Title: Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA
Abstract: Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m22G26) in tRNA. In the reaction, N2-guanine at position 26 (m2G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNACys has an m22G26m2G27 or m22G26m22G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m2G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme. Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m22G26) in tRNA. In the reaction, N2-guanine at position 26 (m2G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNACys has an m22G26m2G27 or m22G26m22G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m2G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme. To date, more than 100 modified nucleosides have been identified in various RNA species with the majority of them having been found in tRNA molecules (1Rozenski J. Crain P.F. McCloskey J.A. Nucleic Acids Res. 1999; 27: 196-197Crossref PubMed Scopus (350) Google Scholar, 2Dunin-Horkawicz S. Czerwoniec A. Gajda M.J. Feder M. Grosjean H. Bujnicki J.M. Nucleic Acids Res. 2006; 34: D145-D149Crossref PubMed Scopus (188) Google Scholar). All of the modified nucleosides are introduced into tRNA by specific tRNA modification enzymes or guide RNA modification systems during the post-transcriptional process. Among them, N2,N2-dimethylguanine at position 26 (m22G26) in tRNA is generated by tRNA (N2,N2-guanine)-dimethyltransferase (tRNA (m22G26) methyltransferase; TrMet (m2,2G26); EC 2.1.1.32) (2Dunin-Horkawicz S. Czerwoniec A. Gajda M.J. Feder M. Grosjean H. Bujnicki J.M. Nucleic Acids Res. 2006; 34: D145-D149Crossref PubMed Scopus (188) Google Scholar, 3Garcia G.R. Goodenough-Lashhua D.M. Grosjean H. Benne R. Modification and Editing of RNA. American Society for Microbiology, Washington, DC1998: 555-560Google Scholar). This enzyme catalyzes the methyl transfer reaction from S-adenosyl-l-methionine (AdoMet) 2The abbreviations used are: AdoMetS-adenosyl-l-methionineLC/MSliquid chromatography/mass spectrometryMS/MStandem mass spectrometry. to the N2 atom of G26, which is located at the junction between the D-arm and the anticodon arm in tRNA. In the reaction, two molecules of AdoMet are consumed, and N2-methylguanine at position 26 (m2G26) is generated as an intermediate (4Reinhart M.P. Lewis J.M. Leboy P.S. Nucleic Acids Res. 1986; 14: 1131-1148Crossref PubMed Scopus (21) Google Scholar). Thus, in some cases, this enzyme catalyzes transfer of only one methyl group and functions as a tRNA (m2G26) methyltransferase (5Constantinesco F. Motorin Y. Grosjean H. J. Mol. Biol. 1999; 291: 375-392Crossref PubMed Scopus (45) Google Scholar). The enzymatic activity was initially detected in rat liver (6Kuchino Y. Nishimura S. Biochem. Biophys. Res. Commun. 1970; 40: 306-313Crossref PubMed Scopus (58) Google Scholar, 7Glick J.M. Averyhart V.M. Leboy P.S. Biochim. Biophys. Acta. 1978; 518: 158-171Crossref PubMed Scopus (25) Google Scholar) and then found in various organisms (8Stange N. Beier H. EMBO J. 1987; 6: 2811-2818Crossref PubMed Scopus (67) Google Scholar, 9Edqvist J. Grosjean H. Stråby K.B. Nucleic Acids Res. 1992; 20: 6575-6581Crossref PubMed Scopus (37) Google Scholar, 10Edqvist J. Blomqvist K. Stråby K.B. Biochemistry. 1994; 33: 9546-9551Crossref PubMed Scopus (32) Google Scholar, 11Grosjean H. Edqvist J. Stråby K.B. Giegé R. J. Mol. Biol. 1996; 255: 67-85Crossref PubMed Scopus (107) Google Scholar, 12Constantinesco F. Motorin Y. Grosjean H. Nucleic Acids Res. 1999; 27: 1308-1315Crossref PubMed Scopus (42) Google Scholar). The most highly purified enzyme from a native source was obtained from Tetrahymena pyriformis (4Reinhart M.P. Lewis J.M. Leboy P.S. Nucleic Acids Res. 1986; 14: 1131-1148Crossref PubMed Scopus (21) Google Scholar). The responsible gene was first determined to be trm1 from Saccharomyces cerevisiae (13Phillips J.H. Kjellin-Stråby K. J. Mol. Biol. 1967; 26: 