Aquifex aeolicus tRNA (Gm18) Methyltransferase Has Unique Substrate Specificity
tRNA RECOGNITION MECHANISM OF THE ENZYME*
Hiroyuki Hori
,
Susumu Kubota,
Kazunori Watanabe,
Jong-Myong Kim
,
Tomio Ogasawara,
Tatsuya Sawasaki and
Yaeta Endo
From the
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan
Received for publication, December 10, 2002
, and in revised form, March 27, 2003.
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ABSTRACT
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Transfer RNA (guanosine-2')-methyltransferase (Gm-methylase) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to 2'-OH of G18 in the D-loop of tRNA. Based on their mode of tRNA recognition, Gm-methylases can be divided into the following two types: type I having broad specificity toward the substrate tRNA, and type II that methylates only limited tRNA species. Protein synthesized by in vitro cell-free translation revealed that Gm-methylase encoded in the Aquifex aeolicus genome is a novel type II enzyme. Experiments with chimeric tRNAs and mini- and micro-helix RNAs showed that the recognition region of this enzyme is included within the D-arm structure of tRNALeu and that a bulge is essentially required. Variants of tRNALeu, tRNASer, and tRNAPhe revealed that a combination of certain base pairs in the D-stem is strongly recognized by the enzyme, that 4 bp in the D-stem enhance methyl acceptance activity, and that the Py16Py17G18G19 sequence is important for efficient methyl transfer. The methyl acceptance activities of all the A. aeolicus tRNA genes, which can be classified into 14 categories on the basis of their D-arm structure, were tested. The results clearly showed that the substrate recognition mechanism elucidated by the variant experiments was applicable to their native substrates.
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INTRODUCTION
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Among the large numbers of modified nucleosides found in tRNAs (13), 2'-O-methylguanosine at position 18 (Gm18) of tRNA is generated by tRNA (guanosine-2')-methyltransferase (Gm-methylase,1 EC 2.1.1.34
[EC]
). This enzyme catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to 2'-OH of the ribose ring of G18 in the D-loop of tRNA (4, 5). G18, one of the highly conserved residues located in the so-called three-dimensional core of tRNA (6, 7), is responsible for the formation of the L-shaped three-dimensional structure by D-loop/T-loop interaction through the tertiary base pairs G18-
55 and G19-C56 (8, 9). Because 2'-O-methylation of ribose stabilizes its C3'-endo form (10), conformational rigidity of the local structure of RNA derived from the G18 modification may affect RNA-RNA and/or RNA-protein interaction. Although Gm18 is found in tRNAs from all three domains, prokaryotes, eukaryotes, and archaea, and plant organella (11), only the enzyme isolated from Thermus thermophilus has been purified (5). Recently, however, the corresponding genes from Escherichia coli (12), Saccharomyces cerevisiae (13), and T. thermophilus (14) have been identified.
Substrate tRNA recognition is one of the important features of tRNA modification enzymes. Although in the last few years some enzymes having multisite specificity have been found (1518), in general, tRNA modification enzymes have strict substrate specificity and act only at one single site (19, 20). Recent genome-wide research and in vitro transcription techniques have accelerated the study of RNA recognition mechanisms (2130). Furthermore, three-dimensional structures of some enzymes have been also reported (3134). In addition, genetic studies and microinjection techniques have enabled the in vivo tRNA recognition mechanisms of several tRNA modification enzymes to be elucidated (3541).
Based on their tRNA recognition, Gm-methylases can be divided into two types. Type I, which includes the enzyme of T. thermophilus, possesses broad specificity toward the substrate tRNA (42), so that all T. thermophilus tRNAs have Gm18 under their optimal temperature (43, 44). Type I enzymes occur in thermophiles. In the case of type II, which includes the enzymes of E. coli (12) and S. cerevisiae (13), only limited tRNA species have the Gm18 residue (43). Almost all type II enzymes occur in mesophiles. In a previous report (42), we described the recognition sites in tRNA of type I Gm-methylase. The type I enzyme recognizes common structures of tRNA; the conserved G18G19 sequence and D-arm are essentially required, and U8, purine 15 (Pu15), pyrimidine 17 (Py17), Pu26, G46, U54, U55, and C56 are important for efficient methyl acceptance activity. On the other hand, the substrate recognition mechanism of type II enzymes is still unknown. How do type II enzymes recognize specific tRNA species? The purpose of this paper is to elucidate this and to clarify the difference(s) between the recognition mechanisms of type I and type II enzymes.
We report that the genome of Aquifex aeolicus, a hyper-thermophile eubacterium, encodes a novel type II Gm-methylase gene. A. aeolicus, which was isolated from a hot spring in Yellowstone National Park, can grow at nearly 95 °C (45). The 16 S rRNA gene of A. aeolicus has been analyzed from the perspective of molecular evolution, and it was suggested that this bacterium is the earliest diverging eubacterium (46). Then in 1998, the complete genome sequence was determined (47). Study of A. aeolicus Gm-methylase will thus contribute to clarifying the origin of RNA modification.
