Differential Modes of Transfer RNASer Recognition in Methanosarcina barkeri
2004; Elsevier BV; Volume: 279; Issue: 47 Linguagem: Inglês
10.1074/jbc.m408753200
ISSN1083-351X
AutoresDragana Korencic, Carla Polycarpo, Ivana Weygand-Đurašević, Dieter Söll,
Tópico(s)Genomics and Phylogenetic Studies
ResumoTwo dissimilar seryl-transfer RNA (tRNA) synthetases (SerRSs) exist in Methanosarcina barkeri, one of bacterial type and the other resembling SerRSs present only in some methanogenic archaea. To investigate the requirements of these enzymes for tRNASer recognition, serylation of variant transcripts of M. barkeri tRNASer was kinetically analyzed in vitro with pure enzyme preparations. Characteristically for the serine system, the length of the variable arm was shown to be crucial for both enzymes, as was the identity of the discriminator base (G73). Moreover, a novel determinant for the specific tRNASer recognition was identified as the anticodon stem base pair G30:C40; its contribution to the efficiency of serylation was remarkable for both SerRSs. However, despite these similarities, the two SerRSs do not possess a uniform mode of tRNASer recognition, and additional determinants are necessary for serylation specificity by the methanogenic enzyme. In particular, the methanogenic SerRS relies on G1:C72 identity and on the number of unpaired nucleotides at the base of the variable stem for tRNASer recognition, unlike its bacterial type counterpart. We propose that such a distinction between the two enzymes in tRNASer identity determinants reflects their evolutionary pathways, hence attesting to their diversity. Two dissimilar seryl-transfer RNA (tRNA) synthetases (SerRSs) exist in Methanosarcina barkeri, one of bacterial type and the other resembling SerRSs present only in some methanogenic archaea. To investigate the requirements of these enzymes for tRNASer recognition, serylation of variant transcripts of M. barkeri tRNASer was kinetically analyzed in vitro with pure enzyme preparations. Characteristically for the serine system, the length of the variable arm was shown to be crucial for both enzymes, as was the identity of the discriminator base (G73). Moreover, a novel determinant for the specific tRNASer recognition was identified as the anticodon stem base pair G30:C40; its contribution to the efficiency of serylation was remarkable for both SerRSs. However, despite these similarities, the two SerRSs do not possess a uniform mode of tRNASer recognition, and additional determinants are necessary for serylation specificity by the methanogenic enzyme. In particular, the methanogenic SerRS relies on G1:C72 identity and on the number of unpaired nucleotides at the base of the variable stem for tRNASer recognition, unlike its bacterial type counterpart. We propose that such a distinction between the two enzymes in tRNASer identity determinants reflects their evolutionary pathways, hence attesting to their diversity. To maintain translational accuracy, aminoacyl-transfer RNA (tRNA) 1The abbreviations used are: tRNA, transfer RNA; RS, tRNA synthetase; SerRS, seryl-tRNA synthetase.1The abbreviations used are: tRNA, transfer RNA; RS, tRNA synthetase; SerRS, seryl-tRNA synthetase. synthetases are highly selective toward their amino acid and tRNA substrates. In the process of tRNA recognition, the cognate and non-cognate substrates are discriminated according to characteristic nucleotides in certain positions of the tRNA, specific for the tRNA/synthetase system. 