FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2004246808> ?p ?o ?g. }

• W2004246808 endingPage "33225" @default.
• W2004246808 startingPage "33217" @default.
• W2004246808 abstract "Aminoacyl-tRNA synthetases are a family of enzymes that are responsible for translating the genetic code in the first step of protein synthesis. Some aminoacyl-tRNA synthetases have editing activities to clear their mistakes and enhance fidelity. Leucyl-tRNA synthetases have a hydrolytic active site that resides in a discrete amino acid editing domain called CP1. Mutational analysis within yeast mitochondrial leucyl-tRNA synthetase showed that the enzyme has maintained an editing active site that is competent for post-transfer editing of mischarged tRNA similar to other leucyl-tRNA synthetases. These mutations that altered or abolished leucyl-tRNA synthetase editing were introduced into complementation assays. Cell viability and mitochondrial function were largely unaffected in the presence of high levels of non-leucine amino acids. In contrast, these editing-defective mutations limited cell viability in Escherichia coli. It is possible that the yeast mitochondria have evolved to tolerate lower levels of fidelity in protein synthesis or have developed alternate mechanisms to enhance discrimination of leucine from non-cognate amino acids that can be misactivated by leucyl-tRNA synthetase. Aminoacyl-tRNA synthetases are a family of enzymes that are responsible for translating the genetic code in the first step of protein synthesis. Some aminoacyl-tRNA synthetases have editing activities to clear their mistakes and enhance fidelity. Leucyl-tRNA synthetases have a hydrolytic active site that resides in a discrete amino acid editing domain called CP1. Mutational analysis within yeast mitochondrial leucyl-tRNA synthetase showed that the enzyme has maintained an editing active site that is competent for post-transfer editing of mischarged tRNA similar to other leucyl-tRNA synthetases. These mutations that altered or abolished leucyl-tRNA synthetase editing were introduced into complementation assays. Cell viability and mitochondrial function were largely unaffected in the presence of high levels of non-leucine amino acids. In contrast, these editing-defective mutations limited cell viability in Escherichia coli. It is possible that the yeast mitochondria have evolved to tolerate lower levels of fidelity in protein synthesis or have developed alternate mechanisms to enhance discrimination of leucine from non-cognate amino acids that can be misactivated by leucyl-tRNA synthetase. Aminoacyl-tRNA synthetases are an ancient family of enzymes that are responsible for carrying out the two-step aminoacylation reaction (1Martinis S.A. Schimmel P. Neidhardt F.C. Escherichia coli and Salmonella Cellular and Molecular Biology. Vol. 1. ASM Press, Washington, D. C.1996: 887-901Google Scholar). In the first step, an amino acid is condensed with a single molecule of ATP to form an aminoacyladenylate intermediate. The activated amino acid is then transferred to the 3′-terminal adenosine of its cognate isoacceptor tRNA (2Carter Jr., C.W. Annu. Rev. Biochem. 1993; 62: 715-748Crossref PubMed Scopus (327) Google Scholar, 3Martinis S.A. Plateau P. Cavarelli J. Florentz C. Biochimie (Paris). 1999; 81: 683-700Crossref PubMed Scopus (76) Google Scholar). Inaccurate aminoacylation could yield infidelities during protein synthesis that would be expected to be detrimental for cell viability and survival (4Hendrickson T.L. de Crecy-Lagard V. Schimmel P. Annu. Rev. Biochem. 2004; 73: 147-176Crossref PubMed Scopus (207) Google Scholar). Linus Pauling had originally predicted that the rate of misactivation of isosteric substrates that differed by one methyl group (for example, alanine and glycine or isoleucine and valine) would be as high as 1 in 5 (5Pauling L. Festschrift fur Prof. Dr. Arthur Stoll. Birkhauser Verlag, Basel, Switzerland1958: 597-602Google Scholar). However, the in vivo rate of valine misincorporation for isoleucine into cellular proteins was measured to be as low as 1 in 3000 (6Loftfield R.B. Biochem. J. 1963; 89: 82-92Crossref PubMed Scopus (116) Google Scholar). This increased fidelity has been attributed to a double sieve mechanism that enhances discrimination and accurate selection of the correct amino acids (7Fersht A.R. Biochemistry. 