509-518Crossref PubMed Google Scholar, 14Ellis S.R. Morales M.J. Li J.M. Hopper A.K. Martin N.C. J. Biol. Chem. 1986; 261: 9703-9709Abstract Full Text PDF PubMed Google Scholar) and then experimentally identified from various eukaryotes (Schizosaccharomyces pombe (15Niederberger C. Gräub R. Costa A. Desgrès J. Schweingruber M.E. FEBS Lett. 1999; 464: 67-70Crossref PubMed Scopus (14) Google Scholar), Caenorhabditis elegans (16Liu J. Zhou G.Q. Stråby K.B. Gene. 1999; 226: 73-81Crossref PubMed Scopus (15) Google Scholar), and human (17Liu J. Strâby K.B. Nucleic Acids Res. 2000; 28: 3445-3451Crossref PubMed Scopus (46) Google Scholar)) and archaea (Pyrococcus furiosus (18Constantinesco F. Benachenhou N. Motorin Y. Grosjean H. Nucleic Acids Res. 1998; 26: 3753-3761Crossref PubMed Scopus (28) Google Scholar), Pyrococcus horikoshii (19Ihsanawati Nishimoto M. Higashijima K. Shirouzu M. Grosjean H. Bessho Y. Yokoyama S. J. Mol. Biol. 2008; 383: 871-884Crossref PubMed Scopus (24) Google Scholar), and Haloferax volcanii (20Grosjean H. Gaspin C. Marck C. Decatur W.A. de Crécy-Lagard V. BMC Genomics. 2008; 9: 470Crossref PubMed Scopus (60) Google Scholar)), as is consistent with the distribution of the m22G26 (or m2G26) modification in tRNA. S-adenosyl-l-methionine liquid chromatography/mass spectrometry tandem mass spectrometry. The m2G and m22G modifications in tRNA can be found not only at position 26 but also at positions 6, 7, 9, 10, 18, and 27 in various organisms (21Auffinger P. Westhof E. Grosjean H. Benne R. Modification and Editing of RNA. American Society for Microbiology, Washington, DC1998: 569-576Google Scholar). However, only m22G10 formation enzymes (the Trm11-Trm112 complex in yeast (22Purushothaman S.K. Bujnicki J.M. Grosjean H. Lapeyre B. Mol. Cell. Biol. 2005; 25: 4359-4370Crossref PubMed Scopus (90) Google Scholar) and Trm-m22G10 enzyme in Pyrococcus abyssi (23Armengaud J. Urbonavicius J. Fernandez B. Chaussinand G. Bujnicki J.M. Grosjean H. J. Biol. Chem. 2004; 279: 37142-371452Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 24Urbonavicius J. Armengaud J. Grosjean H. J. Mol. Biol. 2006; 357: 387-399Crossref PubMed Scopus (35) Google Scholar)) have been identified thus far. These tRNA (m22G10) methyltransferases are structurally different from tRNA (m22G26) methyltransferase (Trm1). Recently, we determined the x-ray crystal structure of P. horikoshii Trm1 (19Ihsanawati Nishimoto M. Higashijima K. Shirouzu M. Grosjean H. Bessho Y. Yokoyama S. J. Mol. Biol. 2008; 383: 871-884Crossref PubMed Scopus (24) Google Scholar); this protein includes N-terminal and C-terminal domains with class I methyltransferase (25Schubert H.L. Blumenthal R.M. Cheng X. Trends Biochem. Sci. 2003; 28: 329-335Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar) and novel folds, respectively. In contrast, amino acid sequence alignment, computational modeling, and deletion mutant experiments have revealed that the Trm-m22G10 enzyme contains a THUMP domain in the N-terminal region and a catalytic domain with a class I methyltransferase fold in the C-terminal portion (26Gabant G. Auxilien S. Tuszynska I. Locard M. Gajda M.J. Chaussinand G. Fernandez B. Dedieu A. Grosjean H. Golinelli-Pimpaneau B. Bujnicki J.M. Armengaud J. Nucleic Acids Res. 2006; 34: 2483-2494Crossref PubMed Scopus (26) Google Scholar). For the past several years, we have studied RNA modification enzymes from Aquifex aeolicus (27Hori H. Kubota S. Watanabe K. Kim J.M. Ogasawara T. Sawasaki T. Endo Y. J. Biol. Chem. 2003; 278: 25081-25090Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 28Okamoto H. Watanabe K. Ikeuchi Y. Suzuki T. Endo Y. Hori H. J. Biol. Chem. 2004; 279: 49151-49159Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 29Takeda H. Toyooka T. Ikeuchi Y. Yokobori S. Okadome K. Takano F. Oshima T. Suzuki T. Endo Y. Hori H. Genes Cells. 