2Numbering of nucleotides in tRNAs was according to M. Sprinzl, K. S. Vassilenko, J. Emmerich, and F. Bauer Compilation of tRNA Sequences and Sequences of tRNA Genes (www.uni-bayreuth.de/departments/biochemie/trna/).2Numbering of nucleotides in tRNAs was according to M. Sprinzl, K. S. Vassilenko, J. Emmerich, and F. Bauer Compilation of tRNA Sequences and Sequences of tRNA Genes (www.uni-bayreuth.de/departments/biochemie/trna/). These recognition (identity) elements are commonly located in the tRNA anticodon, the acceptor stem, and the discriminator base at position 73 (1Giege R. Sissler M. Florentz C. Nucleic Acids Res. 1998; 26: 5017-5035Crossref PubMed Scopus (628) Google Scholar, 2Pallanck L. Pak M. Schulman L.H. Söll D. RajBhandary U.L. tRNA: Structure, Biosynthesis, and Function. American Society of Microbiology, Washington, DC1995: 371-394Google Scholar, 3McClain W.H. J. Mol. Biol. 1993; 234: 257-280Crossref PubMed Scopus (140) Google Scholar, 4McClain W.H. FASEB J. 1993; 7: 72-78Crossref PubMed Scopus (57) Google Scholar, 5Saks M.E. Sampson J.R. Abelson J.N. Science. 1994; 263: 191-197Crossref PubMed Scopus (150) Google Scholar). However, as some systems show little or no conservation among anticodons of their tRNA isoacceptors (the six-codon families Arg, Leu, and Ser), additional parts of the tRNA are necessary for specific recognition (6Normanly J. Ollick T. Abelson J. Proc. Natl. Acad. Sci. U. S. 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Nucleic Acids Res. 1995; 23: 3633-3637Crossref PubMed Scopus (57) Google Scholar).Identity elements required for serylation have been studied in a number of organisms, providing insights into tRNASer recognition in the different domains of life (6Normanly J. Ollick T. Abelson J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5680-5684Crossref PubMed Scopus (84) Google Scholar, 14Himeno H. Hasegawa T. Ueda T. Watanabe K. Shimizu M. Nucleic Acids Res. 1990; 18: 6815-6819Crossref PubMed Scopus (129) Google Scholar, 15Normanly J. Masson J.M. Kleina L.G. Abelson J. Miller J.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6548-6552Crossref PubMed Scopus (126) Google Scholar, 16Schatz D. Leberman R. Eckstein F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6132-6136Crossref PubMed Scopus (96) Google Scholar, 17Sampson J.R. Saks M.E. Nucleic Acids Res. 1993; 21: 4467-4475Crossref PubMed Scopus (76) Google Scholar, 18Saks M.E. Sampson J.R. 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Contrary to the identity requirements of the majority of aminoacyl-tRNA synthetases, Escherichia coli seryl-tRNA synthetase (SerRS) was found to recognize neither the anticodon nor the discriminator base of tRNASer (27Asahara H. Himeno H. Tamura K. Nameki N. Hasegawa T. Shimizu M. J. Mol. Biol. 1994; 236: 738-748Crossref PubMed Scopus (74) Google Scholar). Instead, the length of the variable arm and the characteristic tRNASer tertiary structure were shown to be crucial for serylation. In vitro studies and foot-printing experiments using yeast SerRS and tRNASer (19Dock-Bregeon A.C. Garcia A. Giege R. Moras D. Eur. J. Biochem. 