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• W1966175091 abstract "Transfer RNA (guanosine-2′-)-methyltransferase (Gm-methylase, EC 2.1.1.32) from Thermus thermophilus HB27 is one of the tRNA ribose modification enzymes. The broad substrate specificity of Gm-methylase has so far been elucidated using various species of tRNAs from native sources, suggesting that the common structures in tRNAs are recognized by the enzyme. In this study, by using 28 yeast tRNAPhe variants obtained by transcription with T7 RNA polymerase, it was revealed that the nucleotide residues G18 and G19 and the D-stem structure are essentially required for Gm-methylase recognition, and that the key sequence for the substrate is pyrimidine (Py)17G18G19. The other conserved sequences were found not to be essential, but U8, G15, G26, G46, U54, U55, and C56 considerably affected the methylation efficiency. These residues are located within a limited space embedded in the L-shaped three-dimensional structure of tRNA. Therefore, disruption of the three-dimensional structure of the substrate tRNA is necessary for the catalytic center of Gm-methylase to be able to access the target site in the tRNA, suggesting that the interaction of Gm-methylase with tRNA consists of multiple steps. This postulation was confirmed by inhibition experiments using nonsubstrate tRNA variants which functioned as competitive inhibitors against usual substrate tRNAs. Transfer RNA (guanosine-2′-)-methyltransferase (Gm-methylase, EC 2.1.1.32) from Thermus thermophilus HB27 is one of the tRNA ribose modification enzymes. The broad substrate specificity of Gm-methylase has so far been elucidated using various species of tRNAs from native sources, suggesting that the common structures in tRNAs are recognized by the enzyme. In this study, by using 28 yeast tRNAPhe variants obtained by transcription with T7 RNA polymerase, it was revealed that the nucleotide residues G18 and G19 and the D-stem structure are essentially required for Gm-methylase recognition, and that the key sequence for the substrate is pyrimidine (Py)17G18G19. The other conserved sequences were found not to be essential, but U8, G15, G26, G46, U54, U55, and C56 considerably affected the methylation efficiency. These residues are located within a limited space embedded in the L-shaped three-dimensional structure of tRNA. Therefore, disruption of the three-dimensional structure of the substrate tRNA is necessary for the catalytic center of Gm-methylase to be able to access the target site in the tRNA, suggesting that the interaction of Gm-methylase with tRNA consists of multiple steps. This postulation was confirmed by inhibition experiments using nonsubstrate tRNA variants which functioned as competitive inhibitors against usual substrate tRNAs. tRNA (guanosine-2′-)-methyltransferase purine pyrimidine. To date, more than 80 modified nucleosides in tRNAs have been isolated and characterized (1Limbach P.A. Crain P.F. McCloskey J.A. Nucleic Acids Res. 1994; 22: 2138-2196Crossref Scopus (476) Google Scholar). These nucleosides are post-transcriptionally formed at specific positions of tRNA by specific tRNA modification enzymes and are presumed to play important roles in the structure and function of tRNA (2Bjork G., R. Soll D. RajBhandary U., L. tRNA: Structure, Biosynthesis and Function. American Society for Microbiology, Washington, D. C.1995: 165-205Google Scholar, 3Bjork G.R. Ericson J.U. Gustafsson C.E.D. Hargervall T.G. Jonsson Y.H. Wikstrom P.M. Annu. Rev. Biochem. 1987; 56: 263-287Crossref PubMed Google Scholar, 4Bjork G.R. Hatfield D.L. Lee B.J. Pirtle R.M. Transfer RNA in Protein Synthesis. CRC Press, Boca Raton, FL1992: 23-85Google Scholar, 5Soll D. Kline L.K. Enzymes. 