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Genes to Cells (2006) 11, 1353-1365. doi:10.1111/j.1365-2443.2006.01022.x
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The substrate specificity of tRNA (m1G37) methyltransferase (TrmD) from Aquifex aeolicus

Hiroshi Takeda1, Takashi Toyooka1, Yoshiho Ikeuchi2, Shin-ichi Yokobori3, Kan Okadome1, Fuyumi Takano1, Tairo Oshima3,a, Tsutomu Suzuki2, Yaeta Endo1,4,5 and Hiroyuki Hori1,4,*

1 Department of Applied Chemistry, Faculty of Engineering, Ehime University, Bunkyo 3, Matsuyama 790-8577, Japan
2 Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656, Japan
3 Department of Molecular Biology, School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-1, Hachioji 192-0392, Japan
4 Venture Business Laboratory, Ehime University, Bunkyo 3, Matsuyama 790-8577, Japan
5 Cell-free Science and Technology Research Center, Ehime University, Matsuyama, 790-8577, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Transfer RNA (m1G37) methyltransferase (TrmD) catalyzes methyl-transfer from S-adenosyl-L-methionine to the N1 atom of G37 in tRNA. In Escherichia coli cells, TrmD methylates tRNA species possessing a G36G37 sequence. It was previously believed that G36 was the positive determinant of TrmD recognition. In the current study, we demonstrate that TrmD from Aquifex aeolicus methylates tRNA transcripts possessing an A36G37 sequence as well as tRNA transcripts possessing a G36G37 sequence. In contrast, tRNA transcripts possessing pyrimidine36G37 were not methylated at all. These substrate specificities were confirmed by an in vitro kinetic assay using 16 tRNA transcripts. The modified nucleoside and the position in yeast tRNAPhe transcript were confirmed by LC/MS. Furthermore, nine truncated tRNA molecules were tested to clarify the additional recognition site. Unexpectedly, A. aeolicus TrmD protein efficiently methylated the micro helix corresponding to the anti-codon arm. Because the disruption of the anti-codon stem caused the complete loss of the methyl group acceptance activity, the anti-codon stem is essential for the recognition. Moreover, the existence of the D-arm structure inhibited the activity. Recently, it was reported that E. coli TrmD methylates yeast tRNAPhe harboring a sequence A36G37. Thus, recognition of the purine36G37 sequence is probably common to eubacteria TrmD proteins.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
To date, more than 100 modified nucleosides have been identified in various RNA species with the majority of them having been found in tRNAs (McCloskey & Crain 1998; Motorin & Grosjean 1998; Rozenski et al. 1999). All of the modified nucleosides introduced into tRNA are done so by specific tRNA modification enzymes during the post-transcriptional process (Björk 1992, 1995; Garcia & Goodenough-Lashhua 1998). Although some enzymes with multisite specificity have been found (Lecointe et al. 1998; Motorin et al. 1998; Motorin & Grosjean 1999; Pintard et al. 2002; Behm-Ansmant et al. 2003), in general, tRNA modification enzymes have strict substrate specificity and act only at a single site. Several modified nucleosides such as N1-methylguanine at position 37 (m1G37) are present in a subset of tRNA species (Sprinzl et al. 1998). Therefore, the modification enzymes catalyzing the formation of this type of modified nucleoside should be able to distinguish a subset of tRNA species as the substrate. The m1G37 modification is introduced by tRNA (m1G37) methyltransferase [EC 2.1.1.31 [EC] ] (TrmD; the product of the trmD gene) (Byström & Björk 1982; Byström et al. 1983; Hjalmarsson et al. 1983): TrmD catalyzes methyl-transfer from S-adenosyl-L-methionine (AdoMet) to the N1 atom of guanine at position 37 (G37) (Hjalmarsson et al. 1983). In Escherichia coli cells, the m1G37 modification is found in tRNA species, which recognize only codons beginning with C such as those for leucine, proline and arginine. Therefore, in these tRNAs, nucleotides at positions 36 and 37 in their anti-codon loop are conserved as G36G37 (Sprinzl et al. 1998). Thus, it was believed that TrmD recognized G36 as a positive determinant in addition to G37. The recognition mechanism of TrmD for substrate tRNA has been studied in vitro (Holmes et al. 1992, 1995; Redlak et al. 1997) as well as in vivo (Grosjean et al. 1990; Li & Björk 1999).