1990; 188: 283-290Crossref PubMed Scopus (40) Google Scholar, 22Soma A. Kumagai R. Nishikawa K. Himeno H. J. Mol. Biol. 1996; 263: 707-714Crossref PubMed Scopus (57) Google Scholar, 23Himeno H. Yoshida S. Soma A. Nishikawa K. J. Mol. Biol. 1997; 268: 704-711Crossref PubMed Scopus (45) Google Scholar) revealed the discriminator base to be unimportant and, while the variable arm functions as the major identity element, discrimination in yeast, in contrast to E. coli, is believed to be more sequence- and less structure-dependent (14Himeno H. Hasegawa T. Ueda T. Watanabe K. Shimizu M. Nucleic Acids Res. 1990; 18: 6815-6819Crossref PubMed Scopus (129) Google Scholar, 22Soma A. Kumagai R. Nishikawa K. Himeno H. J. Mol. Biol. 1996; 263: 707-714Crossref PubMed Scopus (57) Google Scholar). Although G73 serves only as an antideterminant in bacteria (9Asahara H. Himeno H. Tamura K. Hasegawa T. Watanabe K. Shimizu M. J. Mol. Biol. 1993; 231: 219-229Crossref PubMed Scopus (104) Google Scholar) and lower eukaryotes (14Himeno H. Hasegawa T. Ueda T. Watanabe K. Shimizu M. Nucleic Acids Res. 1990; 18: 6815-6819Crossref PubMed Scopus (129) Google Scholar, 22Soma A. Kumagai R. Nishikawa K. Himeno H. J. Mol. Biol. 1996; 263: 707-714Crossref PubMed Scopus (57) Google Scholar), it is an essential identity requirement for human tRNASer (13Breitschopf K. Achsel T. Busch K. Gross H. Nucleic Acids Res. 1995; 23: 3633-3637Crossref PubMed Scopus (57) Google Scholar, 25Breitschopf K. Gross H.J. EMBO J. 1994; 13: 3166-3167Crossref PubMed Scopus (43) Google Scholar). Furthermore, recent results show a dual mode of recognition of two unusual tRNASer isoacceptors in mammalian mitochondria, with the T-loop as the main identity target (29Yokogawa T. Watanabe Y. Kumazawa Y. Ueda T. Hirao I. Miura K. Watanabe K. Nucleic Acids Res. 1991; 19: 6101-6105Crossref PubMed Scopus (68) Google Scholar, 30Shimada N. Suzuki T. Watanabe K. J. Biol. Chem. 2001; 276: 46770-46778Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 31Ueda T. Yotsumoto Y. Ikeda K. Watanabe K. Nucleic Acids Res. 1992; 20: 2217-2222Crossref PubMed Scopus (51) Google Scholar, 32Yokogawa T. Shimada N. Takeuchi N. Benkowski L. Suzuki T. Omori A. Ueda T. Nishikawa K. Spremulli L.L. Watanabe K. J. Biol. Chem. 2000; 275: 19913-19920Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Taken together, the variety of tRNASer identity determinants suggests that mechanisms of SerRS recognition may have diverged during evolution.Inspection of the available protein sequences reveals two major types of SerRSs (33Woese C.R. Olsen G.J. Ibba M. Söll D. Microbiol. Mol. Biol. Rev. 2000; 64: 202-236Crossref PubMed Scopus (530) Google Scholar): a “standard” enzyme found in most organisms and a highly diverged SerRS confined to some members of the methanogenic archaea (34Kim H.S. Vothknecht U. Hedderich R. Celic I. Söll D. J. Bacteriol. 1998; 180: 6446-6449Crossref PubMed Google Scholar). Compared with the standard bacterial type SerRSs, the methanogenic enzymes are characterized by insertions in their N-terminal part and by a notable gap in the motif 2 loop (34Kim H.S. Vothknecht U. Hedderich R. Celic I. Söll D. J. Bacteriol. 1998; 180: 6446-6449Crossref PubMed Google Scholar). Because crystallographic studies on E. coli and Thermus thermophilus tRNA·SerRS complexes show both direct involvement of the N-terminal coiled coil in the variable arm recognition and interaction of the motif 2 loop with the major groove of tRNASer (7Biou V. Yaremchuk A. Tukalo M. Cusack S. Science. 1994; 263: 1404-1410Crossref PubMed Scopus (422) Google Scholar, 35Price S. Cusack S. Borel F. Berthet-Colominas C. Leberman R. FEBS Lett. 1993; 324: 167-170Crossref PubMed Scopus (35) Google Scholar), such distinction in the sequences raises questions as to how methanogenic SerRSs recognize their tRNA substrates.Our interest in tRNASer identity focused on Methanosarcina barkeri, whose genome contains two SerRS genes of different origin: its bacterial type SerRS is related to the SerRSs found in some Gram-positive bacteria (36Korencic D. Ahel I. Söll D. Food Technol. Biotechnol. 