2006; 11: 1353-1365Crossref PubMed Scopus (36) Google Scholar, 30Tomikawa C. Ochi A. Hori H. Proteins. 2008; 71: 1400-1408Crossref PubMed Scopus (15) Google Scholar, 31Toyooka T. Awai T. Kanai T. Imanaka T. Hori H. Genes Cells. 2008; 13: 807-816Crossref PubMed Scopus (9) Google Scholar). A. aeolicus is a hyper-thermophilic eubacterium, which grows at close to 95 °C. The 16 S rRNA gene has been analyzed from the perspective of molecular evolution, and it has been suggested that this bacterium is the earliest diverging eubacterium (32Burggraf S. Olsen G.J. Stetter K.O. Woese C.R. Syst. Appl. Microbiol. 1992; 15: 353-356Crossref Scopus (191) Google Scholar), although there is debate concerning the diverging point of this bacterium (33Griffiths E. Gupta R.S. Microbiology. 2001; 147: 2611-2622Crossref PubMed Scopus (40) Google Scholar, 34Griffiths E. Gupta R.S. Int. J. Syst. Evol. Microbiol. 2006; 56: 99-107Crossref PubMed Scopus (32) Google Scholar). The complete genome has been determined, and a putative trm1 gene was found (35Deckert G. Warren P.V. Gaasterland T. Young W.G. Lenox A.L. Graham D.E. Overbeek R. Snead M.A. Keller M. Aujay M. Huber R. Feldman R.A. Short J.M. Olsen G.J. Swanson R.V. Nature. 1998; 392: 353-358Crossref PubMed Scopus (964) Google Scholar). In this study, we have focused on this eubacterial trm1 gene product. We demonstrate that this gene product is genuinely expressed in A. aeolicus cells and has a novel tRNA methyltransferase activity, which is completely different from eukaryotic and archaeal Trm1 enzymes reported thus far. Moreover, we report a long distance recognition mechanism of this enzyme. Abbreviations of the modified nucleosides in this paper are listed in supplemental Table 1. [methyl-14C]AdoMet (1.95 GBq/mmol) and [methyl-3H]AdoMet (2.47 TBq/mmol) were purchased from ICN. Cold AdoMet was obtained from Sigma. DE52 is a product of Whatman. CM-Toyopearl 650M was from Tosoh. DNA oligomers were bought from Invitrogen, and T7 RNA polymerase was from Toyobo. Other chemical reagents were of analytical grade. The culture source of A. aeolicus and 100 ml of culture medium in controlled gas (H2/CO2 (v/v, 4:1) mixed gas pressurized by air to 2 atm) were kindly provided by Dr. Harald Huber (Universitat Regensburg, Germany). The culture was performed at 85 °C for 24 h. The standard assay used during the purification was measured incorporation of 14C-methyl groups from [methyl-14C]AdoMet to A. aeolicus tRNAHis transcript; 0.1 μm enzyme, 11 μm transcript, and 38 μm [methyl-14C]AdoMet in 45 μl of buffer A (50 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 6 mm 2-mercaptoethanol, and 50 mm KCl) were incubated for 15 min at 55 °C. An aliquot (30 μl) was then used for the filter assay. RNA transcripts were prepared as reported previously (29Takeda H. Toyooka T. Ikeuchi Y. Yokobori S. Okadome K. Takano F. Oshima T. Suzuki T. Endo Y. Hori H. Genes Cells. 2006; 11: 1353-1365Crossref PubMed Scopus (36) Google Scholar). If discrimination between the m2G and m22G formation activities was necessary, we employed two-dimensional TLC (36Keith G. Biochimie. 1995; 77: 142-144Crossref PubMed Scopus (133) Google Scholar). The 14C-methylated RNA was dissolved in 5 μl of 50 mm ammonium acetate (pH 5.0) and digested with 2.5 units of nuclease P1, and then 2 μl of standard nucleotides containing 0.05 A260 unit each of pA, pG, pC, and pU was added. 2 μl of the sample was spotted onto a thin layer plate (Merck code number 1.05565, cellulose F) and separated using the following solvent systems: first dimension, isobutyric acid/concentrated ammonia/water, 66:1:33, v/v; second dimension, isopropyl alcohol/HCl/water, 70:15:15, v/v. Incorporation of 14C-methyl groups was monitored with a Fuji Photo Film BAS2000 imaging analyzer. Standard nucleotides were marked by UV 260 nm irradiation. To visualize the methyl transfer reaction, we used 10% PAGE (7 m urea) and an imaging analyzer system as described previously (28Okamoto H. Watanabe K. Ikeuchi Y. Suzuki T. Endo Y. Hori H. J. Biol. Chem. 2004; 279: 49151-49159Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 29Takeda H. Toyooka T. Ikeuchi Y. Yokobori S. Okadome K. Takano F. Oshima T. Suzuki T. Endo Y. Hori H. Genes Cells. 