCl2, 200 ;m phenylmethylsulfonyl fluoride (PMSF), 0.1% Triton X-100, and 7 mm ;-mercaptoethanol (;ME)) and sonicated on ice for 7 min with 10-s bursts followed by 50-s cooling periods. The cell lysate was centrifuged at 15,000 rpm for 30 min at 4 °C. E. coli MetRS was purified from the supernatant using 400 ;l of a 50% nickel-nitrilotriacetic acid (Qiagen) slurry in wash buffer (100 mm Tris-HCl, pH 7.6, 1 m KCl, 10 mm MgCl2, 10 mm imidazole, 200 ;m PMSF, and 7 mm ;ME). The resin was washed with 200 ml of wash buffer. The protein was eluted with 4 ml of elution buffer (100 mm Tris-HCl, pH 7.6, 50 mm KCl, 10 mm MgCl2, 150 mm imidazole, 200 ;m PMSF, and 7 mm ;ME). The protein sample was dialyzed against 2 volumes of 500 ml of dialysis buffer (50 mm Tris-HCl, pH 7.6, 50 mm KCl, 2.5 mm MgCl2, 0.1 mm EDTA, 10% glycerol and 7 mm ;ME) for 1 h.Cells carrying a plasmid encoding the His6-tagged human mitochondrial MetRS were grown as described (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar). The cells were lysed as described above, and the hmMetRS was further purified as described (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar).Purification of Bovine Mitochondrial Methionyl-tRNA Transformylase (MTFmt)—E. coli BL21 cells, carrying the pET15-bovine MTFmt plasmid construct, were grown as described (19Spencer A.C. Spremulli L.L. Nucleic Acids Res. 2004; 32: 5464-5470Crossref PubMed Scopus (50) Google Scholar). Cells were harvested and lysed as described for E. coli MetRS above. The protein was purified as described for the E. coli MetRS except that the buffers contained 50 mm Tris-HCl, pH 7.6. The purified protein sample was dialyzed against 2 volumes of 500 ml of MTF dialysis buffer (20 mm Tris-HCl, pH 7.6, 100 mm KCl, 10% glycerol, and 3 mm ;ME) for 1 h, fast-frozen, and stored at -80 °C.Assay for the Aminoacylation of Human Mitochondrial tRNAMet—The aminoacylation reactions for both the normal and 8U→C mutated tRNAMet transcripts were performed essentially as described (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar). Reaction mixtures (100 ;l) contained 50 mm Tris-HCl, pH 7.6, 2.5 mm MgCl2, 2.5 mm ATP, 0.2 mm spermine, 200 ;g/ml bovine serum albumin, 0.2 units/;l SUPERase·In RNase inhibitor, 40 ;m [35S]methionine (4,000 cpm/pmol), 50 nm human mitochondrial MetRS or 8 nm E. coli MetRS, and 1 ;m U8 or 8U→C hmtRNAMet. The amount of aminoacylated tRNA formed was determined by trichloroacetic acid-precipitable counts at the indicated times.Preparative Aminoacylation of [35S]Met-tRNAMet—Reaction mixtures (2 ml) were prepared as described above except that 20 ;m [35S]methionine (20,000 cpm/pmol), 0.5 ;m U8, or 8U→C hmtRNAMet, and saturating amounts of human mitochondrial MetRS were used. Reactions were incubated for 15 min at 37 °C, followed by phenol/chloroform extraction. The tRNAMet was collected by ethanol precipitation and then dissolved in 10 mm potassium succinate, pH 6.0, before use.Formylation of Human Mitochondrial Met-tRNAMet—Formylation reactions (5 ;l) contained 20 mm Tris-HCl, pH 7.6, 100 ;m EDTA, 150 mm KCl, 7 mm MgCl2, 10 mm ;ME, 125 ;m folinic acid (Sigma), 100 nm normal or 8U→C mutated [35S]Met-hmtRNAMet and 8 nm MTFmt. Reactions were performed at 37 °C for 0-8 min (0-min time point was taken in the absence of enzyme). At the indicated time, 83 mm NaOH (1 ;l of 500 mm) was added, and incubation was continued at 37 °C for 30 min. The [35S]Met and [35S]fMet in 5 ;l of each reaction were separated on Partisil LK5D TLC plates (Whatman) with a butanol:acetic acid:water (4:1:1) mixture. TLC plates were visualized by phosphorimaging (GE Healthcare) and the spots were analyzed using the ImageQuant program.Binding of Human Mitochondrial Met-tRNAMet to Bovine Mitochondrial EF-Tu (EF-Tumt)—EF-Tumt was prepared as described (26Bullard J.M. Cai Y.