Recent computational research has reported that bacterial TrmD proteins and some RNA methyltransferases such as SpoU (TrmH) (Persson et al. 1997; CavailIé et al. 1999; Hori et al. 2002, 2003) might share a common evolutionary origin and form a single "SPOUT" (SpoU-TrmD) super-family (Anantharaman et al. 2002). Work in this laboratory over a number of years has helped delineate the properties of tRNA (Gm18) methyltransferase [SpoU (trmH)] (Hori et al. 1989, 1998, 2002, 2003; Matsumoto et al. 1990; Nureki et al. 2004; Watanabe et al. 2005). TrmH catalyzes methyl-transfer from AdoMet to the 2'-OH of ribose of the conserved G18 (Hori et al. 1998). Recently, we determined the crystal structure of T. thermophilus TrmH (Nureki et al. 2004), and three groups independently reported the structures of TrmD proteins from Haemophilus influenzae (Ahn et al. 2003), E. coli (Elkins et al. 2003) and Aquifex aeolicus (Liu et al. 2003). These structural studies reveal that both TrmH and TrmD proteins have a deep trefoil knot structure (Nureki et al. 2002). Thus, structural similarities of these proteins were established.

To investigate the origin of SPOUT proteins, we have studied the RNA modification enzymes from A. aeolicus (Hori et al. 2003), an organism which was isolated from a hot spring in Yellowstone National Park, and which can grow at nearly 95 °C. The 16S rRNA gene of Aquifex has been analyzed from the perspective of molecular evolution, and it has been suggested that this bacterium may be the earliest diverging eubacterium (Burggraf et al. 1992). The complete genome of this organism was determined in 1998 (Deckert et al. 1998). Because the m1G37 modification prevents frameshift errors during protein synthesis on the ribosome (Björk et al. 1989; Hagervall et al. 1990; 1993; Farabaugh & Björk 1999), this modification is essentially required in all living organisms (Björk et al. 2001). In fact, trmD genes have been reported from all of the eubacterial genomes analyzed. In this paper, we demonstrate the substrate tRNA recognition mechanism of A. aeolicus TrmD, which catalyzes the methyl-transfer to G37 not only in a G36G37 sequence but also in an A36G37 sequence. Further, the molecular evolution of this modification is discussed.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Expression and purification of recombinant A. aeolicus TrmD in E. coli

To characterize A. aeolicus TrmD, we amplified a DNA fragment containing the ORF by PCR and expressed it as a recombinant protein in E. coli. The yield of protein from this expression system in the E. coli BL21(DE3) strain was 100 µg per 1 L culture. In order to improve the protein yield, we exchanged the DNA fragment between SacI and XhoI sites for an artificial DNA fragment containing codons preferentially used in E. coli (see Experimental procedures). The resultant plasmid was introduced into E. coli BL21(DE3)-Codonplus-RIL strain. This alteration resulted in a remarkable fivefold improvement in the yield of recombinant protein.

We purified the TrmD protein to homogeneity. The purified protein is shown on a 15% SDS-polyacrylamide gel in Fig. 1.


Figure 1
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  		Figure 1  SDS-polyacrylamide gel electrophoresis analysis of A. aeolicus TrmD. Purified TrmD protein (1 µg) was analyzed by 15% SDS-polyacrylamide gel electrophoresis. The gel was stained with Coomassie brilliant blue. Markers (1 µg each) are prestained protein markers from New England Bio Laboratories.