2006; 11: 1353-1365Crossref PubMed Scopus (36) Google Scholar). Briefly, tRNA (0.1 A260 units) was incubated with 0.1 μm Trm1 and 38 μm [methyl-14C]AdoMet for 10 min at 55 °C in 30 μl of buffer A and then loaded onto a 10% polyacrylamide gel (7 m urea). The gel was stained with methylene blue and dried. The incorporation of 14C-methyl groups into tRNA was monitored with a Fuji Photo Film BAS2000 imaging analyzer. First, we carried out time course experiments at 55 °C with 0.1 μm Trm1, 11 μm tRNA transcript, and 38 μm [methyl-14C]AdoMet in 210 μl of buffer A. The aliquots (30 μl each) were taken at appropriate times (0, 2, 5, 7.5, 10, and 15 min) and formations of 14C-pm2G and 14C-pm22G monitored by two-dimensional TLC. Under these conditions, only pm2G linearly increased for the first 10 min, and m22G formation was barely observable; pm22G content was less than 5% of the pm2G content in the sample at 10 min. Kinetic parameters for tRNATyr transcripts were determined by incorporation of 3H-methyl groups into tRNA. Both Km and Vmax values for tRNATyr transcripts were small, and activity measurement with low concentrations of enzyme and tRNA transcripts was very difficult. Therefore, we determined approximate kinetic parameters under unusual conditions as follows: 0.1 μm Trm1, 40 μm [methyl-3H]AdoMet, and various concentrations (0.06, 0.1, 0.15, 0.3, 0.6, 0.9, and 1.5 μm) of tRNATyr transcript were incubated at 55 °C for 10 min, and then the filter assay was performed. 3′-Biotin-labeled DNA oligomer (5′-TGC AGT CCC CTG CCT AAC CGC TC-biotin 3′) was used as a probe. Because we expected the G26 in the native tRNACys to be modified to m22G26, the nucleotide at position 11 in the DNA was designed as T instead of C to form an m22G26-T11 base pair. 100 μl of slurry of streptavidin-Sepharose high performance resin (GE Healthcare) was poured into a 1.5-ml Amicon Ultrafree-MC tube (0.22 μm), and then the buffer was removed by centrifugation at 500 × g for 10 s. The resin was equilibrated by addition of 400 μl of 20 mm Tris-HCl (pH 7.5), and the buffer was removed by centrifugation at 500 × g for 10 s. This treatment was repeated two times. 0.8 A260 unit of DNA probe dissolved in 400 μl of 20 mm Tris-HCl (pH 7.5) was poured into the tube, mixed with the resin, incubated for 10 min at room temperature, and then the buffer removed by centrifugation at 500 × g for 10 s. The DNA immobilized on the resin in the tube, 2× hybridization buffer (40 mm Tris-HCl (pH 7.5), 1.8 m tetramethylammonium chloride, 200 μm EDTA), and 10.0 A260 units of A. aeolicus total RNA dissolved in 200 μl of water were preincubated at 69 °C for 5 min. The resin, total RNA (200 μl), and 200 μl of 2× hybridization buffer were mixed and then incubated at 69 °C for 5 min. The hybridization was performed by cooling from 69 to 65 °C for 4 min, and incubation was at 65 °C for 5 min. Unbound RNA was removed by centrifugation at 500 × g for 10 s, and then the resin was washed with 400 μl of 20 mm Tris-HCl (pH 7.5). This treatment was repeated five times. The elution of tRNACys was performed as follows. The resin and 400 μl of the elution buffer (20 mm Tris-HCl (pH 7.5)) were preincubated at 65 °C for 5 min, mixed, incubated at 65 °C for 5 min, and then quickly centrifuged at 500 × g for 10 s. The eluted RNA was collected by ethanol precipitation. In this study, tRNACys was further purified by 10% PAGE (7 m urea). Detailed procedures to the other tRNA cases will be published. 