2002; 40: 255-260Google Scholar), whereas its methanogenic homolog clusters with SerRSs from Methanothermobacter thermautotrophicus, Methanopyrus kandleri, Methanocaldococcus jannaschii, and Methanococcus maripaludis. As tRNASer determinants have not been established for a methanogenic or any archaeal SerRS, it was of interest to define the nature of tRNASer recognition in a representative of this group of organisms. Moreover, the coexistence of two distantly related SerRS enzymes in M. barkeri provides a favorable system for evaluation of the evolutionary aspects of tRNA discrimination.EXPERIMENTAL PROCEDURESMaterials—Oligonucleotides were synthesized and DNAs were sequenced by the Keck Foundation Biotechnology Resource Laboratory at Yale University. [3H]Serine (26 Ci/mmol) and [14C]serine (155 mCi/mmol) were from Amersham Biosciences. Restriction enzymes were from New England Biolabs. NTPs were purchased from Sigma. Expand High Fidelity polymerase and inorganic pyrophosphatase were from Roche Applied Science. pET15b vector was from Novagen. NAP-5 columns were from Amersham Biosciences. Plasmid T7–911 for preparation of the His6-tagged recombinant T7 RNA polymerase was a gift from Dr. Thomas Shrader, Department of Biochemistry, Albert Einstein College of Medicine, New York, NY. Genomic DNA from M. barkeri Fusaro was a gift from Dr. Kevin Sowers, University of Maryland Biotechnology Institute, Baltimore, MD. Nickel-nitrilotriacetic acid matrix and the HiSpeed plasmid maxi kit were from Qiagen. Nitrocellulose filters (0.45 μm) were from Schleicher & Schuell.tRNA Cloning and Preparation—tRNASer genes were identified in the M. barkeri genomic DNA sequence at the U.S. Department of Energy Joint Genome Institute using tRNAScan-S.E. (www.geneticwustl.edu/eddy). Wild-type and mutant tRNA genes were constructed from the synthetic oligomers carrying the tRNA gene under the T7 promoter sequence. The genes were ligated into the BamHI/HindIII restriction sites of pUC18. Following plasmid maxi preparation, the DNA template was prepared by digestion with NsiI or BstNI, phenol/chloroform extraction, and ethanol precipitation. 50 μg of template was incubated for 1 h at 37 °C in a 0.5-ml reaction mixture that included 40 mm Tris-HCl, pH 8.1, 44 mm MgCl2, 0.1% Triton, 2 mm spermidine, 10 mm dithiothreitol, 4 mm each of the nucleoside triphosphates, 10 mm GMP or AMP, and 10 μg of pure T7 RNA polymerase. (Because of the low yield, some of the transcription reactions were proportionally increased 10-fold.) Reactions were stopped by phenol/chloroform extraction, precipitated with ethanol, and resuspended in gel loading buffer (8 m urea, 20% sucrose, 0.1% bromphenol blue, 0.1% xylene cyanol). Transcripts were purified on a denaturing polyacrylamide gel (12% acrylamide:bisacrylamide (19:1), 8 m urea, 89 mm Tris borate, pH 8.3, 2 mm EDTA) and extracted in buffer containing 1 m sodium acetate, pH 6, 1 mm MgCl2, and 0.1% SDS. Finally, the tRNA samples were desalted on NAP-5 columns and refolded (5 min at 95 °C followed by a gradual reduction of temperature; 5 mm MgCl2 was added to the refolding mixture at 55 °C). tRNA concentration was determined spectrophotometrically at 260 nm.Enzyme Cloning and Preparation—Methanogenic and bacterial type SerRS genes were identified in the preliminary M. barkeri Fusaro genomic DNA sequence using JGI BLAST analysis (www.jgi.doe.gov). The DNA sequences