1977; 16: 1025-1030Crossref PubMed Scopus (201) Google Scholar, 8Fersht A.R. Dingwall C. Biochemistry. 1979; 18: 2627-2631Crossref PubMed Scopus (120) Google Scholar, 9Fersht A.R. Science. 1998; 280: 541Crossref PubMed Scopus (41) Google Scholar). The canonical aminoacylation core acts as a coarse sieve where activation of the cognate amino acid as well as smaller non-cognate amino acids occurs. A second fine sieve excludes the correctly charged amino acid and hydrolyzes misaminoacylated tRNA. The homologous LeuRS, isoleucyl-tRNA synthetase (IleRS), and valyl-tRNA synthetase (ValRS) contain these two active sites to enhance fidelity (10Silvian L.F. Wang J. Steitz T.A. Science. 1999; 285: 1074-1077Crossref PubMed Scopus (352) Google Scholar, 11Schmidt E. Schimmel P. Biochemistry. 1995; 34: 11204-11210Crossref PubMed Scopus (78) Google Scholar, 12Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-582Crossref PubMed Scopus (315) Google Scholar, 13Lincecum Jr., T.L. Tukalo M. Yaremchuk A. Mursinna R.S. Williams A.M. Sproat B.S. van Den Eynde W. Link A. Van Calenbergh S. Grøtli M. Martinis S.A. Cusack S. Mol. Cell. 2003; 11: 951-963Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Each of their amino acid editing active sites is located in a discretely folded domain of ∼200 amino acids called CP1 (14Lin L. Hale S.P. Schimmel P. Nature. 1996; 384: 33-34Crossref PubMed Scopus (92) Google Scholar, 15Mursinna R.S. Lincecum Jr., T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar, 16Starzyk R.M. Webster T.A. Schimmel P. Science. 1987; 237: 1614-1618Crossref PubMed Scopus (141) Google Scholar). The CP1 domain is inserted into the main body of the enzyme via two β-strand linkers (10Silvian L.F. Wang J. Steitz T.A. Science. 1999; 285: 1074-1077Crossref PubMed Scopus (352) Google Scholar, 12Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-582Crossref PubMed Scopus (315) Google Scholar, 17Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 18Cusack S. Yaremchuk A. Tukalo M. EMBO J. 2000; 19: 2351-2361Crossref PubMed Scopus (229) Google Scholar). The editing mechanisms of ValRS and IleRS primarily target threonine and valine, respectively, in the cell (11Schmidt E. Schimmel P. Biochemistry. 1995; 34: 11204-11210Crossref PubMed Scopus (78) Google Scholar, 19Baldwin A.N. Berg P. J. Biol. Chem. 1966; 241: 839-845Abstract Full Text PDF PubMed Google Scholar, 20Nangle L.A. de Crecy Lagard V. Doring V. Schimmel P. J. Biol. Chem. 2002; 277: 45729-45733Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 21Freist W. Pardowitz I. Cramer F. Biochemistry. 1985; 24: 7014-7023Crossref PubMed Scopus (41) Google Scholar, 22Eldred E.W. Schimmel P.R. J. Biol. Chem. 1972; 247: 2961-2964Abstract Full Text PDF PubMed Google Scholar). However, LeuRS misactivates many non-leucine amino acids, including isoleucine, valine, methionine, and also structurally similar metabolic cellular intermediates (e.g. norleucine, norvaline, α-aminobutyrate, and homocysteine (23Martinis S.A. Fox G.E. Nucleic Acids Symp. Ser. 1997; 36: 125-128PubMed Google Scholar, 24Englisch S. Englisch U. von der Haar F. Cramer F. Nucleic Acids Res. 1986; 14: 7529-7539Crossref PubMed Scopus (78) Google Scholar, 25Apostol I. Levine J. Lippincott J. Leach J. Hess E. Glascock C.B. Weickert M.J. Blackmore R. J. Biol. Chem. 1997; 272: 28980-28988Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 26Lincecum Jr., T.L. Martinis S.A. Ibba M. Francklyn C. Cusack S. Aminoacyl-tRNA Synthetases. Landes Bioscience, Georgetown, TX2005: 36-46Google Scholar, 27Xu M.G. Li J. Du X. Wang E.D. Biochem. Biophys. Res. Commun. 2004; 318: 11-16Crossref PubMed Scopus (29) Google Scholar)) as well as synthetic hydroxylated (24Englisch S. Englisch U. von der Haar F. Cramer F. Nucleic Acids Res. 1986; 14: 7529-7539Crossref PubMed Scopus (78) Google Scholar) and fluorinated (28Tang Y. Tirrell D.A. J. Am. Chem. Soc. 2001; 123: 11089-11090Crossref PubMed Scopus (150) Google Scholar) leucine derivatives. Biochemical investigations and co-crystal structures of LeuRSs have identified a number of key elements of the LeuRS amino acid editing active site (13Lincecum Jr., T.L. Tukalo M. Yaremchuk A. Mursinna R.S. Williams A.M. Sproat B.S. van Den Eynde W. Link A. Van Calenbergh S. Grøtli M. Martinis S.A. Cusack S. Mol. Cell. 2003; 11: 951-963Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 18Cusack S. Yaremchuk A. Tukalo M. EMBO J. 2000; 19: 2351-2361Crossref PubMed Scopus (229) Google Scholar, 27Xu M.G. Li J. Du X. Wang E.D. Biochem. Biophys. Res. Commun. 