3T. Yokogawa, S. Ohno, and K. Nishikawa, manuscript in preparation. Transfer RNACys transcript (50 μg) was methylated with 0.5 μm Trm1 and 670 μm nonradioisotope-labeled AdoMet for 12 h at 55 °C in 100 μl of buffer A. The RNA was purified by 10% PAGE (7 m urea). The RNA was visualized by UV (254 nm) irradiation on a thin layer plate (Funacell P-254, Japan), excised, and extracted with 400 μl of gel elution buffer (0.5 m ammonium acetate, 10 mm MgCl2, 1 mm EDTA, and 0.1% SDS). The extracted sample was passed through a Steradisc 13 filter unit (0.2 μm, Kurabo, Japan), and the RNA was recovered by ethanol precipitation. About 0.05 A260 units of methylated tRNACys transcript was used for total nucleoside analysis, and about 200 fmol of the transcript and native tRNACys were digested with RNase A or RNase T1 for RNA fragment analysis. Mass spectrometric analysis was carried out as described previously (37Suzuki T. Ikeuchi Y. Noma A. Suzuki T. Sakaguchi Y. Methods Enzymol. 2007; 425: 211-229Crossref PubMed Scopus (100) Google Scholar, 38Ikeuchi Y. Kitahara K. Suzuki T. EMBO J. 2008; 27: 2194-2203Crossref PubMed Scopus (66) Google Scholar, 39Noma A. Kirino Y. Ikeuchi Y. Suzuki T. EMBO J. 2006; 25: 2142-2154Crossref PubMed Scopus (158) Google Scholar, 40Ikeuchi Y. Shigi N. Kato J. Nishimura A. Suzuki T. Mol. Cell. 2006; 21: 97-108Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 41Kaneko T. Suzuki T. Kapushoc S.T. Rubio M.A. Ghazvini J. Watanabe K. Simpson L. Suzuki T. EMBO J. 2003; 22: 657-667Crossref PubMed Scopus (94) Google Scholar) with the following modifications. For total nucleoside analysis, we used yeast total tRNA as control for assignment of modified nucleosides. For the analyses of RNase A or RNase T1 digests, we employed a linear ion trap-orbitrap hybrid mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific) equipped with a custom-made nanospray ion source and a splitless nano HPLC system (DiNa, KYA Technologies). The concentration of RNase T1 was adjusted to 5 units/μl. The digests mixed with TEAA (triethylamine acetate, pH 7.0) were loaded onto a nano-LC trap column (C18, inner diameter 0.5 × 1.0 mm), desalted, and then concentrated with 0.1 m TEAA (pH 7.0). RNA fragments were eluted from the trap column and directly injected into a C18 capillary column (HiQ Sil; 3-μm C18, 100 Å pore size; inner diameter 0.1 × 100 mm, KYA Technologies). The chromatography was carried out in the same condition as described (37Suzuki T. Ikeuchi Y. Noma A. Suzuki T. Sakaguchi Y. Methods Enzymol. 2007; 425: 211-229Crossref PubMed Scopus (100) Google Scholar), and the eluent was sprayed from a sprayer tip attached to the capillary column. The ionization voltage was set to −1.9 kV, and ions were scanned in the negative polarity mode. The following sections are described in the supplemental material: construction of A. aeolicus Trm1 expression system in Escherichia coli; purification of the recombinant Trm1 protein, and Western blotting. In 1998, the complete genome sequence of A. aeolicus was determined, and the existence of a putative trm1 gene was reported (35Deckert G. Warren P.V. Gaasterland T. Young W.G. Lenox A.L. Graham D.E. Overbeek R. Snead M.A. Keller M. Aujay M. Huber R. Feldman R.A. Short J.M. Olsen G.J. Swanson R.V. Nature. 1998; 392: 353-358Crossref PubMed Scopus (964) Google Scholar). All trm1 gene products experimentally analyzed thus far have a tRNA (m22G26) methyltransferase activity. This enzyme activity is only found in eukaryotes and archaea, consistent with the distribution of the trm1 genes. Thus, although A. aeolicus belongs to the eubacteria, a putative trm1 gene is encoded. We compared the amino acid sequence of the A. aeolicus trm1 gene product with those of experimentally identified tRNA (m22G26) methyltransferases (Trm1 proteins) (Fig. 1). As shown in Fig. 1, the A. aeolicus trm1 gene product has many of the amino acid residues conserved among the Trm1 proteins. In fact, during the course of this study, we determined the crystal structures of P. horikoshii Trm1 (19Ihsanawati Nishimoto