1982; 15: 557-566Crossref Scopus (4) Google Scholar, 6Kline L.K. Soll D. Enzymes. 1982; 15: 567-582Crossref Scopus (9) Google Scholar, 7Nishimura S. Schimmel P.R. Soll D. Abelson J.N. Transfer RNA: Structure, Properties and Recognition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1979: 59-79Google Scholar). Among tRNA modification enzymes, tRNA (guanosine-2′-)-methyltransferase (Gm-methylase,1 EC 2.1.1.34), one of the ribose modification enzymes, specifically catalyzes the transfer of a methyl group fromS-adenosyl-l-methionine to the 2′-OH of the ribose ring of guanosine at position 18 (G18) in the D-loop (8Kumagai I Watanabe K. Ohsima T. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1922-1926Crossref PubMed Scopus (36) Google Scholar, 14Persson B.C. Jager G. Gustafsson C. Nucleic Acids Res. 1997; 25: 4093-4097Crossref PubMed Scopus (81) Google Scholar). G18 is one of the hyperconserved residues located in the so-called three-dimensional core of tRNA (9Giege R. Puglisi J.D. Florentz C. Prog. Nucleic Acids Res. Mol. Biol. 1993; 45: 129-206Crossref PubMed Scopus (218) Google Scholar, 10Dirheimer G. Keith G. Dumas P. Westhof E. Soll D. RajBhandary U., L. tRNA: Structure, Biosynthesis and Function. American Society for Microbiology, Washington, D. C.1995: 93-126Google Scholar) and is responsible for the formation of the L-shaped three-dimensional structure by D-loop/T-loop interaction through the tertiary base pair G18-Ψ55 and G19-C56 (11Robertus J.D. Ladner J.E. Finch J.T. Rhodes D. Brown R.S. Clark B.F.C. Klug A. Nature. 1974; 250: 546-551Crossref PubMed Scopus (803) Google Scholar,12Kim S.H. Sussman J.L. Suddath F.L. Quigley G.J. McPherson A. Wang A.H.J. Seeman N.C. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4970-4974Crossref PubMed Scopus (233) Google Scholar). Although 2′-O-methylguanosine at position 18 (Gm18) is distributed widely in tRNAs of prokaryotes, eukaryotes, archaea, and plant mitochondria (13Steinberg S. Misch A. Sprinzl M. Nucleic Acids Res. 1993; 21: 3011-3015Crossref PubMed Scopus (234) Google Scholar), purification of the enzyme has been reported solely from Thermus thermophilus (7Nishimura S. Schimmel P.R. Soll D. Abelson J.N. Transfer RNA: Structure, Properties and Recognition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1979: 59-79Google Scholar). Recently, theEscherichia coli spoU gene has been reported to be essential for Gm18 modification, suggesting that spoUencodes E. coli Gm-methylase (14Persson B.C. Jager G. Gustafsson C. Nucleic Acids Res. 1997; 25: 4093-4097Crossref PubMed Scopus (81) Google Scholar). With respect to the physiological role of the methylation of the ribose of the G18 residue, it is known that the resistance of tRNA against RNases is increased by this modification, thus probably prolonging the half-life of the tRNA (15Kumagai I. Watanabe K. Ohshima T. J. Biol. Chem. 1982; 257: 7388-7395Abstract Full Text PDF PubMed Google Scholar). In higher plants, a relationship between Gm18 methylation and the transport of tRNALeu into mitochondria has also been reported (16Marechal-Drouard L. Neuburger M. Guillemaut P. Douce R. Weil J.-H. Dietrich A. FEBS Letts. 1990; 262: 170-172Crossref PubMed Scopus (17) Google Scholar). The mechanisms of interactions between tRNAs and modification enzymes are of interest not only physiologically but also biochemically as typical examples of RNA-protein interaction. However, there are only a few reports on tRNA recognition by modification enzymes. The following purified enzymes have been studied: tRNA-(m1G37)-methyltransferase (17Holmes W.M. Andraos-Selim C. Roberts I. Wahab S.Z. J. Biol. Chem. 1992; 267: 13440-13445Abstract Full Text PDF PubMed Google Scholar), tRNA-guanine transglycosylase for producing Q34 (18Curnow A.W. Kung F.