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EXPERIMENTAL PROCEDURES
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Materials[methyl-14C]AdoMet (5560 Ci/mol) was purchased from Amersham Biosciences. DNA oligomers were bought from Invitrogen, and DNA-modifying enzymes and human placenta RNase inhibitor were from Takara Bio. T7 RNA polymerase expression system (E. coli BL21/pAR1219) was kindly provided by Dr. F. W. Studier (Brookhaven National Laboratory) (48). T7 RNA polymerase was purified by the method of Grodberg and Dunn (49). Other chemical reagents were of analytical grade.
Transfer RNAs from Native SourcesPurified yeast tRNAPhe (GmAA) was kindly supplied by Dr. C. Takemoto (RIKEN Genome Science Center) and purified E. coli
(CAU) by Dr. T. Suzuki (Tokyo University).
Preparation of tRNA TranscriptsSynthesized E. coli tRNASer (CGA),
(GUA), and tRNALeu (CAG) genes with T7 promoter were kindly provided by Dr. H. Himeno (Hirosaki University). The other tRNA genes with the T7 promoter were constructed by synthetic oligomers as reported previously (42). Readthrough transcription was carried out, and transcripts were purified by Qiagen Tip20 column chromatography. The purities of the transcripts were analyzed by 10% polyacrylamide gel (7 M urea) electrophoresis. Because T7 RNA polymerase has the activity to add extra residues to the 3' end, the amount of the by-product derived from this reaction is
50% of the correct length of transcript in the case of yeast tRNAPhe. The by-product was purified by 15% PAGE (7 M urea). When the methyl acceptance activity of the by-product was compared with that of the correct length of transcript, the two activities were found to coincide (data not shown), confirming that addition of residues to the 3' end does not affect the activity of tRNA (Gm18) methyltransferase. We therefore used run-off transcripts purified on a Qiagen column.
Synthesis, Purification, and Assay of Gm-methylaseA. aeolicus Gm-methylase was synthesized by the method reported previously (50). The sample was heated at 65 °C for 30 min and then centrifuged. The supernatant fraction was passed through a DE52 column in the presence of 100 mM KCl. After the sample was centrifuged, the supernatant was frozen by liquid nitrogen and stored at 80 °C. T. thermophilus recombinant Gm-methylase was purified as described previously (14). The quantity of protein was measured with a Bio-Rad protein assay kit using bovine serum albumin as the standard. The standard assay was carried out with 0.38 µM the enzyme, 11 µM E. coli tRNALeu transcript, and 25 µM [14C]AdoMet in 30 µl of a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2,6mM 2-mercaptoethanol, and 50 mM KCl at 60 °C for 10 min.
Kinetic Parameter MeasurementThe apparent kinetic parameters Km and Vmax were determined by a Lineweaver-Burk plot of the reaction with [3H]methyl-AdoMet. The standard reaction was carried out with 0.1 µM purified enzyme, 50 µM [3H]methyl-AdoMet diluted with cold AdoMet, and the appropriate substrate RNA in 30 µl of a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 6 mM 2-mercaptoethanol, and 50 mM KCl at 60 °C for 10 min. The ranges of tRNA concentrations and incubation times were changed according to the tRNA species. In the case of the relatively good substrate (E. coli tRNALeu (CAG), A. aeolicus tRNAPro (GGG), and so on), RNA concentrations were basically 0, 0.2, 0.25, 0.33, 0.5, 1, 2, 3, 5, or 7.5 µM. In the case of the relatively poor substrate (A. aeolicus tRNAAsp (GUC), tRNAArg (ACG), and so on), RNA concentrations in the reaction mixture were 0, 1, 3, 5, 7.5, 10, 15, or 20 µM. In the latter case, incubation time was changed to 20 or 30 min. When 0.1 µM the enzyme and 7.5 µM tRNALeu were used,
10% of tRNA was methylated for 10 min. The linearity of the reaction could be observed for 30 min. When incorporation of the [3H]methyl group was below 500 dpm, [14C]methyl group incorporation was measured using a gel electrophoresis-imaging analyzer system. A mixture of 0.38 µM purified enzyme, 25 µM [14C]AdoMet, and the appropriate substrate RNA in 30 µl of buffer (50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 6 mM 2-mercaptoethanol, and 50 mM KCl) was incubated at 60 °C for 1 h. RNA concentrations were basically 0, 10, 15, 20, 30, or 40 µM. In this case, the turnover of the enzyme-tRNA complex was very slow. We monitored the reaction for 24 h to check the linear increase of the methyl group incorporation into the tRNA. Since the linear reaction could be observed for 6 h, we confirmed the turnover of the enzyme-tRNA complex. The reaction mixture (5 µl) was then loaded onto a 10% PAGE (7 M urea). The gel was stained with methylene blue and dried. [14C]Methyl group incorporations were measured with a Fuji Photo Film BAS2000 imaging analyzer.