were amplified by PCR using Expand High Fidelity polymerase and cloned into the pET15b vector for expression of N-terminal His6-tagged proteins. Purification on nickel affinity columns was performed as published (37Ahel I. Stathopoulos C. Ambrogelly A. Sauerwald A. Toogood H. Hartsch T. Söll D. J. Biol. Chem. 2002; 277: 34743-34748Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar).Aminoacylation Assay—To determine the amount of the active enzyme in the preparation, active site titration was performed. The assay is based on quantifying the amount of the complex between the enzyme and radioactively labeled amino acid adenylate retained on the nitrocellulose filters (38Salazar J.C. Zuniga R. Lefimil C. Söll D. Orellana O. FEBS Lett. 2001; 491: 257-260Crossref PubMed Scopus (11) Google Scholar). The reaction was performed in 0.1 ml of 0.5× EAP buffer (50 mm Tris-HCl, pH 7.5, 5 mm KCl, 5 mm MgCl2) containing 0.5 units of inorganic pyrophosphatase, 4 mm ATP, and 20 μm [14C]serine (300 cpm/pmol) in the presence of varying concentrations of SerRSs (0.2–5 μm). After 1–10 min of incubation at 37 °C, 30-μl aliquots were spotted onto nitrocellulose filters, filtered, and washed twice with 5 ml of 0.5× EAP buffer. The filters were dried, and the radioactivity was measured by liquid scintillation counting. The amount of active sites varied between 35 and 42%.Aminoacylation was performed at 37 °C in 50 mm HEPES-KOH, pH 7.2, 50 mm KCl, 15 mm MgCl2, 5mm dithiothreitol with 10 mm ATP, 300 μm [3H]serine (200 cpm/pmol), and varying concentrations of tRNA transcripts (1–200 μm). The enzyme concentrations were experimentally determined for each tRNA transcript in order to obtain linear velocities (4–17 nm for the wild-type transcripts and 4–170 nm for the mutant transcripts). Radioactive aminoacyl-tRNA synthesized after 2–8 min was quantified as described previously (37Ahel I. Stathopoulos C. Ambrogelly A. Sauerwald A. Toogood H. Hartsch T. Söll D. J. Biol. Chem. 2002; 277: 34743-34748Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Experimental kinetic data are based on three measurements. The kinetic constants were derived from Hanes-Woolf plots using five different tRNA concentrations. In exceptional cases where Km values were too high to be determined, kcat/Km values were obtained from the slope of the plot of initial rates against substrate concentrations (39Takeuchi N. Vial L. Panvert M. Schmitt E. Watanabe K. Mechulam Y. Blanquet S. J. Biol. Chem. 2001; 276: 20064-20068Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). For ATP kinetics, 30 or 50 μm of tRNASerGCU were used for the bacterial type SerRS or the methanogenic enzyme, respectively. Both enzymes were used at a final concentration of 150 nm as determined by linear velocity.RESULTSM. barkeri Possesses Two Functional SerRS Enzymes—Both the bacterial type and the methanogenic type M. barkeri SerRS showed efficient serylation activity with similar affinities for serine (or ATP) (Km values of 25 ± 4 (13.6 ± 1.6) and 34 ± 4 (13.8 ± 1.5) μm for the methanogenic and bacterial type SerRS, respectively) determined in the aminoacylation reaction. Furthermore, both enzymes successfully aminoacylated transcripts of all M. barkeri tRNASer isoacceptors (tRNASerGCU, tRNASerCGA, tRNASerGGA; Fig. 1), although with different efficiencies (Table I). tRNASerCGA and tRNASerGGA isoacceptors were similar in their kinetic properties toward the methanogenic SerRS. The same is true for the bacterial type enzyme; however, whereas the methanogenic enzyme