 
A. aeolicus TrmD catalyzes methyl-transfer to G37 not only in a G36G37 sequence but also in an A36G37 sequence

To investigate the substrate RNA specificity of A. aeolicus TrmD, we analyzed methyl-transfer to various tRNA transcripts (Fig. 2). tRNA transcript (0.3 A260 units) was incubated with 300 ng of the purified TrmD protein (5 pmol of the dimer enzyme) and 38 µM [methyl-14C]-AdoMet for 10 min at 60 °C. The RNA was loaded on to a 10% polyacrylamide gel (7 M urea). The gel was stained with methylene blue (Fig. 3, left) to visualize RNA and dried. The 14C-incorporation into the RNA was monitored using a Fuji Photo Film BAS2000 imaging analyzer (Fig. 3, right). The kinetic parameters measured at 55 °C are given in Table 1. As expected, A. aeolicus TrmD methylated tRNA transcripts possessing a G36G37 sequence in their anti-codon loop, a result consistent with the recognition mechanism of E. coli TrmD (Holmes et al. 1992, 1995; Redlak et al. 1997) (Fig. 3, lanes 1–7, and Table 1). We also tested tRNA transcripts possessing an A36G37 sequence (yeast tRNAPhe (GAA), Haloferax volcanii tRNALeu (CAA), etc.) (see Fig. 2). Surprisingly, methyl-transfer to these tRNAs was observed (Fig. 3, lanes 8–12, and Table 1). The methyl-transfer activity to these tRNAs was considerably stronger compared to that to tRNAs possessing a G36G37 sequence (Fig. 3, lanes 1–7, and Table 1). In Table 1, E. coli tRNALeu, yeast tRNAPhe, H. volcanii tRNATrp and tRNATyr were methylated slower than the other tRNA transcripts. These tRNA transcripts have many A–U pairs and/or mismatch pairs (G–U and A–C pairs) in the stem structure as compared to the other transcripts. Because we measured the kinetic parameters at 55 °C, the stability of the stem structure may influence the methyl acceptance activities of these transcripts. We also tested yeast tRNAAsp (GUC) and H. volcanii tRNAGlu (UUC) possessing a C36G37 sequence, yeast mutant tRNAPhe (GAU) possessing an U36G37 sequence, and E. coli tRNASer (UGA) possessing an A36A37 sequence (Fig. 2). These tRNA species were not methylated at all (Fig. 3, lanes 13–16, and Table 1). From these results, we conclude that A. aeolicus TrmD catalyzes the methyl-transfer to tRNA possessing a purine36G37 sequence.


Figure 2
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  		Figure 2  Partial nucleotide sequence of tRNA transcript used in this paper. Sequences of anti-codon arm of tRNA transcripts used in this paper are shown. They are arranged according to the nucleotide sequence at positions 36 and 37. Particular classification of tRNAs is given in Table 1. The number of each tRNA corresponds to the lane number in Fig. 3. Asterisks show the positions 30 and 40.

 

Figure 3
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  		Figure 3  Polyacrylamide gel (7 M urea) analysis of methyl-transfer activities of A. aeolicus TrmD to various tRNA transcripts. Left. Transcripts visualized with methylene blue staining. Transcripts were incubated with purified TrmD and [methyl-14C]-AdoMet as described in Experimental procedures and then analyzed by 10% polyacrylamide gel (7 M urea) electrophoresis. The gels were stained with methylene blue after electrophoresis. The 16 tRNAs transcripts used in this experiment are classified according to the nucleotides in positions 36 and 37. Each lane number corresponds to the tRNA number in Fig. 2 and Table 1: 1, E. coli tRNALeu (CAG); 2, A. aeolicus tRNALeu (CAG); 3, A. aeolicus tRNAHis (GUG); 4, A. aeolicus tRNAPro (GGG); 5, A. aeolicus tRNAGln (UUG); 6, A. aeolicus tRNAArg (CCG); 7, A. aeolicus tRNAArg (ACG); 8, yeast tRNAPhe (GAA); 9, H. volcanii tRNALeu (CAA); 10, H. volcanii tRNATrp (CCA); 11, H. volcanii tRNATyr (GUA); 12, H. volcanii tRNACys (GCA); 13, yeast tRNAAsp (GUC); 14, H. volcanii tRNAGlu (UUC); 15, yeast tRNAPhe A36U mutant (GAU); 16, E. coli tRNASer (UGA). Right. Autoradiogram of the same gel. Autoradiography of the gel shown in the left panel was performed. Incorporation of 14C-methyl group into the transcripts was monitored with a Fuji Photo Film BAS2000 imaging analyzer.