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RESULTS
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A. aeolicus spoU Gene Product Is a Type II Gm-methylaseA. aeolicus putative spoU (trmH, Gm-methylase gene) was retrieved from the NCBI data base by a BLAST search (13, 14). The putative gene was encoded on a DNA fragment (accession number AE000479
[GenBank]
). As shown in Fig. 1A, this gene shares considerable homology with T. thermophilus trmH, a typical type I Gm-methylase gene. Three conserved motifs (motifs 13) were also found. These motifs, which are conserved among RNA ribose 2'-O-methyltransferases, are thought to be involved in methyl transfer activity (51). In a recent study (14), we compared the amino acid sequence of T. thermophilus HB8 Gm-methylase with sequences of previously identified or putative genes for RNA ribose 2'-O-methyltransferases. Several amino acids residues were found to be conserved exclusively among eubacterial Gm-methylases. For example, general motif 1 has been reported as an "N-G- -R" sequence; however, the conserved Gly residue in the motif is replaced by Ser residue in eubacterial enzymes. Therefore, we expected this gene product to be Gm-methylase. However, since we could not judge from the amino acid sequence whether or not it is a type I enzyme, we cloned the DNA fragment by PCR and tested the expression of the gene product with a pET-30a E. coli expression system. Unfortunately, this strategy was not successful because of severe proteolysis (data not shown).

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FIG. 1. Analysis of putative spoU gene product. A, comparison of amino acid sequence of T. thermophilus Gm-methylase (T. th HB8) and A. aeolicus putative spoU gene product (AE000749). Asterisks indicate the same amino acid residues; conserved motifs (motifs 13) are boxed. B, synthesis of A. aeolicus putative spoU gene product by wheat germ in vitro cell-free translation system. The sample was analyzed by 12.5% SDS-PAGE and autoradiography. The synthesized protein is shown by an arrow. C, 15% SDS-PAGE analysis of spoU gene product. The partially purified protein (1 µg) was analyzed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. Standard markers were Bio-Rad prestained protein markers (1 µg each). D, substrate tRNA specificity of spoU gene product. The methylation reaction was carried out with [14C]AdoMet, and tRNAs (0.04 A260 unit) were analyzed by 10% PAGE (left, methylene blue (MB) staining) and imaging analyzer (right, autoradiogram (IP)). Lane 1, native E. coli (CAU); lane 2, yeast tRNAPhe (GAA) transcript; lane 3, E. coli tRNALeu (CAG) transcript; lane 4, E. coli tRNASer (CGA) transcript.
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We next tried expressing the gene using a wheat germ in vitro cell-free translation system (50). Fig. 1B shows the time course of the synthesis of the target protein with [14C]Leu. As shown in the figure, this time no proteolysis was observed. We synthesized the protein with cold amino acids and partially purified it by heat treatment and DE52 column chromatography. We attempted to purify a sample by S-adenosyl-L-homo-cysteine affinity column chromatography (5, 42), but the enzyme bound to the column tightly and was difficult to recover in a fully active form. Therefore, we decided to use the partially purified enzyme, which had a purity of more than 90% (Fig. 1C). In this fraction, the activities of nucleases and the other RNA methyltransferases were not observed (Fig. 1D). Large scale synthesis (15 ml) provided
300 µg of the protein. This cell-free translation system is a very powerful tool for the expression of heterogeneous proteins (50), and it enabled us to obtain sufficient A. aeolicus Gm-methylase for the biochemical investigations carried out in this study.