-L. Kock K. Garcia G.A. Biochemistry. 1993; 32: 5239-5246Crossref PubMed Scopus (71) Google Scholar, 19Nakanishi S. Ueda T. Hori H. Yamazaki N. Okada N. Watanabe K. J. Biol. Chem. 1994; 269: 32221-32225Abstract Full Text PDF PubMed Google Scholar), and tRNA-(m5U54)-methyltransferase (20Gu X. Santi D.V. Biochemistry. 1991; 30: 2999-3002Crossref PubMed Scopus (59) Google Scholar, 21Gu X. Ofengand J. Santi D.V. Biochemistry. 1994; 33: 2255-2261Crossref PubMed Scopus (26) Google Scholar) from E. coli; tRNA-(m5C48)-methyltransferase (22Keith J. Winters E.M. Moss B. J. Biol. Chem. 1980; 255: 4636-4644Abstract Full Text PDF PubMed Google Scholar) from HeLa cell line; and Gm18-methylase (23Matsumoto T. Ohta T. Kumagai I. Oshima T. Murao K. Hasegawa T. Ishikura H. Watanabe K. J. Biochem. 1987; 101: 1191-1198Crossref PubMed Scopus (13) Google Scholar, 24Hori H. Saneyoshi M. Kumagai I. Miura K. Watanabe K. J. Biochem. 1989; 106: 798-802Crossref PubMed Scopus (23) Google Scholar, 25Matsumoto T. Nishikawa K. Hori H. Ohta T. Miura K. Watanabe K. J. Biochem. 1990; 107: 331-338Crossref PubMed Scopus (25) Google Scholar) and tRNA-(m1A58)-methyltransferase (26Yamazaki N. Hori H. Ozawa K. Nakanishi S. Ueda T. Kumagai I. Watanabe K. Nishikawa K. Biosci. Biotechnol. Biochem. 1994; 58: 1128-1133Crossref PubMed Google Scholar) from T. thermophilus. A crude extract, tRNA-(Ψ35)-synthase from a higher plant (27Szweykowska-Kulinska Z. Beier H. EMBO J. 1992; 11: 1907-1912Crossref PubMed Scopus (29) Google Scholar), has also been investigated. In addition, several tRNA modification enzymes from Xenopus laevis (28Melton D.A. Robertis E.M. Cortese R. Nature. 1980; 284: 143-148Crossref PubMed Scopus (143) Google Scholar, 29Carbon P. Haumont E. Fournier M. de Henau S. Grosjean H. EMBO J. 1983; 2: 1093-1097Crossref PubMed Scopus (34) Google Scholar, 30Droogmans L. Haumont E. de Henau S. Grosjean H. EMBO J. 1986; 5: 1105-1109Crossref PubMed Scopus (32) Google Scholar, 31Edqvist J. Straby K.B. Grosjean H. Nucleic Acids Res. 1993; 21: 413-417Crossref PubMed Scopus (15) Google Scholar, 32Edqvist J. Grosjean H. Straby K.B. Nucleic Acids Res. 1992; 20: 6575-6581Crossref PubMed Scopus (37) Google Scholar, 33Grosjean H. Edqvist J. Straby K.B. Giege R. J. Mol. Biol. 1996; 255: 67-85Crossref PubMed Scopus (107) Google Scholar) and yeast (34Johnson P.F. Abelson J. Nature. 1983; 302: 681-687Crossref PubMed Scopus (102) Google Scholar) have been reported using in vivo assay systems. Two technical difficulties have hindered investigations using purified enzymes. First, most tRNA modification enzymes are labile and only very scanty amounts are able to be purified. Second, special RNAs are usually required as substrates because the enzymes are highly specific for a particular nucleoside(s), sequence(s) and/or three-dimensional structure, and such tRNAs are not easy to prepare. Fortunately, the modification enzymes from T. thermophilus, one of which we used in this work, are relatively stable compared with those from other species. To overcome the second problem, we employed a T7 RNA polymerase system. A synthetic gene of yeast tRNAPhe was chosen as the template DNA of T7 RNA polymerase, because yeast tRNAPhe is one of the best substrate tRNAs for Gm-methylase, and its three-dimensional structure is well established (11Robertus J.D. Ladner J.E. Finch J.T. Rhodes D. Brown R.S. Clark B.F.C. Klug A. Nature. 1974; 250: 546-551Crossref PubMed Scopus (803) Google Scholar, 12Kim S.H. Sussman J.L. Suddath F.L. Quigley G.J. McPherson A. Wang A.H.J. Seeman N.C. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4970-4974Crossref PubMed Scopus (233) Google Scholar). In this report, the essential regions in the tRNA for recognition by Gm-methylase and its recognition mechanism are discussed. The methyl-14C-labeledS-adenosyl-l-methionine (55–60 Ci/mol) was purchased from Amersham Pharmacia Biotech. DNA oligomers were synthesized by an Applied Biosystems model 381 DNA synthesizer. DNA modifying enzymes and human placenta RNase inhibitor were obtained from Takara (Ohtsu, Japan). A T7 RNA polymerase expression system (E. coli BL21/pAR1219) was kindly provided by Dr. F. W. Studier (Brookhaven National Laboratory) (35Studier F.W. Rosenberg A.H. Dunn J.J. Dubendroff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar). T7 RNA polymerase was purified by the method of Grodberg and Dunn (36Grodberg J. Dunn J.J. J. Bacteriol. 1988; 170: 1245-1253Crossref PubMed Scopus (576) Google Scholar). Other chemical reagents were of analytical grade. Purified E. colitRNA2Glu, tRNA3Ser, and tRNA2Tyr were kindly provided by Dr. N. Hayashi (Tokyo Institute of Technology); Hallobacterium volcanii tRNAiMettRNA was a gift of Dr. Y. Kuchino (National Cancer Research); Bacillus subtilistRNAGly was supplied by Dr. K. Murao (Jichi Medical School). Yeast tRNAPhe was purchased from Boehringer Mannheim. A synthetic wild-type yeast tRNAPhe gene with a T7 promoter were constructed between the EcoRI and BamHI sites in the multicloning linker of pUC18, and the insert was then subcloned into the SalI and HindIII sites in the multicloning linker of pUC118 for site-directed mutagenesis. In the resultant plasmid, the transcriptional initiation site was designed to be G at the 5′ terminal, the first position of yeast tRNAPhe. Yeast tRNAPhe gene variants were produced by site-directed mutagenesis using a Muta-Gene phagemid mutation kit (Bio-Rad). The sequences of all the tRNA genes were analyzed using a Sequenase version 2.0 DNA sequencing kit (U.S. Biochemical Corp.). Read-through transcription was carried out with 18 μg of T7 RNA polymerase at 37 °C for 3 h using 20 μg of BstNI-digested plasmids encoding the yeast tRNAPhe gene variants as the template in a buffer containing 40 mm Tris-HCl (pH 8.0), 10 mm MgCl2, 5 mmdithiothreitol, 2 mm ATP, 2 mm GTP, 2 mm CTP, 2 mm UTP, 20 mm GMP, 1 mm spermidine, 5 μg of bovine serum albumin, 50 units of human placenta RNase inhibitor, and 3% glycerol in a total volume of 100 μl. The reaction mixture was extracted with phenol-chloroform (1:1, w/w) and then with chloroform-isoamylalcohol(25:1, v/v). Transcripts were recovered from the aqueous phase by ethanol precipitation. The dried pellet was dissolved in 50 μl of buffer containing 10 mm Tris-HCl (pH 8.0) and 1 mmEDTA, and the transcript was purified by 10% polyacrylamide (7m urea) gel electrophoresis. The melting profiles of yeast tRNAPhe and the wild-type transcript were measured by monitoring the change in the absorbance at 260 nm at a heating rate of 0.5 °C/min with a Gilford Response II spectrophotometer using 0.32 A 260 unit RNA in 400 μl of buffer containing 50 mm Tris-HCl (pH 7.5), 5 mmMgCl2, and 100 mm NaCl. The melting temperatures were determined by the first derivative of the melting curve. Gm-methylase was purified by the method previously reported (8Kumagai I Watanabe K. Ohsima T. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1922-1926Crossref PubMed Scopus (36) Google Scholar). The homogeneity of the purified enzyme was confirmed by SDS-polyacrylamide gel electrophoresis (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar). The quantity of the protein was measured with a Bio-Rad protein assay kit using bovine serum albumin as the standard. The standard assay for Gm-methylase activity was used according to our previous report (8Kumagai I Watanabe K. Ohsima T. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1922-1926Crossref PubMed Scopus (36) Google Scholar), except that the assays with transcripts were carried out at 60 °C. The apparent kinetic parameters, K m and V max, were determined by Lineweaver-Burk plots of the methylation reaction in which incorporations of the14C methyl group into the transcripts was measured for 20 min. In tRNA of the extreme thermophile T. thermophilus, Gm18 is one of the generally existing modified nucleosides, and is produced by Gm-methylase (8Kumagai I Watanabe K. Ohsima T. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1922-1926Crossref PubMed Scopus (36) Google Scholar, 38Watanabe K. Shinma M. Oshima T. Nishimura S. Biochem. Biophys. Res. Commun. 1976; 72: 1137-1144Crossref PubMed Scopus (100) Google Scholar), which catalyzes the transfer of methyl groups to various kinds of tRNAsin vitro as well as in vivo. The tRNAs that have so far been identified as substrates of Gm-methylase in an in vitro methylation reaction are listed in Table I. The only tRNA in the table that cannot be methylated is E. colitRNA2Tyr, because it already contains the Gm18 residue in the E. coli cells. This indicates that Gm-methylase does not catalyze the exchange reaction of the methyl group, which is in line with the reaction mechanisms of E. coli tRNA-(m5U54)-methyltransferase (39Kealy J.T. Santi D.V. Biochemistry. 1995; 34: 2441-2446Crossref PubMed Scopus (13) Google Scholar) and T. thermophilus tRNA-(m1A58)-methyltransferase (26Yamazaki N. Hori H. Ozawa K. Nakanishi S. Ueda T. Kumagai I. Watanabe K. Nishikawa K. Biosci. Biotechnol. Biochem. 1994; 58: 1128-1133Crossref PubMed Google Scholar). Since not only Class I but also Class II tRNAs (E. colitRNA3Ser and B. subtilistRNALeu) are methylated by Gm-methylase, the structural diversities derived from the sizes of the D-arm and the variable arm do not affect recognition by the enzyme. Moreover, the tRNA from an archaean, H. volcaniitRNAiMet, was a good substrate for Gm-methylase, suggesting that the recognition sites of Gm-methylase are common for tRNAs from three kingdoms, eukarya, prokarya, and archaea.Table INatural tRNAs that can be substrates for Gm-methylasetRNARelative initial velocity of CH3incorporationK mV maxAssay temperatureReference%nmnmol/mg protein/h° CProkaryotes E. colitRNAfMet69.0578657Nishimura S. Schimmel P.R. Soll D. Abelson J.N. Transfer RNA: Structure, Properties and Recognition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1979: 59-79Google Scholar,13Steinberg S. Misch A. Sprinzl M. Nucleic Acids Res. 1993; 21: 3011-3015Crossref PubMed Scopus (234) Google Scholar, 22Keith J. Winters E.M. Moss B. J. Biol. Chem. 1980; 255: 4636-4644Abstract Full Text PDF PubMed Google ScholartRNAmMet46.0——657Nishimura S. Schimmel P.R. Soll D. Abelson J.N. Transfer RNA: Structure, Properties and Recognition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1979: 59-79Google ScholartRNAPhe36.01079657Nishimura S. Schimmel P.R. Soll D. Abelson J.N. Transfer RNA: Structure, Properties and Recognition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1979: 59-79Google Scholar, 13Steinberg S. Misch A. Sprinzl M. Nucleic Acids Res. 1993; 21: 3011-3015Crossref PubMed Scopus (234) Google ScholartRNAmajorIle67.467265This worktRNA3Ser4.7——65This worktRNA2Glu14.3——65This worktRNA2TyrNot methylated——65This work B. subtilistRNAVal68.0——6021Gu X. Ofengand J. Santi D.V. Biochemistry. 1994; 33: 2255-2261Crossref PubMed Scopus (26) Google ScholartRNAAsp65.7——6021Gu X. Ofengand J. Santi D.V. Biochemistry. 1994; 33: 2255-2261Crossref PubMed Scopus (26) Google ScholartRNAThr54.6——5521Gu X. Ofengand J. Santi D.V. Biochemistry. 