 

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  		Table 1  Kinetic parameters for various tRNA transcripts
 
Formation of m1G at position 37 in yeast tRNAPhe transcript

In order to check the modified nucleotide, we first performed the 5'-mononucleotide analysis by two-dimensional thin layer chromatography (Keith 1995). Yeast tRNAPhe was methylated with [methyl-14C]-AdoMet, and then completely digested with nuclease P1. The 5'-mononucleotides were separated by two-dimensional TLC and the mobility of the labeled spot was compared to those of cold markers: unlabeled pA, pU, pG, and pC were separated on the same thin-layer plate and marked by UV (254 nm) irradiation. As a result, [14C]-pm1G could be identified as a single labeled spot (data not shown).

To identify the modified nucleoside and the position of the methylated site precisely, we employed LC/MS using ESI/iontrap mass spectrometry (Figs 4 and 5). In these experiments, we used yeast tRNAPhe transcript, because the elution profile of the yeast tRNAPhe fragments by RNase T1 digestion was investigated in our previous studies (Hori et al. 2002; Okamoto et al. 2004). Fig. 4 shows the result of the nucleoside analysis. Yeast tRNAPhe transcript was methylated by the TrmD protein with cold AdoMet at 60 °C for 4 h and then completely digested with nuclease P1 and bacterial alkaline phosphatase. The m1G nucleoside eluted at 25 min (Fig. 4A), and the mass signals of the protonated molecule of m1G nucleoside (m/z = 298), the fragment ion derived from the base moiety (m/z = 166) and the sodium adduct (m/z = 320) were clearly identified (Fig. 4B).


Figure 4
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  		Figure 4  LC/MS analysis of nucleosides derived from methylated tRNAPhe. (A) Chromatograms of the nucleosides derived from methylated tRNAPhe. The upper panel shows the UV chromatogram. The peak of m1G nucleoside is indicated by an arrow. The lower panel shows the mass chromatogram representing the mass range m/z 297.5–298.5 for the methylated guanine. (B) Mass spectra of the m1G in the methylated tRNAPhe transcript. Mass spectra recorded at 25.0 min is shown. The protonated molecule ([M + H]+, m/z 298), and the fragment ion derived from the base moiety (BH2+, m/z 166) are indicated.

 

Figure 5
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  		Figure 5  RNase T1-mapping of methylated tRNAPhe. (A) Chromatograms of oligonucleotides obtained by RNase T1 digestion of methylated tRNAPhe. The upper panel is the UV trace at 254 nm. Peak numbers represent RNA fragments derived from the methylated tRNA: the nucleotide sequence of each fragment is shown in (C). The lower panel shows the mass chromatogram and shows the doubly charged ion of the fragment 14 (AAm1GAUCUGp; m/z = 1312.3). (B) Mass spectra of fragment 14. Mass spectra recorded at 38.0 min is shown. Charge states are indicated in parentheses. (C) Cloverleaf structure of yeast tRNAPhe. Solid lines show the cleavage sites of RNase T1. A broken line between G37 and A38 indicates that this site is protected from RNase T1 by m1G37 modification (shown by an arrow). All fragments eluted in (A) are indicated: the peak numbers and the nucleotide sequences are shown.