-C. Zhang Y. Spremulli L.L. Biochim. Biophys. Acta. 1999; 1446: 102-114Crossref PubMed Scopus (21) Google Scholar), except that the cells were lysed as described above for E. coli MetRS, and the high speed centrifugation step was omitted. Where indicated the normal U8 or 8U→C mutated hmtRNAs were phosphorylated with cold ATP using polynucleotide kinase (New England Biolabs) prior to large scale aminoacylation.To measure ternary complex formation, reaction mixtures (50 ;l) were prepared as reported (27Hunter S.E. Spremulli L.L. RNA Biol. 2004; 2: 95-102Crossref Scopus (8) Google Scholar) except that 20 mm Hepes-KOH, pH 7, and the indicated amounts of EF-Tumt were used. The reactions were incubated for 15 min at 0 °C or 6 min at 37 °C as indicated. Free [35S]Met-hmtRNAMet was digested by a 30-s incubation with 10 ;g of RNase A, and the reaction was terminated by the addition of cold 5% trichloroacetic acid. Following a 10-min incubation on ice, the [35S]Met-hmtRNAMet precipitate was collected on nitrocellulose filters and quantified by liquid scintillation counting. Determination of the Kd for ternary complex formation was carried out as described previously (28Cai Y.-C. Bullard J.M. Thompson N.L. Spremulli L.L. J. Biol. Chem. 2000; 275: 20308-20314Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar).Degradation of hmtRNAMet in a Mitochondrial Extract—Bovine mitoplasts (0.2 g) were prepared as described (29Schwartzbach C. Farwell M. Liao H.-X. Spremulli L.L. Methods Enzymol. 1996; 264: 248-261Crossref PubMed Google Scholar). Mitoplasts were lysed in buffer (2 ml) containing 15 mm Tris-HCl, pH 7.6, 40 mm KCl, 6 mm MgCl2, 6 mm ;ME, 0.8 mm EDTA, and 1.6% Triton X-100 by hand homogenization. The extract was clarified by centrifugation at 10,000 rpm for 15 min at 4 °C. Normal U8 and 8U→C mutated hmtRNAMet were 32P-end-labeled using polynucleotide kinase (New England Biolabs) according to the manufacturer's instructions. Reaction mixtures (10 ;l) contained 100 nm normal U8 or 8U→C mutated [32P]hmtRNAMet and the indicated amount of the extract in the lysis buffer above. Incubation was for 10 min at 37 °C, and cold trichloroacetic acid precipitation was used to quantitate the amount of [32P]hmtRNAMet remaining.Selective 2′-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE) Analysis of Normal U8 and 8U→C Mutated hmtRNAMet Transcripts—Normal U8 or 8U→C mutated hmtRNAMet (12 pmol, 0.33 ;m) in 36 ;l of nuclease-free water (Ambion) was incubated at 50 °C for 2 min and then cooled on ice for 2 min. The RNA was divided into 2 aliquots of 4 pmol (12 ;l) and 8 pmol (24 ;l). Folding buffer (6 ;l; 333 mm Hepes-KOH, pH 8, 333 mm NaCl) was added to the 4 pmol of RNA, and folding buffer with 20 mm Mg2+ (12 ;l) was added to the 8 pmol of RNA, and the two samples were incubated at 37 °C for 20 min. To 1 ;l of 100 mm 1-methyl-7-nitroisotoic anhydride (1M7) in anhydrous DMSO or 1 ;l of anhydrous DMSO (control), 9 ;l (2 pmol) of folded RNA was added and allowed to react at 37 °C for 70 s (5 half-lives). The balance of the RNA folded in the presence of Mg2+ (18 ;l) was divided into 2 aliquots of 2 pmol (9 ;l) each and stored at 37 °C for sequencing. MgCl2 (1 ;l; 64 mm) was added to the RNA treated with the folding buffer in the absence of Mg2+. Radiolabeled oligonucleotide (0.3 ;m;3 ;l; 5′-32P-GAACCGGACCGAAGCCCG, obtained from the Nucleic Acids Core Facility at University of North Carolina) was added to the 1M7-treated, DMSO-treated, or untreated RNA (2 pmol), and the samples were incubated at 65 °C for 5 min and then at 35 °C for 20 min for primer annealing. To each reaction, reverse transcription buffer (6 ;l; 250 mm KCl, 167 mm Tris-HCl, pH 8.3, 17 mm dithiothreitol, and 0.42 mm each