First, we analyzed the initial velocities of methyl transfer to various native tRNAs and tRNA transcripts from [14C]AdoMet. Measurements were done at 60 °C to stabilize the L-shaped structure of the transcripts. To compare substrate specificities, T. thermophilus HB8 Gm-methylase, a typical type I enzyme, was used. The results are summarized in Table I. In the table, the 100% values were the initial velocity of the methyl group incorporation into 11 µM tRNALeu for 10 min at 60 °C. The amounts of T. thermophilus and A. aeolicus enzymes in the reaction mixture were 150 and 80 ng, respectively. Because the initial velocities of T. thermophilus enzyme for native yeast tRNAPhe and E. coli
were very fast, we calculated these velocities by the other assay, in which 20 ng of the T. thermophilus enzyme was used. Unexpectedly, A. aeolicus Gm-methylase methylated specific tRNAs efficiently; for example, E. coli tRNALeu (CAG) transcript was a good substrate, whereas methyl transfer to some tRNAs such as E. coli native
(CAU) was not detected by the conventional filter assay (Table I). We further analyzed methyl transfer to tRNAs by gel electrophoresis and an imaging analyzer (see Fig. 1D). In this experiment, long term incubation was carried out with excess enzyme (see "Experimental Procedures"). As a result, very slow methyl transfer to the tRNASer (UGA) transcript could be observed (Fig. 1D, lane 4). The initial velocity of the methylation was calculated to be 0.2% of tRNALeu (CAG). The kinetic parameters are given in Table II. However, we could not detect methyl transfer to native E. coli
(CAU) (Fig. 1D, lane 1) nor to tRNATyr (GUA) and tRNASer (GCU) transcripts, despite using imaging analyzer. Their relative initial velocities are estimated to be below 0.05% that of the tRNALeu (CAG) transcript. In contrast, the T. thermophilus enzyme clearly methylated all of the tRNAs tested. Thus, the A. aeolicus spoU gene product is a type II Gm-methylase, even though A. aeolicus is a hyper-thermophile. This is the first reported finding of type II Gm-methylase from a thermophile. The substrate specificity of A. aeolicus Gm-methylase is clearly different from those of other type II enzymes because E. coli tRNASer (GCU) and tRNATyr (GUA) are methylated in the E. coli cell, whereas yeast tRNAPhe (GmAA) is not methylated in the yeast cell. Furthermore, the difference in methyl transfer efficiency between the native yeast tRNAPhe (GmAA) and its transcript suggests that several modified nucleotides in the native tRNA support the methyl transfer reaction. We determined the methylation target site by the method reported previously (14), and we confirmed Gm18 formation. We could not find methyl transfer to any other site (data not shown). Consequently, we renamed A. aeolicus spoU (AE000479
[GenBank]
) as trmH in accordance with the nomenclature established for the gene of E. coli Gm18-methylase (12).

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FIG. 2. Cloverleaf structures and kinetic parameters of E. coli and chimeric tRNAs. A, E. coli tRNALeu (CAG); B, E. coli tRNASer (CGA). Residues of E. coli tRNASer (CGA) are shown by shadowed letters. C and D, two chimeric tRNAs were made by shuffling of D-arm regions. E, measured kinetic parameters. Unfilled and filled circles show [3H]methyl group incorporation into E. coli tRNALeu (CAG) and chimera D, respectively.
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A. aeolicus Gm-methylase Recognizes D-arm Structure of tRNATo clarify the recognition region in the tRNA structure, we constructed two chimeric tRNA transcripts (Fig. 2). In this paper, nucleotide positions are numbered according to Sprinzl et al. (43): conserved Pu26 is the 27th nucleotide from 5' end due to the insertion of one nucleotide into the D-loop. As noted above, the E. coli tRNALeu (CAG) transcript (Fig. 2A) is a good substrate, whereas the E. coli tRNASer (UGA) transcript (Fig. 2B) is a very poor one. We exchanged their D-arm structures and made two chimera transcripts as shown in Fig. 2, C and D. The kinetic parameter measurements are shown in Fig. 2E and Table II. As expected, tRNASer (UGA) possessing the D-arm of tRNALeu (CAG) (Chimera D, Fig. 2D) was a good substrate for A. aeolicus Gm-methylase, whereas tRNALeu (CAG) possessing the D-arm of tRNASer (UGA) (Chimera C, Fig. 2C) was a very poor one. We measured the melting profiles of chimera transcripts to analyze the interaction of their T- and D-arms. The melting profile of chimera C transcript was very similar to tRNALeu, and the melting temperature was determined as 62 °C in the buffer (50 mM Tris-HCl (pH 7.6), 5 mM MgCl2, and 100 mM NaCl) (data not shown). This experimental result suggested that chimera C transcript formed the tertiary base pairs between T- and D-arms. The kinetic parameters (Fig. 2E) clearly show that the essential recognition sites are located within D-arm structure of the tRNA.
Experiments were next carried out with micro- and mini-helix RNAs (Fig. 3). A micro-helix corresponding to the D-arm structure (from U8 to G26, see Fig. 2) of tRNALeu (CAG) (Fig. 3A) was not methylated at all. Because measurement of enzyme activity for the micro- and mini-helices was done at 50 °C, we also examined this micro-helix at 25 and 37 °C to stabilize the D-arm structure. However, methyl transfer to the micro-helix was still not observed. Because the enzyme activity at 25 °C was only 1/20 that at 60 °C (data not shown), it was difficult to detect the methyl transfer to micro- or mini-helices. Therefore, we added artificial stem structures to the D-arm of tRNALeu (CAG) as shown in Fig. 3, B and C. These fragments were methylated, although the initial velocities were very slow. These findings clearly showed that the essential recognition sites were included in the D-arm structure of tRNALeu (CAG) and that the added sequences themselves were not recognized by the enzyme. When the bulge structure corresponding to U8, G9, and G26 was deleted (Fig. 3D) or the artificial stem structure was disrupted (Fig. 3E), methyl acceptance activity was completely lost. Thus, this bulge structure is an essential requirement for substrate recognition by A. aeolicus Gm-methylase. However, as demonstrated in the next section, the nucleosides themselves (U8, G9, and G26) are not essentially required (see Fig. 4), suggesting that the ribose-phosphate backbone structure formed by this bulge is required. These micro- and mini-helix experimental results with A. aeolicus Gm-methylase (type II) are in line with that of short fragment experiments with T. thermophilus Gm-methylase (type I); T. thermophilus Gm-methylase methylated a half-fragment of yeast tRNAPhe (GmAA) (52) but did not methylate synthesized RNA (19-mer) corresponding to the D-arm extending from U8 to G26 (42). Modified nucleosides in the half-fragment (m2G10 and m22G26) seemed to affect the formation of the ribose-phosphate backbone structure around the D-arm. Mini-helix experiments clearly showed that tertiary base pairs are not essentially required for substrate recognition by A. aeolicus Gm-methylase.