M. Higashijima K. Shirouzu M. Grosjean H. Bessho Y. Yokoyama S. J. Mol. Biol. 2008; 383: 871-884Crossref PubMed Scopus (24) Google Scholar). In the study, it was found that two phenylalanine residues (corresponding to Phe-27 and Phe-134 in A. aeolicus Trm1) form a pocket, which is predicted to be a part of the G26-binding site (19Ihsanawati Nishimoto M. Higashijima K. Shirouzu M. Grosjean H. Bessho Y. Yokoyama S. J. Mol. Biol. 2008; 383: 871-884Crossref PubMed Scopus (24) Google Scholar). Furthermore, an aspartic acid (corresponding to Asp-132 in A. aeolicus Trm1) was expected to be a catalytic center. These Trm1-specific amino acid residues are conserved in A. aeolicus Trm1 in addition to the amino acid sequence motifs conserved among methyltransferases (42Bujnicki J.M. Leach R.A. Debski J. Rychlewski L. J. Mol. Microbiol. Biotechnol. 2002; 4: 405-415PubMed Google Scholar). Thus, the amino acid sequence alignment strongly suggested the A. aeolicus trm1 gene product to be a tRNA (m22G26) methyltransferase. To investigate whether the A. aeolicus putative trm1 gene product has tRNA methyltransferase activity, we performed PCR cloning and attempted expression in E. coli. Although the expression level in E. coli was very low (10 μg of recombinant protein/1 liter of culture), we could detect weak but clear m22G formation activity in the yeast tRNAPhe transcript in the supernatant of crude extract (data not shown). However, the expression level was so low as to make purification of the enzyme difficult. To improve the yield of the recombinant protein, the coding sequence of the N terminus was optimized for translation in E. coli (see supplemental material). This alteration did not cause any changes in amino acid sequence. The expression level was dramatically increased (2 mg of recombinant protein/1 liter of culture), and we could purify the recombinant protein as shown in Fig. 2. It is noteworthy that this purified Trm1 fraction does not contain any nucleic acids. In the case of purification of P. furiosus Trm1, it has been reported that RNA bound tightly to the recombinant protein, and that RNase A treatment is effective for removal of the RNA (18Constantinesco F. Benachenhou N. Motorin Y. Grosjean H. Nucleic Acids Res. 1998; 26: 3753-3761Crossref PubMed Scopus (28) Google Scholar). We also observed contamination by RNA in the A. aeolicus Trm1 fraction during the purification steps; however, we could separate the protein and RNA by repeated ion-exchange chromatography (see supplemental material). The enzyme assay revealed that the purified Trm1 protein has strong tRNA methyltransferase activity (data not shown). Although we could show the tRNA methyltransferase activity of the recombinant Trm1 protein, an important question remained, namely whether there was expression of the putative trm1 gene in living A. aeolicus cells. To analyze the expression of the Trm1 protein in A. aeolicus cells, we prepared an anti-A. aeolicus Trm1 polyclonal antibody and performed Western blotting analysis (Fig. 3A). As shown in Fig. 3A, a single band corresponding to the Trm1 protein was clearly detected, demonstrating that the trm1 gene is really expressed in living A. aeolicus cells. Furthermore, we examined whether m2G and m22G formation activity could be observed in the cell extract. In this experiment, we used an E. coli tRNA mixture as the substrate. Because the E. coli tRNA mixture is already modified by tRNA modification enzymes of E. coli cells, limited methyltransferase activities of the A. aeolicus extract could be detected. The E. coli tRNA mixture, [14C]AdoMet, and A. aeolicus cell extract were incubated at 55 °C overnight, and 14C-methylated nucleotides were analyzed by two-dimensional TLC (Fig. 3B). As shown in Fig. 3B, 14C-pm2G and 14C-pm22G spots could be observed, demonstrating that tRNA (m2G and m22G) methyltransferase activities exist in the A. aeolicus cells. In