comparatively showed a notable decrease in the efficiency of tRNASerGCU aminoacylation, this isoacceptor seemed to be a preferred substrate for the bacterial type SerRS.Table ISerylation kinetics of tRNASer isoacceptors by the two M. barkeri SerRSsTranscriptMethanogenic SerRSBacterial type SerRSKmkcatkcat/KmKmkcatkcat/KmtRNASerCGA3 ± 0.2183 ± 8.6612.9 ± 0.264 ± 3.122tRNASerGGA4.7 ± 0.1263 ± 17.655.92.6 ± 0.154 ± 3.120.7tRNASerGCU5.3 ± 0.468 ± 3.612.81.3 ± 0.155 ± 3.642.3 Open table in a new tab Identity Determinants of tRNASer—To elucidate tRNASer identity determinants for the two M. barkeri SerRS enzymes, mutant tRNASerCGA species were produced (Fig. 2) based on the conservation of the nucleotides among tRNASer isoacceptors and on the known identity requirements of bacterial and eukaryotic serine systems. Kinetic analysis of their serylation efficiency revealed that a number of mutations remarkably affected the relative kcat/Km values for both enzymes (Table II). Contributions of the specific positions and structural elements in different tRNA domains are discussed below.Fig. 2Mutations in tRNASerCGA. Outlined symbols represent mutated positions. Numbers in parentheses denote mutated variants.View Large Image Figure ViewerDownload (PPT)Table IISerylation kinetics of tRNASer variants by the two SerRSs from M. barkeritRNAMethanogenic SerRSBacterial type SerRSKmkcatrel kcat/KmkcatKmrel kcat/KmG73→U (1)4.4 ± 0.24.7 ± 0.70.024.7 ± 0.60.9 ± 0.10.009G73→A (2)9.2 ± 0.79 ± 0.80.022.1 ± 0.20.2 ± 0.0090.005G73→C (3)18 ± 1.66.6 ± 0.60.0061.6 ± 0.10.1 ± 0.0060.005G1:C72→A:U (4)5.6 ± 0.54.7 ± 0.50.015.2 ± 0.637 ± 4.80.3C2:G71→U:A (5)3.3 ± 0.2182 ± 60.93.9 ± 0.2139 ± 13.61.6C3:G70→G:C (6)3 ± 0.1166 ± 5.60.92.6 ± 0.193 ± 4.91.6C3:G70→A:U (7)4.4 ± 0.1168 ± 50.63 ± 0.359 ± 6.50.9C3:G70→U:A (8)6.3 ± 0.6274 ± 15.30.75.9 ± 0.385 ± 7.10.7G6:C67→U:A (9)2.2 ± 0.2239 ± 11.51.80.8 ± 0.327 ± 1.91.5A9→C (10)7.4 ± 0.747 ± 4.20.116 ± 2.4111 ± 5.80.3G10:C25→A:U (11)NDaND, not determined. Because of the high Km values of mutants 11, 13, and 14 for the methanogenic SerRS, kcat/Km values were obtained from the slope of the plot of initial rates against substrate concentrations.NDaND, not determined. Because of the high Km values of mutants 11, 13, and 14 for the methanogenic SerRS, kcat/Km values were obtained from the slope of the plot of initial rates against substrate concentrations.0.18.4 ± 0.841 ± 3.80.2CC addition in D-loop (12)9.8 ± 1135 ± 6.40.23.7 ± 0.327 ± 2.20.3G30:C40→A:U (13)NDaND, not determined. Because of the high Km values of mutants 11, 13, and 14 for the methanogenic SerRS, kcat/Km values were obtained from the slope of the plot of initial rates against substrate concentrations.NDaND, not determined. Because of the high Km values of mutants 11, 13, and 14 for the methanogenic SerRS, kcat/Km values were obtained from the slope of the plot of initial rates against substrate concentrations.0.0534 ± 4.443 ± 1.40.06G30:C40→C:G (14)NDaND, not determined. Because of the high Km values of mutants 11, 13, and 14 for the methanogenic SerRS, kcat/Km values were obtained from the slope of the plot of initial rates against substrate concentrations.NDaND, not determined. Because of the high Km values of mutants 11, 13, and 14 for the methanogenic SerRS, kcat/Km values were obtained from the slope of the plot of initial rates against substrate concentrations.0.0223 ± 2.830 ± 1.80.06Anticodon→UAG (15)6.7 ± 0.5346 ± 27.70.84.1 ± 0.473 ± 5.80.8Ue1→G (16)5.4 ± 0.3263 ± 28.90.83.4 ± 0.244 ± 4.20.6Ue2 deletion (17)21 ± 