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FIG. 3. Micro-helix and mini-helices used for methyl transfer assays. The results are shown in the lower panel. The initial velocity for the full-length tRNALeu (CAG) transcript at 50 °C is expressed as 100%. Not methylated means that the relative initial velocity was less than 0.5%.
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FIG. 4. Mutations introduced into the recognition sites of type I Gm-methylase in tRNALeu (CAG). Arrows indicate the site and the substituted residue. Substitution of G10C11G12 by A10A11A12 is shown by a box. The results are summarized in the right panel. Not detectable means that no methyl transfer was observed using imaging analyzer system, in which the limit of methyl transfer detection was 0.02% of the initial velocity for the wild-type transcript.
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Recognition Sites of A. aeolicus Gm-methylase (Type II) Are the Sum of Those of the T. thermophilus Enzyme (Type I) and Additional Site(s)As described above, the recognition sites of A. aeolicus Gm-methylase are included within the D-arm structure of tRNALeu (CAG). In order to clarify whether the recognition sites of this type II Gm-methylase are different from those of the type I T. thermophilus enzyme, we introduced point mutations into the recognition sites of the type I enzyme in tRNALeu (CAG) (Fig. 4). One variant with a disrupted D-stem structure was also obtained (Fig. 4). Although the kinetic parameters of methyl transfer to these variants differed slightly between the type I and type II enzymes, the essential and important sites for the two enzymes completely coincided (Fig. 4). For example, G18G19 sequence and D-stem structures were essentially required, whereas the U8, Py17, and Pu26 sequences were important for efficient methyl transfer. These results strongly suggested that the recognition sites of A. aeolicus Gm-methylase are basically in common with those of the T. thermophilus enzyme but that an additional recognition site(s) is required for the A. aeolicus enzyme.
Additional Requirements Are a Combination of D-stem Base Pairs and Py16 To determine the additional requirement(s), we compared the D-arm structures of the tested tRNAs from good substrate tRNAs to those not methylated. However, we could not find any specific sequence or secondary structure among them. Therefore, we introduced mutations systematically; for example, the D-loop size, the location of the GG sequence in the D-loop, the D-stem size, and the D-stem sequence were changed (Fig. 5). Among these variants, we found one clue. When the C11-G24 base pair was changed to G11-C24, methyl acceptance activity was completely lost; however, when the adjacent base pair G12-C23 was additionally substituted by C12-G23, methyl acceptance activity was partially recovered (Fig. 5). The same recovery of methyl acceptance activity was also observed with the double substitution of C10-G25 and G11-C24 base pairs (Fig. 5). Thus, one of the additional requirements for the recognition is a combination of certain base pairs in the D-stem sequence. Furthermore, since the variant U11-A24 base pair had methyl acceptance activity (Fig. 5), the sequences themselves are not essentially required, suggesting that the ribose phosphate backbone derived from combination of particular D-stem base pairs is important for the recognition by A. aeolicus Gm-methylase. Among the base pairs in the D-stem, it is concluded that Py11-Pu24 plays a key role in methyl acceptance activity.

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FIG. 5. Systematic mutations introduced into tRNALeu (CAG). Arrows shows the site and substituted residue. Because this tRNA has one extra nucleotide in the -region, U20a and A20b exist. In the cases of base pairs in the D-stem, two nucleotides were substituted. Essential residues (G18 and G19) and an important base pair (C11-G24) are shown by shadowed letters. The results are summarized in the right panel. Not detectable means that no methyl transfer was observed by using imaging analyzer system, in which the limit of methyl transfer detection was 0.02% of the initial velocity for the wild-type transcript.
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The above findings prompted us to construct variants of E. coli tRNASer (UGA) (Fig. 6), which is a very poor substrate for A. aeolicus Gm-methylase (Table I). We changed the C12-G23 base pair of E. coli tRNASer (UGA) to G12-C23 so that the resultant variant had the same D-stem sequence as E. coli tRNALeu (CAG). As expected, a marked alteration in the methyl acceptance activity was observed (Fig. 6). Thus, it was confirmed that a combination of the recovery base pairs in the D-stem base pairs is very important for substrate recognition by A. aeolicus Gm-methylase.