this experiment, we detected 14C-labeled pm1A, pm6A, pm7G, and pCm, as well as pm2G and pm22G. Of these the pm1A and pm6A were probably derived from tRNA (m1A58) methyltransferase (TrmI) activity (43Droogmans L. Roovers M. Bujnicki J.M. Tricot C. Hartsch T. Stalon V. Grosjean H. Nucleic Acids Res. 2003; 31: 2148-2156Crossref PubMed Scopus (82) Google Scholar), because pm6A can be generated from pm1A nonenzymatically. The pm7G was probably generated by tRNA (m7G46) methyltransferase (TrmB) (28Okamoto H. Watanabe K. Ikeuchi Y. Suzuki T. Endo Y. Hori H. J. Biol. Chem. 2004; 279: 49151-49159Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 30Tomikawa C. Ochi A. Hori H. Proteins. 2008; 71: 1400-1408Crossref PubMed Scopus (15) Google Scholar, 44De Bie L.G. Roovers M. Oudjama Y. Wattiez R. Tricot C. Stalon V. Droogmans L. Bujnicki J.M. J. Bacteriol. 2003; 185: 3238-3243Crossref PubMed Scopus (63) Google Scholar), suggesting that G46 in the E. coli tRNA mixture is not completely modified to m7G46 in E. coli cells. The Cm modification seemed to occur mainly at C32, because C32 in native tRNACys is modified to Cm32 as described below. Based on these experimental results, we concluded that the trm1 gene product is expressed in living A. aeolicus cells, and the m2G and m22G formation activities exist in the cell extract. The substrate tRNA specificities of both eukaryotic and archaeal Trm1 proteins have been reported (9Edqvist J. Grosjean H. Stråby K.B. Nucleic Acids Res. 1992; 20: 6575-6581Crossref PubMed Scopus (37) Google Scholar, 10Edqvist J. Blomqvist K. Stråby K.B. Biochemistry. 1994; 33: 9546-9551Crossref PubMed Scopus (32) Google Scholar, 11Grosjean H. Edqvist J. Stråby K.B. Giegé R. J. Mol. Biol. 1996; 255: 67-85Crossref PubMed Scopus (107) Google Scholar, 12Constantinesco F. Motorin Y. Grosjean H. Nucleic Acids Res. 1999; 27: 1308-1315Crossref PubMed Scopus (42) Google Scholar). Although there are several differences in the tRNA specificity of eukaryote and archaea Trm1 proteins, they all recognize the D-stem and variable region of tRNA. To compare the substrate tRNA specificity of the A. aeolicus Trm1 to those of eukaryotic and archaeal enzymes, we prepared 22 tRNA transcripts (Fig. 4A). Fig. 4B shows the results of the assay at 15-min periods. These results revealed that the tRNA specificity of the A. aeolicus Trm1 is completely different from those of eukaryotic and archaeal Trm1 proteins. For example, H. volcanii tRNAVal (CAC) transcript was well methylated by A. aeolicus Trm1, although the G26 in this tRNA is not modified in living H. volcanii cells (45Gupta R. J. Biol. Chem. 1984; 259: 9461-9471Abstract Full Text PDF PubMed Google Scholar). Furthermore, to our surprise, A. aeolicus tRNATyr transcript was well methylated; the nucleotide at position 26 in this tRNA is not G but A. Moreover, we found that more than two methyl groups were incorporated into the A. aeolicus tRNACys transcript by the time course experiment (data not shown). We checked the template DNA sequences for in vitro transcription and repeated the time course experiments. However, we could not find any mistakes in these experiments. Thus, we found an unexpected tRNA specificity of the A. aeolicus Trm1; however, we could not rationally explain the mechanism through these experiments. A. aeolicus tRNATyr transcript was well methylated, although this tRNA contains A26 instead of G26. This result suggested that A. aeolicus Trm1 methylates a nucleotide(s) at another position in addition to the G26. Initially, we suspected methylation of G10 in the D-stem. However, the A. aeolicus tRNAGln transcript was not methylated at all, and this tRNA has G10 and A26 (Fig. 4). Thus, the additional modification site(s) did not seem to be G10. Furthermore, comparison of substrate tRNA sequences did not throw up a key sequence that enabled us to solve this puzzle. These experimental results prompted us