3.6560 ± 49.80.43.7 ± 0.557 ± 4.60.7U addition in variable loop (18)5 ± 0.5481 ± 44.71.63.1 ± 0.294 ± 61.4Ce17:Ge27→A:U (19)6.4 ± 0.472 ± 130.25.1 ± 0.413 ± 1.10.1Ce12:Ge22 deletion (20)13 ± 1.64.8 ± 0.60.00626 ± 4.264 ± 4.70.1Ue11:Ae21 and Ce12:Ge22 deletion (21)7.3 ± 0.32.3 ± 0.20.0055.5 ± 0.41.4 ± 0.10.01U:A addition in variable arm (22)8.8 ± 0.8163 ± 13.90.32 ± 0.168 ± 6.51.5G46 substitution by UU (23)6.8 ± 0.36.3 ± 0.40.022.9 ± 0.212 ± 10.2C48→U (24)20 ± 1.6329 ± 250.33.9 ± 0.3118 ± 11.11.4G49:C65→C:G (25)21 ± 2268 ± 180.211 ± 1.359 ± 100.2Wild-type tRNASerCGA3 ± 0.2183 ± 8.612.9 ± 0.264 ± 3.11a ND, not determined. Because of the high Km values of mutants 11, 13, and 14 for the methanogenic SerRS, kcat/Km values were obtained from the slope of the plot of initial rates against substrate concentrations. Open table in a new tab The importance of the discriminator base and of nucleotides from the first three base pairs in the acceptor stem has been shown in E. coli by in vivo identity conversion of a leucine to a serine suppressor tRNA (6Normanly J. Ollick T. Abelson J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5680-5684Crossref PubMed Scopus (84) Google Scholar) and emphasized by work on tRNASer minihelices (18Saks M.E. Sampson J.R. EMBO J. 1996; 15: 2843-2849Crossref PubMed Scopus (67) Google Scholar). The nucleotides in this part of the M. barkeri tRNASer species are most conserved, which suggests their involvement in specific tRNASer recognition. Accordingly, mutational analysis shows a dramatic drop of the aminoacylation efficiencies for variants of G73 for both enzymes, as well as for the first base pair position in the case of the methanogenic SerRS (mutants 1–4). Inability of these mutants to be efficiently serylated results primarily from the decrease in their kcat values. However, the contribution of the acceptor stem is limited to its upper part, as mutations of the conserved base pairs C2:G71, C3:G70 and G6:C67 (mutants 5–9) either retained the wild-type activity or resulted in a slight stimulation of serylation.The D-arm and the D-loop have been identified as elements contributing to tRNASer identity. In addition to the changes in the acceptor stem and the discriminator base, the in vivo identity conversion from bacterial tRNALeu to tRNASer required the change from G11:C24 to C:G (6Normanly J. Ollick T. Abelson J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5680-5684Crossref PubMed Scopus (84) Google Scholar). This base pair, however, is not conserved among tRNASer isoacceptors in M. barkeri. Furthermore, bacterial tRNAsSer share a specific D-loop feature, marked by a deletion at position 17 and a double insertion of the 20a and 20b nucleotides. The significance of this structural element is manifested through its involvement in the tertiary interactions with the variable arm, whose orientation is thus defined (7Biou V. Yaremchuk A. Tukalo M. Cusack S. Science. 1994; 263: 1404-1410Crossref PubMed Scopus (422) Google Scholar). On the other hand, deletion of position 17 is rather infrequent in archaeal serine tRNAs, as are the insertions at positions 20a and 20b in the D-loop. tRNASer isoacceptors from M. barkeri do not share a uniform structure: although tRNASerCGA and tRNASerGGA possess deletion 17, tRNASerGCU contains a double insertion at the same position. When a CC insertion was introduced into tRNASerCGA, the resulting variant showed a somewhat reduced serylation efficiency (mutant 12) as did the mutation of A9 (mutant 10). Possibly, these alterations affected the tertiary structure of tRNASer, because the D-loop interacts with the T-loop and because position 9 participates in formation of base triplets in yeast tRNAPhe, tRNAAsp, and E. coli tRNASer (7Biou V. Yaremchuk A. Tukalo M. Cusack S. Science. 