The low methyl acceptance activity of this tRNASer (UGA) variant suggested the existence of another requirement, which is not essential but is important for efficient methyl transfer. To ascertain this, we compared the D-arm of tRNASer (UGA) with that of tRNALeu (CAG) and found a difference at position 16; tRNASer (UGA) lacks a nucleotide at position 16. We constructed two position 16 variants of tRNASer (UGA) as shown in Fig. 6; in one we simply added an extra C at position 16, whereas the other was a double mutation. Addition of C16 did not have any effect on methyl acceptance activity, but the double mutation resulted in strong methyl acceptance (Fig. 6). Thus, the presence of Py16 enhanced methyl acceptance activity, although it was not essentially required. In view of this, we concluded that another preferred requirement for efficient methyl transfer is a combination of certain D-stem base pairs and the Py16Py17G18G19 sequence in the D-loop.
D-stem Structure with 4 bp Enhances Methyl Acceptance ActivityWe have shown that in the case of 3 bp in the D-stem, a combination of G10-C25, C11-G24, and G12-C23 in tRNALeu (CAG) provided the best structure for methyl acceptance activity among the various sequence variations tested. However, the yeast tRNAPhe (GAA) transcript had considerable methyl acceptance activity (Table I), even though its D-stem consists of G10-C25, C11-G24, U12-A23, and C13-G22 (Fig. 7); Pu10-Py25 is conserved in almost all tRNAs, and Py11-Pu24 is conserved in all elongator tRNAs, but Pu12-Py23 is very rare. It appeared difficult to apply the findings obtained from a 3-bp stem structure to the case of a 4-bp stem. How then does A. aeolicus Gm-methylase recognize the D-stem of tRNAPhe (GAA)? Among our variants, we noticed that a D-stem structure with 4 bp enhanced methyl acceptance activity; the A22C variant of tRNALeu (CAG), whose D-stem is composed of 4 bp because the alternative G13-C22 base pair is newly formed, had strong methyl acceptance activity (Fig. 5). Based on this observation, we constructed mutants of yeast tRNAPhe (GAA) (Fig. 7). When the C13-G22 base pair was disrupted (C13A and G22A variant in Fig. 7), the methyl acceptance activity dramatically decreased. These results clearly showed that a stem structure with 4 bp enhanced methyl acceptance activity and enabled it to serve as a substrate structure without the G10-C25, C11-G24, and G12-C23 base pair combination found in tRNALeu (CAG). However, this base pair combination still seemed to be important even when there are 4 bp, because E. coli
(CAU) was not methylated at all, although it has a 4-bp stem structure (Table I). Therefore, we constructed a variant in which the D-stem of yeast tRNAPhe (GAA) was substituted by the D-stem of
(CAU) (Fig. 7). Expectedly, this variant was scarcely methylated. Thus, it was confirmed that combination of base pairs in the D-stem is important, even in the case of 4-bp tRNAs.

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FIG. 7. Mutations introduced into yeast tRNAPhe (GAA). To change 4 bp in the D-stem to 3 bp, C13 and G22 substitutions by A were carried out. Arrows show the site and the substituted residue. The D-stem of yeast tRNAPhe (GAA) was also substituted by that of E. coli . The D-stem sequences are boxed and connected by an arrow. The kinetic parameters for these variants are shown below the cloverleaf structure. The relative Vmax/Km values are expressed with respect to that of the wild type, which is taken as 1.00.
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Methyl Acceptance Activities of A. aeolicus tRNA TranscriptsIn the experiments described above, we used artificial substrate tRNAs from different sources, for example, E. coli tRNALeu (CAG) and yeast tRNAPhe (GAA). We next examined the methyl acceptance activities of A. aeolicus tRNAs. The A. aeolicus genome encodes 42 tRNA genes without tRNASec and tmRNA genes, and 40 tRNA species were probably transcribed; tRNAAla (UGC) and tRNAIle (GAU) were duplicated at separate sites in the genome (47). Also, judging from its sequence, one tRNAMet (CAU) is probably modified to tRNAIle (k2CAU) (53). As shown in Fig. 8,Fig. 8, these 40 tRNAs can be classified into 14 categories (AN) based on their D-arm structure; the D-loop has variable regions between Pu15 and G18 (the
region) and between G19 and A21 (the
region) in addition to D-stem variations (43). The methyl acceptance activities of the majority were easily estimated by the results from artificial substrates. For example, since the D-arm of A. aeolicus tRNALeu (CAG) is completely the same as that of E. coli tRNALeu (CAG) (category K), we could speculate that tRNALeu (CAG) would be a good substrate. In fact, as shown in Fig. 8,Fig. 8, the A. aeolicus tRNALeu (CAG) transcript was confirmed to be well methylated. However, it was difficult to estimate the methyl acceptance activities of some tRNAs (for example, categories H, I, and J), because of their unique D-arm structures. Therefore, we chose typical tRNAs from all of categories, and their methyl acceptance activities were examined (Fig. 8,Fig. 8). In this experiment, full-length transcripts were used. All the transcripts were biologically active. The methyl acceptance activities by other RNA methyltransferases such as T. thermophilus tRNA (m1A58) methyltransferase and E. coli tRNA (m5U54) methyltransferase were also checked (data not shown). Fig. 8,Fig. 8 shows that the A. aeolicus tRNA transcripts could be divided into four classes on the basis of