2004; 318: 11-16Crossref PubMed Scopus (29) Google Scholar, 29Fukunaga R. Yokoyama S. Nat. Struct. Mol. Biol. 2005; 12: 915-922Crossref PubMed Scopus (89) Google Scholar, 30Fukunaga R. Yokoyama S. J. Mol. Biol. 2005; 346: 57-71Crossref PubMed Scopus (78) Google Scholar, 31Liu Y. Liao J. Zhu B. Wang E.D. Ding J. Biochem. J. 2006; 394: 399-407Crossref PubMed Scopus (29) Google Scholar). A conserved threonine-rich region within the CP1 domain comprises a portion of the editing active site pocket in Escherichia coli LeuRS and confers specificity and activity (15Mursinna R.S. Lincecum Jr., T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar, 27Xu M.G. Li J. Du X. Wang E.D. Biochem. Biophys. Res. Commun. 2004; 318: 11-16Crossref PubMed Scopus (29) Google Scholar, 32Mursinna R.S. Martinis S.A. J. Am. Chem. Soc. 2002; 124: 7286-7287Crossref PubMed Scopus (57) Google Scholar, 33Mursinna R.S. Lee K.W. Briggs J.M. Martinis S.A. Biochemistry. 2004; 43: 155-165Crossref PubMed Scopus (33) Google Scholar, 34Zhai Y. Martinis S.A. Biochemistry. 2005; 44: 15437-15443Crossref PubMed Scopus (33) Google Scholar). Significantly, a key specificity discriminator at a conserved threonine in the editing active site blocks cognate leucine from binding (15Mursinna R.S. Lincecum Jr., T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar) while allowing this wide array of amino acids to bind for hydrolytic editing. LeuRS shares a universally conserved aspartic acid that is also found in the CP1 domains of IleRS and ValRS. The aspartate forms a hydrogen bond with the amino moiety of the bound amino acid and is essential for hydrolytic editing. Mutations of this conserved aspartate abolish the editing activity of LeuRS, IleRS, and ValRS in vitro (13Lincecum Jr., T.L. Tukalo M. Yaremchuk A. Mursinna R.S. Williams A.M. Sproat B.S. van Den Eynde W. Link A. Van Calenbergh S. Grøtli M. Martinis S.A. Cusack S. Mol. Cell. 2003; 11: 951-963Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 35Bishop A.C. Nomanbhoy T.K. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 585-590Crossref PubMed Scopus (51) Google Scholar, 36Fukunaga R. Yokoyama S. J. Biol. Chem. 2005; 280: 29937-29945Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). We hypothesized that cell viability and growth would be significantly affected by editing-defective LeuRS enzymes. Statistical protein mutations that are introduced via mischarged tRNALeu would be expected to alter or abolish the function of critical proteins. Our results show that amino acid editing is critical to bacterial survival. In contrast, the yeast mitochondrion appears to tolerate LeuRS infidelities. Thus, different organisms have varied thresholds for fidelity. It is also possible that these organisms have evolved alternate mechanisms to enhance fidelity that extend beyond the LeuRS CP1 domain. Materials—Oligonucleotides were obtained from MWG Biotech (High Point, NC). All 3H-labeled amino acids were purchased from Amersham Biosciences. Restriction endonucleases were acquired from Promega (Madison, WI), except BstNI, which was from New England Biolabs, Inc. (Beverley, MA). Cloned Pfu DNA polymerase and commercially mixed dNTPs were obtained from Stratagene (La Jolla, CA). The E. coli temperature-sensitive strain KL231 (F–, thy-35, str-120, leuS31) was obtained from the E. coli Genetic Stock Center (Yale University, New Haven, CT) (37Low B. Gates F. Goldstein T. Söll D. J. Bacteriol. 1971; 108: 742-750Crossref PubMed Google Scholar). The plasmid pGP1-2 (38Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1074-1078Crossref PubMed Scopus (2452) Google Scholar) encoding the T7 RNA polymerase was a gift from Dr. Tracy Palmer (University of East Anglia). Site-directed Mutagenesis—The plasmid pYM3–17 (39Rho S.B. Martinis S.A. RNA (Cold Spring Harbor). 