1994; 263: 1404-1410Crossref PubMed Scopus (422) Google Scholar). A similar effect was observed for mutant 11, although the relative contributions of its kinetic parameters to serylation efficiency appear to differ between the two enzymes. These results imply that conservation of the G10:C25 base pair among M. barkeri and a significant number of other archaeal tRNAsSer is not coincidental.The anticodon was expectedly shown not to contribute to serine identity (mutant 15), in accordance with the published results of other serine systems (27Asahara H. Himeno H. Tamura K. Nameki N. Hasegawa T. Shimizu M. J. Mol. Biol. 1994; 236: 738-748Crossref PubMed Scopus (74) Google Scholar). However, mutation of G30:C40 in the anticodon stem resulted in a significant loss of serylation activity (mutants 13 and 14) for both enzymes with notable Km effect. Although the upper part of the anticodon stem has been suggested to possess discriminatory function by footprinting experiments in the yeast system (base pair A27: U43) (19Dock-Bregeon A.C. Garcia A. Giege R. Moras D. Eur. J. Biochem. 1990; 188: 283-290Crossref PubMed Scopus (40) Google Scholar), none of the anticodon stem nucleotides have directly been identified as recognition determinants in a mutational study. Support of such a notion comes from the fact that high conservation of nucleotides in this region of bacterial tRNAsSer cannot be detected; contrary to that, the G30:C40 base pair is absolutely conserved among archaeal tRNASer species. This position has been identified as an identity determinant for human phenylalanyl-tRNA synthetase (40Nazarenko I.A. Peterson E.T. Zakharova O.D. Lavrik O.I. Uhlenbeck O.C. Nucleic Acids Res. 1992; 20: 475-478Crossref PubMed Scopus (51) Google Scholar). Our preliminary results have indicated that the anticodon arm of tRNASer also participates in specific recognition by M. maripaludis SerRS. 3I. Gruic-Sovulj, personal communication.tRNAs with a variable arm of at least 11 nucleotides (type 2 tRNAs) are restricted to three families: tRNATyr, tRNALeu, and tRNASer. Regardless of the fact that the variable arm of tRNASer differs in both length and sequence within isoacceptors, it makes the largest contribution to serine identity, as confirmed by different experimental approaches (9Asahara H. Himeno H. Tamura K. Hasegawa T. Watanabe K. Shimizu M. J. Mol. Biol. 1993; 231: 219-229Crossref PubMed Scopus (104) Google Scholar, 17Sampson J.R. Saks M.E. Nucleic Acids Res. 1993; 21: 4467-4475Crossref PubMed Scopus (76) Google Scholar, 41Himeno H. Hasegawa T. Asahara H. Tamura K. Shimizu M. Nucleic Acids Res. 1991; 19: 6379-6382Crossref PubMed Scopus (44) Google Scholar). Despite direct contacts that have been observed between T. thermophilus SerRS and the variable arm stem (7Biou V. Yaremchuk A. Tukalo M. Cusack S. Science. 1994; 263: 1404-1410Crossref PubMed Scopus (422) Google Scholar), sequence alterations of the variable arm only insignificantly affected the serylation efficiency (27Asahara H. Himeno H. Tamura K. Nameki N. Hasegawa T. Shimizu M. J. Mol. Biol. 1994; 236: 738-748Crossref PubMed Scopus (74) Google Scholar). It was therefore concluded that the variable arm of tRNASer is not recognized sequence specificall
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