dNTP) was added. Then either ddCTP or ddTTP (1 ;l; 5 mm; Amersham Biosciences) was added to the untreated RNA. After heating to 52 °C, reverse transcriptase (1 ;l; 200 units; Superscript III, Invitrogen) was added, and the primer extension reactions were performed at 52 °C for 5 min. Reactions were quenched with 4 m NaOH (1 ;l) and heated at 95 °C for 5 min. For gel analysis, a gel loading solution (29 ;l; 138 mm unbuffered Tris-HCl, 73% (v/v) formamide, 2 mm Tris borate, 86 mm EDTA, pH 8, with xylene cyanol and bromphenol blue) was added, and the samples were heated at 95 °C for an additional 5 min. The cDNA products from the + and - 1M7 and sequencing reactions were separated by denaturing gel electrophoresis (10% polyacrylamide). Samples on gels (21 cm × 40 cm × 0.4 mm) were subjected to electrophoresis at 1400 V for ∼2.5 h. Gels were visualized by phosphorimaging (GE Healthcare). The + and - 1M7 band intensities were quantified using SAFA (30Das R. Laederach A. Pearlman S.M. Herschlag D. Altman R.B. RNA (N. Y.). 2005; 11: 344-354Crossref PubMed Scopus (254) Google Scholar) and corrected for signal drop-off (31Badorrek C.S. Weeks K.M. Biochemistry. 2006; 45: 12664-12672Crossref PubMed Scopus (37) Google Scholar). SHAPE reactivities were normalized by subtracting intensities for the -1M7 control from the +1M7 reaction and dividing each by the average reactivity of the most reactive 7% of the nucleotides. To facilitate comparison of the normal U8 and 8U→C mutant tRNAs, the two data sets were normalized to the reactivity of the -CCA end nucleotides. The reactivity of each nucleotide was assigned a value between 0 and 1. Nucleotides fall into one of four categories (32Wilkinson K.A. Merino E.J. Weeks K.M. J. Am. Chem. Soc. 2005; 127: 4659-4667Crossref PubMed Scopus (125) Google Scholar, 33Merino E.J. Wilkinson K.A. Coughlan J.L. Weeks K.M. J. Am. Chem. Soc. 2005; 127: 4223-4231Crossref PubMed Scopus (551) Google Scholar) as follows: unreactive (0.000-0.055), low reactivity (0.055-0.110), moderately reactive (0.110-0.220), or highly reactive (0.220-1.000).Structural Studies of hmtRNAMet Half-molecules—Reconstitution of hmtRNAMet from U8 and 8U→C D-half-molecules with T-half-molecules required Mg2+ and was assessed by gel mobility shift assays using native 15% PAGE in Tris borate buffer (89 mm Tris base, 89 mm boric acid, pH 8.3) with and without 3 mm Mg2+ at 4 °C (25Jones C. Spencer A.C. Hsu J. Spremulli L.L. Martinis S.A. DeRider M. Agris P.F. J. Mol. Biol. 2006; 362: 771-786Crossref PubMed Scopus (24) Google Scholar). The concentration of the D-half-molecule was held constant at 31.2 ;m, whereas the concentration of the T-half-molecule was varied from 4.2 to 112 ;m.UV-Monitored Thermodynamic Experiments—The half-molecule RNA samples were dissolved in the above Tris borate buffer used for the PAGE experiments to obtain an RNA concentration of 1.2 ;m. MgCl2 was added to a concentration of 3 mm. UV-monitored thermal denaturations and re-naturations were replicated 10 times and monitored by measuring UV absorbance (260 nm) using a Cary 3 spectrophotometer as published (34Ashraf S.S. Guenther R.H. Ansari G. Malkiewicz A. Sochacka E. Agris P.F. Cell Biochem. Biophys. 