2000; 6: 1882-1894Google Scholar) expressing the wild-type yeast mitochondrial LeuRS was used as a template to introduce mutations including T263V/T264V (TT/VV; pVK081), R265A (pVK064), and D357A (pVK078) for overexpression and purification of each protein for in vitro studies. Likewise, the plasmid ymLEURST (40Houman F. Rho S.B. Zhang J. Shen X. Wang C.C. Schimmel P. Martinis S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13743-13748Crossref PubMed Scopus (24) Google Scholar), encoding the wild-type yeast mitochondrial LeuRS, was used as a template to generate the same set of mutations, T263V/T264V (TT/VV; pVK042), R265A (pVK100), and D357A (pVK102), and the plasmid pKIRAN, which also encoded wild-type E. coli LeuRS, was used as a template for generating homologous mutations T247V/T248V (TT/VV; pVK105), R249A (pVK106), or D345A (pVK108) for complementation assays in yeast null strains. The LeuRS mutations were made via PCR-based mutagenesis. A 50-μl PCR reaction mixture containing 50 ng of plasmid DNA template, 100 ng of each forward and reverse primer, 200 μm dNTPs, and 2.5 units of Pfu DNA polymerase in commercial buffer was heated at 95 °C for 1 min. The DNA was then amplified by PCR for 16 cycles under the following conditions: 95 °C for 1 min, 55 °C for 1 min, and 68 °C for 20 min. The reaction was then restriction-digested with 20 units of DpnI for 3 h at 37 °Cand used for transformation of E. coli DH5α (Stratagene, La Jolla, CA). The DNA sequence of the mutant LeuRS genes were confirmed by SeqWright Laboratories (Houston, TX). Expression and Purification of Yeast Mitochondrial LeuRS—For protein overexpression, wild-type or mutant plasmids were used to transform the E. coli BL21 (DE3) codon PLUS (Stratagene). A single colony was used to inoculate 3 ml of Luria-Bertani medium containing 100 μg/ml ampicillin and 35 μg/ml chloramphenicol (LB-Amp/Cm) and grown overnight at 37 °C. The overnight culture was transferred to 500 ml of LB-Amp/Cm and grown at 30 °C to an A600 of 0.6–0.8. Expression of LeuRS was induced with 1 mm isopropyl β-d-thiogalactopyranoside for 2 h. The cells were harvested by centrifugation at 6000 revolutions/min (JLA-10.500 rotor) for 15 min in a Beckman J2-HS centrifuge (Beckman Coulter, Inc., Fullerton, CA). The cell pellet was resuspended in 10 ml of HA-1 (20 mm NaPi, 10 mm Tris, pH 8.0, 100 mm NaCl, 5 mm imidazole, and 5% glycerol) and sonicated at 60 A for 2 min using a Sonics Vibra Cell sonicator (Sonics, Newtown, CT). The lysate was separated from the cell debris by low speed centrifugation at 7500 revolutions/min (JA-17) for 30 min at 4 °C. The lysate was gently mixed for 1 h with HIS-Select HF nickel affinity resin (Sigma) that was pre-equilibrated with HA-1. The lysate was separated from the resin by low speed centrifugation in a clinical centrifuge. The resin was then washed with HA-2 (20 mm NaPi, 10 mm Tris, pH 7.0, 500 mm NaCl, 5 mm imidazole, and 5% glycerol) six times for 10 min each. The N-terminal His6-tagged LeuRS was eluted using 1 ml of HA-1 that contained 200 mm imidazole. Protein was concentrated using a centricon-50 (Amicon, Bedford, MA) and its concentration measured via the Bio-Rad protein assay according to the commercial protocol. Preparation of tRNALeu—The plasmid pymtDNAleu1 containing the gene for yeast mitochondrial tRNALeu was digested for 6 h at 60°C with 25 units of BstNI and then used as the template for in vitro transcription (41Milligan J.F. Groebe D.R. Witherell G.W. Uhlenbeck O.C. Nucleic Acids Res. 1987; 15: 8783-8798Crossref PubMed Scopus (1882) Google Scholar, 42Sampson J.R. Uhlenbeck O.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1033-1037Crossref PubMed Scopus (612) Google Scholar). A 1-ml reaction contained 450 μg of plasmid template, 40 mm Tris, pH 8.0, 30 mm MgCl2, 5 mm dithiothreitol, 0.01% Triton X-100, 50 μg/ml bovine serum albumin, each dNTP at 4 mm, 80 mg/ml polyethylene glycol 8000, 0.02 units/μl RNase inhibitor (Eppendorf, Hamburg, Germany), 2 mm spermidine, 0.01 mg/ml E. coli pyrophosphatase (Sigma), and 500 nm T7 RNA polymerase. The reaction mixture was incubated for 3 h at42 °C followed by a second addition of 500 nm T7 RNA polymerase and then incubated for an additional 3 h at 42°C. The RNA product was purified on a 10% polyacrylamide (19:1) 8 m urea gel. The