 
To clarify the methylation site, the methylated tRNA was digested with RNase T1. The fragments were separated by LC (Fig. 5A), and introduced directly into the mass-spectrometer. When the guanine base is modified to m1G, RNase T1 does not cleave the site. As shown in Fig. 5A,B, the methylated fragment AAm1GAUCUGp (fragment 14 at 37.5 min) can be clearly identified as a doubly charged negative ion (m/z = 1312.3). This fragment corresponds to the nucleotides between positions 35 and 43 in the tRNAPhe transcript (Fig. 5C). It should also be noted that we also detected the unmodified fragment AAGp (fragment 5 at 29 min), indicating incomplete m1G37 modification of the substrate tRNA. The methylation efficiency was estimated to be around 70% from these experiments. All other fragments derived from yeast tRNAPhe were also analyzed (Fig. 5C) and the m1G modification not found in any of them. Based on these experimental results, we concluded that A. aeolicus TrmD protein has a methyl-transfer activity to the N1-atom of G37 in an A36G37 sequence in tRNA.

Methyl group acceptance activities of the truncated RNA molecules and replacements of the anti-codon stem sequence

To search the additional identity elements recognized by A. aeolicus TrmD, we prepared nine truncated RNA molecules of yeast tRNAPhe (Fig. 6). The methyl acceptance activities of these truncated RNA molecules were tested at 37 and 55 °C. Because both experiments showed the same tendency, the results at 55 °C are shown in Fig. 6. Unexpectedly, the micro helix RNA corresponding to the anti-codon arm was efficiently methylated (Fig. 6B). The kinetic study showed that the Km value for this micro helix was very large compared to that for the full-length yeast tRNAPhe (Table 2). However, because Vmax value for this micro helix was increased, the initial velocity of the micro helix was around 80% of the full-length transcript. When the anti-codon stem structure was disrupted, the methyl group acceptance activity was completely lost (Fig. 6C). This result clearly showed that the anti-codon stem structure is one of the additional identity elements. Furthermore, it is also clear that the A. aeolicus TrmD does not require the L-shaped tRNA structure. Moreover, the kinetic study showed that the deletion of the D-arm structure increased the Vmax value (transcript D, E, and G in Table 2). Indeed, these transcripts were methylated faster than the full-length yeast tRNAPhe transcript (Fig. 6 and Table 2). Because the existence of the nucleotides corresponding to positions 19–25 decreased the Vmax value (transcript F in Table 2), this region seems to affect the methyl group acceptance activity. In the case of E. coli TrmD, it has been reported that the deletions of both T- and D-arms decreased the methyl group acceptance activity severely: the initial velocity to the mutant transcript is 1/100 of that of the full-length transcript (Redlak et al. 1997). This transcript corresponds to the transcript E in the current study (Fig. 6 and Table 2). Thus, the effect of the D-stem structure seems to differ between A. aeolicus and E. coli TrmD proteins.


Figure 6
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  		Figure 6  Truncated RNA molecules. (A) The full-length yeast tRNAPhe transcript is shown by the clover-leaf structure. The positions at 36 and 37 are highlighted. (B–J) The truncated RNA molecules are shown. Each RNA molecules were treated with 14C-AdoMet and the TrmD protein and analyzed by the same method shown in Fig. 3. The left panel shows the RNA molecules visualized by methylene blue (MB) staining. The right panel shows the autoradiogram of the same gel.