2000; 33: 241-252Crossref PubMed Scopus (30) Google Scholar, 35Yarian C.S. Basti M.M. Cain R.J. Ansari G. Guenther R.H. Sochacka E. Czerwinska G. Malkiewicz A. Agris P.F. Nucleic Acids Res. 1999; 27: 3543-3549Crossref PubMed Scopus (88) Google Scholar). The data points were averaged over 20 s and recorded with a temperature change of 1 °C per min from 4 to 90 °C. The one most inconsistent of the 10 melting transitions (either a denaturation or renaturation) was discarded from each set, and the resulting data were averaged on a point-by-point basis.For UV melts of the hmtRNAMet transcripts, the normal U8 and the 8U→C hmtRNAMet were dialyzed against water using 10-kDa cutoff dialysis cups (Stratagene). The U8 and 8U→C hmtRNAMet transcripts were diluted to 0.5 ;m in a buffer containing 10 mm NaCl and 10 mm Hepes-KOH, pH 8.0. The thermal denaturation of the tRNAs was monitored by UV absorbance at 260 nm using a Cary 3 spectrophotometer. Data points were recorded once per min from 4 to 95 °C with a temperature change of 1 °C per min. Following thermal renaturation, 6 mm Mg2+ was added to the U8 and 8U→C transcripts, and the UV-monitored thermal denaturation experiments were repeated.RESULTSAminoacylation of the Normal and 8U→C Mutated tRNAMet—Previous studies (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar) have shown that the transcript of mitochondrial tRNAMet has aminoacylation properties similar to those observed with the native tRNA. Thus, it was possible to use the normal transcript and a transcript containing the 8U→C mutation for studies on the effect of the mutation on the properties of the tRNA. The 8U→C mutation leads to a myopathy presumably arising from a reduction in translational activity in mitochondria. To determine the biochemical consequence of the 8U→C mutation, the abilities of the U8 and 8U→C hmtRNAMet transcripts to be aminoacylated by the human mitochondrial methionyl-tRNA synthetase (hmMetRS) were tested. Aminoacylation is an early step required for the tRNA to be used in either the elongation or initiation phase of protein synthesis and is thus of central importance for protein synthesis in mitochondria. The normal U8 transcript was aminoacylated as expected (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar); however, the 8U→C mutation caused a significant reduction in the rate of aminoacylation of the tRNA by hmMetRS (Fig. 1B). This observation provides one clear rationale for the failure of this tRNA to function in mitochondrial protein biosynthesis.Not unexpectedly, the normal hmtRNAMet was aminoacylated by the E. coli MetRS (Fig. 1C). Interestingly, whereas the 8U→C hmtRNAMet was poorly aminoacylated by the hmMetRS, it was not aminoacylated at all by the E. coli MetRS (Fig. 1C) suggesting that the mutated tRNA had a significantly altered structure. The hmMetRS is believed to be both structurally and functionally homologous to its prokaryotic counter-part (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar). However, this work demonstrates that the hmMetRS is less discriminatory than E. coli MetRS for the structure of the tRNA. Because a major determinant in the recognition of tRNAMet by the MetRS is thought to lie in the anticodon sequence that is unchanged (36Schulman L.H. Pelka H. Science. 1988; 242: 765-768Crossref PubMed Scopus (184) Google Scholar), the weak aminoacylation most likely reflects significant structural alterations in the tRNA as a result of the mutation.Formylation of Normal and 8U→C Mutated Met-tRNAMet—The defective aminoacylation of the 8U→C hmtRNAMet made it difficult to assess the effects of the mutation on additional steps in protein biosynthesis. However, small amounts of the aminoacylated 8U→C mutated hmtRNAMet could be isolated, permitting a limited investigation of additional steps in translation.In the mammalian mitochondrial system, the Met-tRNAMet must be formylated by the mitochondrial transformylase (MTFmt) to be used in initiation (19Spencer A.C. Spremulli L.L. Nucleic Acids Res. 2004; 32: 5464-5470Crossref PubMed Scopus (50) Google Scholar, 37Takeuchi 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 (52) Google Scholar). The abilities of the U8 and 8U→C Met-tRNAMet to be formylated were tested by incubation of the [35S]Met-tRNA with th
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