tRNA band was detected via UV shadowing, excised from the gel, and extracted overnight in 0.5 m NH4OAC and 1 mm EDTA, pH 8.0, at 37 °C. The supernatant was collected and the extraction repeated two more times. The tRNA was concentrated by butanol extraction, and a total of 1 μg/μl glycogen was added to the concentrated tRNA followed by ethanol precipitation. The tRNA pellet was washed twice in 70% ethanol, dried, and then resuspended in 100 μl of nuclease-free water (Ambion, Austin, TX). Purified tRNALeu was denatured at 80 °C for 1 min followed by the addition of 1 mm MgCl2 and quick cooling on ice. The concentration of the tRNA was determined by measuring the absorbance at 260 nm using a calculated extinction coefficient of 878,500 L·mol–1 cm–1 (43Puglisi J.D. Tinoco Jr., I. Methods Enzymol. 1989; 180: 304-325Crossref PubMed Scopus (657) Google Scholar). Plateau values for fully charged tRNALeu transcript indicated that ∼55% of the tRNA pool was competent for leucylation by yeast mitochondrial LeuRS. Aminoacylation Assays—Aminoacylation reactions contained 60 mm Tris, pH 7.5, 10 mm MgCl2, 150 mm KCl, 1 mm dithiothreitol, 4 μm yeast mitochondrial tRNALeu, 22 μm [3H]leucine (152 Ci/mmol), and 50 nm enzyme. The addition of KCl stimulates the enzymatic activity of yeast mitochondrial LeuRS. 2J. L. Hsu and S. A. Martinis, unpublished data. Misaminoacylation assays were carried out similarly, except 22 μm [3H]isoleucine (92 Ci/mmol) and 1 μm enzyme were used. The reactions were initiated with 4 mm ATP and carried out at room temperature. At specific time points, 5 μlof the reaction were quenched on Whatman grade 3 filter pads that were presoaked in 5% trichloroacetic acid. The pads were washed three times with cold 5% trichloroacetic acid, once with 70% ethanol, and then finally anhydrous ether. The pads were dried and quantified in a Beckman LS 6000IC scintillation counter (Beckman Coulter, Inc.). Post-transfer Editing Assays—Yeast mitochondrial tRNALeu was aminoacylated with [3H]isoleucine (92 Ci/mmol) by an editing-defective E. coli LeuRS mutant at 1μm in a reaction containing 60 mm Tris, pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol, 4 mm ATP, and 8 μm yeast mitochondrial tRNALeu. The reaction was incubated for 3 h at room temperature and quenched with 0.18% acetic acid. Protein was removed by extraction with 1 volume of phenol: chloroform:isoamyl alcohol (125:24:1), pH 5.2 (Fisher Biotech, Fair Lawn, NJ). A half-volume of 4.6 m ammonium acetate, pH 5.0, was added, and the mischarged tRNA was ethanol-precipitated. The dried pellet was resuspended in 50 mm KPi buffer, pH 5.0. A total of 140 pmol of mischarged tRNA was obtained. Hydrolysis assays were carried out using 60 mm Tris, pH 7.5, 10 mm MgCl2, 150 mm KCl, and 300 nm mischarged yeast mitochondrial tRNALeu. As indicated above, including KCl increases the post-transfer editing activity of yeast mitochondrial LeuRS.2 The reaction was initiated by the addition of 50 nm enzyme and carried out at room temperature (15Mursinna R.S. Lincecum Jr., T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar). At specific time points, 5 μl of the reaction were quenched on filter pads that were presoaked in 5% trichloroacetic acid and then washed and dried as described above. Yeast Complementation Assays—The yeast null strain HM402 (MATα ade2-1 his3-11,15 leu2-3, 112 trp1-1 ura3-1 can1-100 nam2Δ::LEU2 (Δ introns)) carrying an allelic disruption in the nam2 gene encoding yeast mitochondrial LeuRS (44Li G.Y. Becam A.M. Slonimski P.P. Herbert C.J. Mol. Gen. Genet. 1996; 252: 667-675PubMed Google Scholar, 45Seraphin B. Boulet A. Simon M. Faye G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6810-6814Crossref PubMed Scopus (102) Google Scholar) and expressing a mitochondrial LeuRS via a maintenance plasmid YEpGMC063 (URA3+) was used for complementation assays (40Houman F. Rho S.B. Zhang J. Shen X. Wang C.C. Schimmel P. Martinis S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13743-13748Crossref PubMed Scopus (24) Google Scholar). The HM402 strain contains an ade2 mutation, which enables the cells to develop a red pigment when the mitochondria are functional. Strains were transformed with plasmids encoding wild-type (ymLEURST) or TT/VV (pVK042), R265A (pVK100) or D357A (pVK102) editing-defective mutant yeast mitochondrial LeuRSs, wild-type (pKIRAN) or TT/VV (pVK105), R249A (pVK106) or D345A (pVK108) E. coli LeuRSs (TRP1 marker), as well as an empty vector (pQB153T) for a negative control. The upstream region of each E. coli LeuRS gene encoded a mitochondrial target sequence to allow import of the heterologous protein into the yeast mitochondria. Transformants were selected on synthetic complete medium containing 2% glucose and lacking leucine, tryptophan, and uracil (40Houman F. Rho S.B. Zhang J. Shen X. Wang C.C. Schimmel P. Martinis S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13743-13748Crossref PubMed Scopus (24) Google Scholar). Subsequently, transformants were grown on 5-fluoroorotic acid medium to allow the loss of the maintenance plasmid YEpGMC063. Selected transformants were grown on synthetic complete medium containing either 2% glucose or glycerol medium (Trp–, Leu–) to test for complementation activity (40Houman F. Rho S.B. Zhang J. Shen X. Wang C.C. Schimmel P. Martinis S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13743-13748Crossref PubMed Scopus (24) Google Scholar). Complementation of E. coli KL231—Competent cells of E. coli KL231 were co-transformed with pGP1-2 (KanR) (38Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1074-1078Crossref PubMed Scopus (2452) Google Scholar) and plasmids encoding wild-type (p15ec3-1 (23Martinis S.A. Fox G.E. Nucleic Acids Symp. Ser. 1997; 36: 125-128PubMed Google Scholar)) or T247V/T248V (pYZHAI3 (34Zhai Y. Martinis S.A. Biochemistry. 2005; 44: 15437-15443Crossref PubMed Scopus (33) Google Scholar)), R249A (pMURe6 (15Mursinna R.S. Lincecum Jr., T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar)), or D345A (pHAPPY2-1-1-28 (13Lincecum Jr., T.L. Tukalo M. Yaremchuk A. Mursinna R.S. Williams A.M. Sproat B.S. van Den Eynde W. Link A. Van Calenbergh S. Grøtli M. Martinis S.A. Cusack S. Mol. Cell. 2003; 11: 951-963Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar)) mutant E. coli LeuRSs (AmpR). Transformants were selected on LB medium containing antibiotics, including kanamycin (30 μg/ml), streptomycin (50 μg/ml), and ampicillin (100 μg/ml) as well as 200 μg/ml thymine and 100 μg/ml thiamine (LB-Amp/Kan/Str). Selected E. coli KL231 transformants were grown in 3 ml of minimal standard (MS) 3The abbreviation used is: MS, minimal standard. medium (37Low B. Gates F. Goldstein T. Söll D. J. Bacteriol. 1971; 108: 742-750Crossref PubMed Google Scholar) supplemented with 100 μg/ml of each of the following amino acids: leucine, isoleucine, valine, arginine, histidine, lysine, methionine, phenylalanine, threonine, tryptophan, and tyrosine. It also included 40 μg/ml uracil, 40 μg/ml adenine, 200 μg/ml thymine, 100 μg/ml thiamine, and 2% glucose. Antibiotics including kanamycin (30 μg/ml), streptomycin (50 μg/ml), and ampicillin (100 μg/ml) were also added to generate MS-Amp/Kan/Str. Overnight cultures were transferred to 50 ml of MS-Amp/Kan/Str medium and grown until A600 = 0.8. The cells were shifted to 42 °C for 30 min followed by a 120-min incubation at 40 °C to allow the induction of the T7 RNA polymerase for expression of LeuRSs. The cells were then harvested and resuspended in 5 ml of MS-Amp/Kan/Str medium. A 500-μl aliquot of the cell suspension was mixed with 3 ml of 0.7% soft agar and spread evenly on the MS-Amp/Kan/Str medium plates. An aliquot of 100 μlof100 mm isoleucine was incorporated into a bored out well in the center of the plate. The isoleucine diffused to generate a concentration gradient of isoleucine around the well. Plates were incubated at 30 or 42 °C for 48–72 h. The diameter of the halo was measured, and the diameter of the well (1 cm) was subtracted from the halo diameter. Cell Growth Curves—The yeast null strain HM402 harboring either the wild-type or editing-defective D357A mutant yeast mitochondrial LeuRS was grown in 100 ml of synthetic complete Leu– Trp– medium containing 2% glycerol, 0.1% fluoroorotic acid in the presence or absence of 50 mm excess isoleucine. Cells were grown at 30 °C with constant shaking at 200 revolutions/min for a period of 48 h. Absorbance was measured at specific time points at 600 nm. For E. coli growth curves, 