 

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  		Table 2  Kinetic parameters for truncated tRNAPhe variants (The sequences of the transcripts are shown in Fig. 6)
 
As described above, the micro helix RNA was efficiently methylated by the A. aeolicus TrmD protein and the disruption of the anti-codon stem structure caused the loss of the methyl group acceptance activity. These results clearly showed that the minimal identity elements were contained within the anti-codon arm structure and the anti-codon stem structure is one of the essential elements. We compared the sequences of all methylated RNA molecules (Figs 2 and 6) and found that all of them contained G30-C40 base pair in the anti-codon stem. Therefore, we made three yeast tRNAPhe mutant transcripts, in which the G30-C40 base pair were substituted (Fig. 7). We tested the full-length transcripts; however, the anti-codon arm sequences are highlighted in Fig. 7. The substitution of the G30-C40 base pair by A30-U40 or C30-G40 base pair did not affect the methyl group acceptance activity (Fig. 7B,C). When the G30-C40 base pair was disrupted by introducing A30 and C40 mismatch pair, the initial velocity was decreased through the increase of the Km value (Fig. 7D). These results demonstrated that the G30 and C40 sequences are not the identity element but the base pair itself is required for efficient methylation.


Figure 7
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  		Figure 7  Substitution of the G30-C40 base pair in yeast tRNAPhe transcript. The anti-codon arm sequences of the wild-type yeast tRNAPhe (A) and its mutant transcripts (B–D) are shown. The kinetic parameters are given under the transcript name.

 
Phylogenetic tree analysis

To address the structural differences of eubacterial TrmD proteins, we performed phylogenetic tree analysis. Because the phylogenetic tree in Fig. 8 does not have the root, the tree does not represent the precise evolutionary process followed by TrmD proteins: it simply shows the resemblances of amino acid sequences. This analysis demonstrates that A. aeolicus TrmD is considerably different from the other TrmD proteins of eubacteria. For example, the value of the local bootstrap probability between A. aeolicus and Thermotoga maritima TrmD proteins was calculated as 67, even though T. maritima TrmD is the most similar protein to A. aeolicus TrmD (Fig. 8). As found in Fig. 8, the A. aeolicus TrmD protein was grouped with T. martima and M. capricolum TrmD proteins. In small subunit rRNA based molecular phylogenetic analyses, T. martima and A. aeolicus are placed as the most basal species of eubacteria (Woese et al. 1990; Burggraf et al. 1992). However, the M. capricolum is a member of Firmicutes, so that its phylogenetic position is different from the result of the small subunit rRNA analysis. This result suggests that the evolutionary rate of the M. capricolum TrmD protein may be accelerated. Furthermore, recently, another approach to determine the relative branching order of bacterial divisions has been reported: Griffiths & Gupta (2001) used signature sequences in the Hsp60, Hsp70, CTP synthase and alanyl-tRNA synthetase to determine the phylogenetic placement of Fibrobacter succinogenes. In their work, A. aeolicus was classified in the Cytophaga-Flavobacteria-Bacteroides group based on their signature sequence analyses. Thus, their results suggest that A. aeolicus is not an earliest diverged eubacterium. More recently, the relationship of the species in the Aquificae has been investigated by inserts and deletions (so called "indels") and signature sequences (Griffiths & Gupta 2006).


Figure 8
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  		Figure 8  Phylogenetic tree of eubacterial trmD proteins. The ML tree reconstructed with PROTML in MOLPHY is shown. For each node, the local bootstrap probability estimated by the RELL method (ML analysis)/bootstrap probability of 1000 resamplings (NJ analysis)/bootstrap probability of 100 resamplings (MP analysis) are shown in this order. BP values lower than 50% are not shown (shown with –). For each taxon, the species name and accession number in GENBANK are indicated. The topology of ML tree, NJ tree (not shown), and MP tree (not shown) are not identical.

 
It should also be noted that A. aeolicus TrmD is not derived from archaeal TrmD by the horizontal transfer of the gene. Recently, Christian et al. (2004) have reported that the MJ0883 protein from Methanocaldococcus jannashii is the archaeal counterpart of the bacterial TrmD and the catalytic domain of MJ0883 protein is expected to form a class I fold of the AdoMet-dependent methyltransferases. Thus, archaeal TrmD proteins do not belong to the SPOUT proteins unlike bacterial TrmD proteins.