their methyl acceptance activity. The best substrate was tRNALeu (CAG) (category K), and tRNAs belonging to categories A, B, and C were relatively good. Those in categories E, F, G, H, J, and M, were relatively poor substrates, whereas those belonging to categories D, I, L, and N were very poor. Among all the tRNAs, tRNAPro (GGG) (category B) has a unique D-stem composed of G10-C25, C11-G24, G12-C23, and C13-G22 base pairs. This combination of the D-stem base pairs was expected to serve as a good substrate structure for Aquifex Gm-methylase judging from our results with the artificial tRNA variants, and, in fact, this tRNA had relatively strong methyl acceptance activity. A D-loop sequence containing Pu17 was expected not to be suitable, and it was confirmed that tRNAs possessing Pu17 were not substrates for the Aquifex enzyme (categories D and N). Py16 was expected to be suitable for methyl acceptance activity as compared with Pu16, and this was indeed also confirmed by comparing categories F and G, because the only difference between these two categories is a nucleotide at position 16. Thus, all the features elucidated by artificial tRNA variants were applicable to the Aquifex tRNA transcripts, which are natural substrates for the A. aeolicus enzyme. Furthermore, the results obtained with the Aquifex tRNA transcripts also clarified the importance of the location of the GG sequence in the D-loop. As shown in Fig. 8,Fig. 8, tRNAs possessing the correct G18G19 sequence were efficiently methylated as compared with those having an insertion(s) or deletion(s) in their D-loop, in which the G18G19 sequence corresponds to G17G18 or G19G20 by direct numbering from 5' end, suggesting that the location of the GG sequence in the D-loop is one of the important factors for the efficient methyl transfer. Finally, kinetic parameter analysis revealed that the combination of D-stem base pairs mainly affects Km, whereas the location of the GG sequence influences both Km and Vmax.

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FIG. 8. Classification of A. aeolicus tRNA transcripts. Forty two tRNA genes encoded in the A. aeolicus genome are classified into 14 categories (AN) based on their D-arm sequences. Asterisks indicate that these tRNA genes (Ala (UGC) and Ile (GAU)) are duplicated in the genome. A double asterisk means that this tRNA (Met (CAU)) is probably modified to tRNAIle (k2CAU). The relative Vmax/Km values are expressed with respect to that of tRNALeu (CAG), which is taken as 1.00.
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FIG. 8. Classification of A. aeolicus tRNA transcripts. Forty two tRNA genes encoded in the A. aeolicus genome are classified into 14 categories (AN) based on their D-arm sequences. Asterisks indicate that these tRNA genes (Ala (UGC) and Ile (GAU)) are duplicated in the genome. A double asterisk means that this tRNA (Met (CAU)) is probably modified to tRNAIle (k2CAU). The relative Vmax/Km values are expressed with respect to that of tRNALeu (CAG), which is taken as 1.00.
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DISCUSSION
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We have shown that the genome of A. aeolicus, a hyper-thermophile eubacterium, encodes a novel type II Gm-methylase gene. The gene product has methyl transfer activity toward a limited number of tRNA species. Biochemical experiments clearly proved that the recognition sites of A. aeolicus Gm-methylase (type II) are basically in common with those of T. thermophilus Gm-methylase (type I). The most important difference between the A. aeolicus and T. thermophilus enzymes is the strictness with which they recognize D-stem sequences (i.e. combinations of particular base pairs in the D-stem). We further elucidated that the A. aeolicus enzyme requires Py16 in the D-loop sequence as well as the correct location of the GG sequence for efficient methyl transfer. These findings suggest one general rule for Gm-methylases: the location of the target site (2'-OH of G18 ribose) for these enzymes (type I and type II) is decided by the distance and angle from the ribose phosphate backbone of the D-stem. Fig. 9 shows the locations of G18 and the D-stem in the three-dimensional structure of yeast tRNAPhe (GmAA) (8, 9), which is methylated by both T. thermophilus Gm-methylase (type I) and the A. aeolicus enzyme (type II). G18, nucleotides in the D-stem, and U8, U9, and G26 are shown in red, yellow, and green, respectively. All of the phosphorus atoms are indicated in blue. In our previous paper (42), we proposed a multistep recognition mechanism for type I Gm-methylase; the residues involved in substrate recognition by the type I enzyme are embedded inside the L-shaped tRNA molecule, and disruption of the tertiary structure of the tRNA may be necessary for the enzyme to gain access to these residues. Not only the findings of our present experiments with tRNA variants but also previous data from footprinting (52) and competitive inhibition experiments (42) support this idea. Therefore, the first recognition sites should be located on the tRNA surface before the structure of the tRNA is changed and the methyl transfer is catalyzed. As shown in Fig. 9, 5-phosphate groups C13, G22, A23, G24, C25, and G26 are located on the same side as G18. Therefore, a ribose phosphate backbone may be the first site(s) recognized by the enzyme. Kinetic analysis in this work supports this idea, because the combination of D-stem base pairs was shown to have an effect on Km.