3-ml cultures of E. coli KL231 harboring either wild-type or the editing-defective D345A mutant E. coli LeuRS were grown at 30 °C in MS-Amp/Kan/Str medium. Cells were induced as described above to allow the expression of the enzymes. The cells were transferred to 100 ml MS-Amp/Kan/Str medium that either lacked or contained 10 mm excess isoleucine. The cells were grown at 200 revolutions/min for a period of 48 h at 42 °C. Absorbance measurements were taken at specific time points at 600 nm. Yeast Mitochondrial LeuRS Is Active in Post-transfer Editing—The CP1 domain of LeuRS contains a threonine-rich region that comprises a portion of the editing active site (13Lincecum Jr., T.L. Tukalo M. Yaremchuk A. Mursinna R.S. Williams A.M. Sproat B.S. van Den Eynde W. Link A. Van Calenbergh S. Grøtli M. Martinis S.A. Cusack S. Mol. Cell. 2003; 11: 951-963Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 15Mursinna R.S. Lincecum Jr., T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar, 31Liu Y. Liao J. Zhu B. Wang E.D. Ding J. Biochem. J. 2006; 394: 399-407Crossref PubMed Scopus (29) Google Scholar, 33Mursinna R.S. Lee K.W. Briggs J.M. Martinis S.A. Biochemistry. 2004; 43: 155-165Crossref PubMed Scopus (33) Google Scholar, 46Tang Y. Tirrell D.A. Biochemistry. 2002; 41: 10635-10645Crossref PubMed Scopus (87) Google Scholar). A second downstream region is marked by a universally conserved aspartic acid that forms a hydrogen bond with the amino moiety of the bound amino acid (Fig. 1) (13Lincecum Jr., T.L. Tukalo M. Yaremchuk A. Mursinna R.S. Williams A.M. Sproat B.S. van Den Eynde W. Link A. Van Calenbergh S. Grøtli M. Martinis S.A. Cusack S. Mol. Cell. 2003; 11: 951-963Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). These regions are highly conserved in LeuRSs and also shared with the homologous IleRS and ValRS editing domains. Mutational analysis within these two regions has identified sites that alter or abolish amino acid editing activity of these synthetases (13Lincecum Jr., T.L. Tukalo M. Yaremchuk A. Mursinna R.S. Williams A.M. Sproat B.S. van Den Eynde W. Link A. Van Calenbergh S. Grøtli M. Martinis S.A. Cusack S. Mol. Cell. 2003; 11: 951-963Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 15Mursinna R.S. Lincecum Jr., T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar, 32Mursinna R.S. Martinis S.A. J. Am. Chem. Soc. 2002; 124: 7286-7287Crossref PubMed Scopus (57) Google Scholar, 34Zhai Y. Martinis S.A. Biochemistry. 2005; 44: 15437-15443Crossref PubMed Scopus (33) Google Scholar, 36Fukunaga R. Yokoyama S. J. Biol. Chem. 2005; 280: 29937-29945Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). We introduced mutations into yeast mitochondrial LeuRS, which had been shown to decrease or eliminate editing activity of LeuRSs from other origins. This included a double mutation of two neighboring threonines (Thr-263 and -264) to valine (TT/VV), which abolishes post-transfer editing activity of E. coli LeuRS (34Zhai Y. Martinis S.A. Biochemistry. 2005; 44: 15437-15443Crossref PubMed Scopus (33) Google Scholar). The universally conserved aspartic acid (Asp-357) of yeast mitochondrial LeuRS was also mutated to alanine and was expected to eliminate amino acid editing activity similar to analogous mutations in IleRS, ValRS, and other LeuRSs (13Lincecum Jr., T.L. Tukalo M. Yaremchuk A. Mursinna R.S. Williams A.M. Sproat B.S. van Den Eynde W. Link A. Van Calenbergh S. Grøtli M. Martinis S.A. Cusack S. Mol. Cell. 2003; 11: 951-963Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 35Bishop A.C. Nomanbhoy T.K. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 585-590Crossref PubMed Scopus (51) Google Scholar, 36Fukunaga R. Yokoyama S. J. Biol. Chem. 2005; 280: 29937-29945Abstract Full Text Full Text PDF PubMed Scop" @default.
• W2004246808 created "2016-06-24" @default.
• W2004246808 creator A5009797730 @default.
• W2004246808 creator A5028624856 @default.
• W2004246808 creator A5030728909 @default.
• W2004246808 creator A5047422822 @default.
• W2004246808 date "2006-11-01" @default.
• W2004246808 modified "2023-10-11" @default.
• W2004246808 title "A Viable Amino Acid Editing Activity in the Leucyl-tRNA Synthetase CP1-splicing Domain Is Not Required in the Yeast Mitochondria" @default.