1994; 33: 2255-2261Crossref PubMed Scopus (26) Google ScholartRNALeu41.5——5021Gu X. Ofengand J. Santi D.V. Biochemistry. 1994; 33: 2255-2261Crossref PubMed Scopus (26) Google ScholartRNAGly55.2——65This workEukaryote YeasttRNAPhe100.0<589657Nishimura S. Schimmel P.R. Soll D. Abelson J.N. Transfer RNA: Structure, Properties and Recognition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1979: 59-79Google Scholar, 13Steinberg S. Misch A. Sprinzl M. Nucleic Acids Res. 1993; 21: 3011-3015Crossref PubMed Scopus (234) Google Scholar, 22Keith J. Winters E.M. Moss B. J. Biol. Chem. 1980; 255: 4636-4644Abstract Full Text PDF PubMed Google ScholarArchaean H. volcaniitRNA1Met85.0——65This workInitial velocities are expressed relative to methyl group incorporation into yeast tRNAPhe for 20 min at 65 °C which was taken as 100%.—, not determined.“Not methylated” means that the relative initial velocity was below 0.5%. Open table in a new tab Initial velocities are expressed relative to methyl group incorporation into yeast tRNAPhe for 20 min at 65 °C which was taken as 100%. —, not determined. “Not methylated” means that the relative initial velocity was below 0.5%. The nucleotide residues conserved and semiconserved in the tRNAs of the three kingdoms are shown in Fig. 1 A. To clarify the recognition sites of Gm-methylase, 28 variants of yeast tRNAPhetranscribed by T7 RNA polymerase were employed. Nucleotide substitutions were mainly introduced into the conserved residues in the three- dimensional core region in the tRNA (Fig. 1 B), because these residues interact directly or indirectly with the D-arm, which includes the methylation target site, the 2′-OH group of the G18 ribose. Previous results from foot printing and experiments using half fragments indicated that the essential region for the recognition by Gm-methylase was limited within the sequence G10–G26 in E. coli tRNAfMet (24Hori H. Saneyoshi M. Kumagai I. Miura K. Watanabe K. J. Biochem. 1989; 106: 798-802Crossref PubMed Scopus (23) Google Scholar, 25Matsumoto T. Nishikawa K. Hori H. Ohta T. Miura K. Watanabe K. J. Biochem. 1990; 107: 331-338Crossref PubMed Scopus (25) Google Scholar). Therefore, all the residues of the D-loop were individually substituted by other nucleotides, irrespective of whether they were conserved or nonconserved (Fig. 1 B), by introducing point mutations into the synthetic yeast tRNAPhe gene. Each variant is designated by the original and mutated residues connected byarrows in Fig. 1 B and the position of the substitution (e.g. U8A denotes the variant in which U at position 8 was substituted by A). Some examples of the kinetic analyses of Gm-methylase for these variants are given in Fig. 2, and the summarized results are given in Table II. Fig. 2 A shows the relationship between substrate concentration and the initial velocity of methyl group incorporation by Gm-methylase when the wild-type tRNAPhe transcript and some tRNA variants (U17A, U17G, and G18A) were used as the substrate. Fig. 2 B shows the Lineweaver-Burke plots for wild-type transcript (left) and for U17G and U17A variants, from which the kinetic parameters (K m and V max) were estimated as shown in Table II.Figure 2Methylation profiles (A) and Lineweaver-Burke plots (B) for the wild-type tRNAPhe transcript and its variants, U17A, U17G, and G18A. A, initial velocities of methyl group incorporation were measured with various concentrations of substrate tRNA transcripts: •, wild-type transcript; ○, U17A variant; ▪, U17G variant; ■, G18A variant. B, Lineweaver-Burke plots for wild-type transcript (•; left and right) and for U17G (▪) and U17A (○) variants.