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FIG. 9. Three-dimensional structure of D-arm in yeast tRNAPhe (GmAA). G18 and nucleotides in the D-stem are indicated in red and yellow, respectively. U8, U9, and G26 are indicated in green. Because almost all of U8 and A9 are located behind the D-stem, only the 3'-carbon atom of U8 ribose and the 2-carbon atom of A9 are visible from this side. All of the phosphorus atoms are indicated in blue. This figure was generated by RasMol version 2.6.
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Again, we have shown that A. aeolicus Gm-methylase strictly recognizes the D-stem sequence and requires the presence of Py16 and the correct location of the GG sequence in the D-loop for the efficient methyl transfer. Is this substrate recognition mechanism applicable to other type II enzymes? There are two type II enzymes whose genes have been identified, E. coli trmH (12) and S. cerevisiae trm3 (13). Thus far, the E. coli trmH gene product has not been purified; we have tried to purify it several times, but we have always failed because the E. coli enzyme is very labile (data not shown). Therefore, we must judge the substrate recognition mechanism of this enzyme from patterns of Gm18 modification in native tRNAs (43). By using this approach, the characteristics of A. aeolicus Gm-methylase found in the present work do not appear to explain the Gm18 modification pattern in the E. coli cell (43). However, striking homologies among bacterial Gm-methylases (14) strongly suggest that they do use the same mechanism for tRNA recognition. In terms of the D-arm structure, E. coli enzyme seems to favor a relatively large D-loop. Thus, in the case of E. coli Gm-methylase, the D-loop size and D-stem sequence may play key roles in substrate recognition. On the other hand, the S. cerevisiae Trm3 sequence has a long stretch at the N-terminal region (13); hence, it is difficult to envisage the substrate recognition mechanism of the enzyme from the known characteristics of these bacterial Gm-methylases.
Type II Gm-methylases generally occur in mesophiles. Exceptionally, A. aeolicus is a thermophile having a type II. Although the function of Gm18 modification is still unclear, it probably stabilizes the conformational rigidity around G18. Type I enzymes normally found in thermophiles probably support this stabilization. How then does A. aeolicus stabilize all of its tRNAs at extremely high temperatures (above 85 °C)? Recently, Schimmel and co-workers (54, 55) reported the existence of a novel tRNA-binding protein, Trbp111. In the living Aquifex cell, this protein may have a function to stabilize tRNAs instead of several modified nucleosides. Furthermore, in the present study, all experiments were carried out at below 60 °C to stabilize the L-shaped tRNA structure. In fact, we could not detect methyl transfer activity toward the transcripts at 85 °C (data not shown). This was not due to denaturation of the enzyme, because it did not lose its activity during the 1-h incubation at 85 °C (data not shown). In the living cell, newly transcribed RNAs would be correctly stabilized for recognition(s) by modification enzymes. Probably, some protein factors, polyamines, and/or modified nucleotides are required for Gm18 modification at 85 °C. Although modified nucleotides and polyamines from A. aeolicus have not been investigated yet, Trbp111 may be one of the protein factors. We demonstrated in vitro enzymatic characteristics of A. aeolicus Gm-methylase such as its substrate specificities, kinetic parameters, and recognition mechanism. Our biochemical studies clearly showed that several tRNAs, such as tRNASer, were scarcely methylated by the enzyme. However, these tRNAs may be partially modified under in vivo conditions in the presence of stabilization factors, because these factors have the potential to alter the structure of tRNAs.
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FOOTNOTES
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* This work was supported in part by a Grant-in-aid for Science Research 13680692 from the Ministry of Education, Sports, Science and Technology, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
Present address: RIKEN Genomic Science Center, Yokohama 230-0045, Japan. 
To whom correspondence should be addressed. Tel.: 81-89-927-8548; Fax: 81-89-927-9941; E-mail: hori{at}eng.ehime-u.ac.jp.
1 The abbreviations used are: Gm-methylase, tRNA (guanosine-2')-methyltransferase (tRNA (Gm18) methyltransferase); Pu, purine; Py, pyrimidine; AdoMet, S-adenosyl-L-methionine. 
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ACKNOWLEDGMENTS
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We are grateful to Koichi Sato (Ehime University) for skillful technical assistance in preparing the enzyme. We also thank Dr. Gota Kawai (Chiba Institute of Technology) and Dr. Kimitsuna Watanabe (University of Tokyo) for fruitful discussions.
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