• W2004246808 cites W1027635430 @default.
• W2004246808 cites W1480195325 @default.
• W2004246808 cites W1495386551 @default.
• W2004246808 cites W1523614548 @default.
• W2004246808 cites W1571051346 @default.
• W2004246808 cites W1682642099 @default.
• W2004246808 cites W1965524864 @default.
• W2004246808 cites W1967286437 @default.
• W2004246808 cites W1977493587 @default.
• W2004246808 cites W1979414125 @default.
• W2004246808 cites W1980461282 @default.
• W2004246808 cites W1980902241 @default.
• W2004246808 cites W1981957241 @default.
• W2004246808 cites W1982590059 @default.
• W2004246808 cites W1985888036 @default.
• W2004246808 cites W1986561802 @default.
• W2004246808 cites W1996097229 @default.
• W2004246808 cites W1997779800 @default.
• W2004246808 cites W1999253552 @default.
• W2004246808 cites W2001343943 @default.
• W2004246808 cites W2003897992 @default.
• W2004246808 cites W2004416250 @default.
• W2004246808 cites W2008264319 @default.
• W2004246808 cites W2016340006 @default.
• W2004246808 cites W2021463176 @default.
• W2004246808 cites W2021731125 @default.
• W2004246808 cites W2022574047 @default.
• W2004246808 cites W2028846063 @default.
• W2004246808 cites W2031465355 @default.
• W2004246808 cites W2036078885 @default.
• W2004246808 cites W2044131412 @default.
• W2004246808 cites W2046129360 @default.
• W2004246808 cites W2048111541 @default.
• W2004246808 cites W2053093232 @default.
• W2004246808 cites W2060005245 @default.
• W2004246808 cites W2066768470 @default.
• W2004246808 cites W2066929816 @default.
• W2004246808 cites W2068518785 @default.
• W2004246808 cites W2069520595 @default.
• W2004246808 cites W2073186433 @default.
• W2004246808 cites W2077432477 @default.
• W2004246808 cites W2082627904 @default.
• W2004246808 cites W2082846797 @default.
• W2004246808 cites W2083591638 @default.
• W2004246808 cites W2085272777 @default.
• W2004246808 cites W2085863852 @default.
• W2004246808 cites W2091067056 @default.
• W2004246808 cites W2109526373 @default.
• W2004246808 cites W2110274263 @default.
• W2004246808 cites W2126459625 @default.
• W2004246808 cites W2127749120 @default.
• W2004246808 cites W2130033465 @default.
• W2004246808 cites W2137717307 @default.
• W2004246808 cites W263820944 @default.
• W2004246808 cites W4235003401 @default.
• W2004246808 doi "https://doi.org/10.1074/jbc.m607406200" @default.
• W2004246808 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16956879" @default.
• W2004246808 hasPublicationYear "2006" @default.
• W2004246808 type Work @default.
• W2004246808 sameAs 2004246808 @default.
• W2004246808 citedByCount "36" @default.
• W2004246808 countsByYear W20042468082012 @default.
• W2004246808 countsByYear W20042468082013 @default.
• W2004246808 countsByYear W20042468082014 @default.
• W2004246808 countsByYear W20042468082015 @default.
• W2004246808 countsByYear W20042468082016 @default.
• W2004246808 countsByYear W20042468082023 @default.
• W2004246808 crossrefType "journal-article" @default.
• W2004246808 hasAuthorship W2004246808A5009797730 @default.
• W2004246808 hasAuthorship W2004246808A5028624856 @default.
• W2004246808 hasAuthorship W2004246808A5030728909 @default.
• W2004246808 hasAuthorship W2004246808A5047422822 @default.
• W2004246808 hasBestOaLocation W20042468081 @default.
• W2004246808 hasConcept C104317684 @default.
• W2004246808 hasConcept C153957851 @default.
• W2004246808 hasConcept C185592680 @default.
• W2004246808 hasConcept C2779222958 @default.
• W2004246808 hasConcept C2779315393 @default.
• W2004246808 hasConcept C28859421 @default.
• W2004246808 hasConcept C515207424 @default.
• W2004246808 hasConcept C54458228 @default.
• W2004246808 hasConcept C55493867 @default.
• W2004246808 hasConcept C67705224 @default.
• W2004246808 hasConcept C86803240 @default.
• W2004246808 hasConceptScore W2004246808C104317684 @default.
• W2004246808 hasConceptScore W2004246808C153957851 @default.
• W2004246808 hasConceptScore W2004246808C185592680 @default.
• W2004246808 hasConceptScore W2004246808C2779222958 @default.
• W2004246808 hasConceptScore W2004246808C2779315393 @default.

• next