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIEffect of point mutations in tRNA transcripts on their methyl group acceptance activityVariantConservation of substitution siteDisrupted or altered tertiary base pairK mV maxRelativeV max/K mnmnmol/mg/h%Wild-type8085100 U8AConservedU8-A142407329 U8GConservedU8-A141706737 U8CConservedU8-A142807927 A9USemiconserved as PuA9-A228088104D-loop region A14CConservedU8-A141106758 G15USemiconservedG15-C48903436 U16ANoNone808297 U17ANoNone6035 U17GNoNone6012 U17CNoNone7085115 G18AConservedG18-U55 → A18-U55NDNDND G18CConservedG18-U55NDNDND G19AConservedG19-C56NDNDND G20ANoNone1005855 A21CConservedNone905972 G26AConserved as PuG25-A44 → A26-A447081109 G26UConserved as PuG25-A44 → U26-A441304835 G45CNoNone2107031 G46CSemiconservedG22-G463206419 C48AConservedG15-G481107564 U54ASemiconservedU54-A581608450 U55AConservedG18-U554407817 C56GConservedG19-C56840678 A58GConservedU54-A581109279The wild type is totally unmodified tRNAPhe transcribed by T7 RNA polymerase. The designation given to each variant indicates the position of the point mutation in the tRNA and the original and mutated residues. For example, U8A means the variant in which U at position 8 was substituted by A. Symbols: no, nonconserved; none, the residue is not responsible for the tertiary base pair; ND, methylation was not detected (the initial velocity was below 0.5% of that of the wild-type transcript). When an alternative tertiary base pair was created by point mutation, the disrupted and alternative base pairs are connected by →. For example, G18-U55 → A18-U55 means the base pair G18-U55 was disrupted to form A18-U55. The relative Vmax: Km is expressed in relation to that of the wild type which is taken as 100%. The kinetic parameters indicated in this table are the averages of data obtained in at least two independent experiments. Open table in a new tab The wild type is totally unmodified tRNAPhe transcribed by T7 RNA polymerase. The designation given to each variant indicates the position of the point mutation in the tRNA and the original and mutated residues. For example, U8A means the variant in which U at position 8 was substituted by A. Symbols: no, nonconserved; none, the residue is not responsible for the tertiary base pair; ND, methylation was not detected (the initial velocity was below 0.5% of that of the wild-type transcript). When an alternative tertiary base pair was created by point mutation, the disrupted and alternative base pairs are connected by →. For example, G18-U55 → A18-U55 means the base pair G18-U55 was disrupted to form A18-U55. The relative Vmax: Km is expressed in relation to that of the wild type which is taken as 100%. The kinetic parameters indicated in this table are the averages of data obtained in at least two independent experiments. The methyl group acceptance activity of the wild-type transcript was about 1/20 that of the native yeast tRNAPhe through increase of K m (Tables I and II and Fig. 2 B). The structural flexibility of a transcript can be estimated by its melting curve; the melting temperature of the wild-type transcript was determined to be 69.0 °C, while that of the native yeast tRNAPhe was 76.0 °C. Some of the modified nucleosides in the native tRNA affects the methylation efficiency, probably through the structural stability of the substrate RNA and/or the direct association with the enzyme. Since a suitable temperature for the methylation of the transcript was estimated to be 60 °C (data not shown), all the experiments with transcripts were carried out at that temperature. The difference in methyl group acceptance between native tRNA and its transcripts is more clearly observed with shorter length transcripts corresponding to a part of yeast tRNAPhe; methyl group acceptance activity of the transcript corresponding to the 5′-half fragment (positions 1–33 in yeast tRNAPhe) was hardly detectable at 37–60 °C (data not shown), while that of the native 5′-half fragment could be detected, the initial velocity being about 20% of that of the full-length tRNA (24Hori H. Saneyoshi M. Kumagai I. Miura K. Watanabe K. J. Biochem. 1989; 106: 798-802Crossref PubMed Scopus (23) Google Scholar, 25Matsumoto T. Nishikawa K. Hori H. Ohta T. Miura K. Watanabe K. J. Biochem. 1990; 107: 331-338Crossref PubMed Scopus (25) Google Scholar). In the case of the thermophile Gm-methylase, it was difficult to discern the so-called minimalist substrate by using a totally unmodified short fragment, since the initial velocities for such fragments could not be measured under the standard conditions; at least, the methylation of the chemically synthesized 18-mer corresponding to positions 9–26 was not able to be detected at either 37 or 50 °C in 24-h incubation under the standard conditions (data not shown). Thus, it is assumed that modified nucleosides such as m2G10, D16, D17, m22G26, and Cm32 present in the native 5′-half fragment strongly affect the methylation efficiency, probably through stabilizing the D-loop stem structure or making the enzyme recognition toward the substrate easier. In the point-substituted full-length variants, the only residues essential for Gm-methylase recognition among those conserved or semiconserved were determined to be G18 and G19 (Fig. 2 A and Table II). Substitution of the nonconserved residue U17 by purine (Pu) (U17A and U17G) resulted in a drastic decrease in theV max/K m value. In contrast, no effect was observed when U17 was substituted by C. Analysis of the kinetic parameters indicated that the V maxvalues for U17A and U17G were very small (Fig. 2 and Table II), suggesting that the substitution of U17 by Pu changes the environment of the catalytic center in the Gm-methylase -tRNA complex. Thus, the most appropriate minimal sequence for Gm-methylase was deduced to be Py17G18G19 (Py = pyrimidine). This is supported by the result forE. coli tRNA3Ser possessing an A17G18G19 sequence (Table I), which was the worst substrate for Gm-methylase among the native tRNAs tested. In E. coli tRNAs, the G18 residue in almost all class II tRNAs is modified to Gm18, an exception being tRNA3Ser, which has an unmodified G18 (13Steinberg S. Misch A. Sprinzl M. Nucleic Acids Res. 1993; 21: 3011-3015Crossref PubMed Scopus (234) Google Scholar). Moreover, no prokaryote tRNA possessing a Pu17Gm18 sequence has been reported (13Steinberg S. Misch A. Sprinzl M. Nucleic Acids Res. 1993; 21: 3011-3015Crossref PubMed Scopus (234) Google Scholar). Thus, the optimum sequence deduced for the thermophile Gm-methylase, Py17G18G19, is likely to be applicable to most prokaryotic Gm-methylases. It is also clear that positions 17–19 were distinctly recognized by the enzyme, because the G18C variant with a C18G19G20 sequence was not methylated at all (Table II). Judging from the results with the D-stem variants (discussed in the next section; see Table III), it is likely that the recognition of these positions depends on the steric distance and the angle from the phosphate-ribose backbone of the D-stem structure. On the other hand, it has been reported that the 2′-O-methylation of G34 (the anticodon first letter) in X. laevis is not affected by the nucleotide sequence around position 34 (30Droogmans L. Haumont E. de Henau S. Grosjean H. EMBO J. 1986; 5: 1105-1109Crossref PubMed Scopus (32) Google Sc" @default.
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• W1966175091 title "Substrate Recognition of tRNA (Guanosine-2′-)-methyltransferase from Thermus thermophilus HB27" @default.
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