Distribution of A36 recognition by TrmD

Recently, Brulé et al. (2004) reported that E. coli TrmD catalyzes methyl-transfer to transcripts of yeast tRNAPhe, tRNAAsp A36 mutant and human mitochondria tRNAPro A36 mutant, although the initial velocities of the methyl-transfer to these transcripts were slow compared to the E. coli tRNALeu1 transcript harboring the sequence G36G37. Both our own and their kinetic studies of tRNA transcripts showed that the substrate specificity of A. aeolicus TrmD is rather broad, because A. aeolicus TrmD efficiently methylated all A36 tRNA transcripts as shown in Fig. 3. However, the recognition of A36 is the common feature between E. coli and A. aeolicus TrmD proteins. Furthermore, it has also been reported that E. coli TrmD did not methylate yeast tRNAAsp C36 and U36 mutants (Brulé et al. 2004). E. coli TrmD has this property in common with TrmD of A. aeolicus as demonstrated in this study. Thus, E. coli TrmD recognizes purine36G37 sequence in the same way as A. aeolicus TrmD, suggesting that the purine36G37 recognition by TrmD may be common to the eubacterial domain.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this paper, we demonstrate that A. aeolicus TrmD catalyzes methyl-transfer to G37 in both G36G37 and A36G37 sequences. This specificity is clearly demonstrated in methyl-transfer experiments, which used 16 different tRNA transcripts. Furthermore, the methylation of the N1 atom of G37 in an A36G37 sequence was confirmed by LC/MS. Based on these results, we conclude that A. aeolicus TrmD recognizes a Pu36G37 sequence. The recognition of the Pu36G37 sequence by E. coli TrmD protein has been reported (Brulé et al. 2004) during the course of the current study. Thus, the recognition of the Pu36G37 sequence seems to be a common feature of the bacteria TrmD proteins. In their study, Brulé et al. (2004) reported that human mitochondrial tRNAPro mutant possessing A36G37 sequence was methylated by E. coli TrmD very slowly: the relative Vmax/Km was calculated as 0.6% of that for E. coli tRNALeu. Furthermore, it has been reported that yeast tRNAAsp mutant possessing A36G37 sequence was methylated and its relative Vmax/Km was 8.9% of that for E. coli tRNALeu. As compared to the E. coli TrmD protein, the A. aeolicus TrmD protein efficiently methylated tRNA transcripts possessing an A36G37 sequence (Fig. 3 and Table 1).

In the eubacteria domain, tRNA with an A36G37 sequence is very rare (Sprinzl et al. 1998). In fact, the A. aeolicus genome codes 42 tRNA genes except for tRNASec and tmRNA; however, tRNA with an A36G37 sequence is not found (Deckert et al. 1998). Thus, A. aeolicus TrmD does not need to methylate tRNA species with an A36G37 sequence in the living cell. However, it is noteworthy that several tRNA genes with an A36G37 sequence can be found in eubacterial genomes (Sprinzl et al. 1998). Furthermore, it has been reported that tRNAPhe (GAA) from Mycoplasma sp. (Kid) has an A36m1G37 modification (Kimball et al. 1974). It therefore seems that there is widespread distribution of the A36 specificity of TrmD proteins.

A. aeolicus and E. coli TrmD proteins do not recognize a pyrimidine36G37 sequence. In general, it is difficult to distinguish the structures of purine and pyrimidine precisely by hydrogen bonds from amino acid residues. Therefore, the size and/or hydrophobicity of the purine ring at position 36 should be a positive determinant for bacterial TrmD proteins. A. aeolicus may diverge at the earliest period in eubacterial evolution and the amino acid sequence of A. aeolcius TrmD is considerably different from those of other eubacterial TrmD proteins. Therefore, this determinant may be a remnant derived from the common ancestor of eubacterial TrmD proteins. Furthermore, the experiments using the truncated RNA molecules showed that the RNA recognition mechanism of the A. aeolicus TrmD is considerably different from that of E. coli TrmD: the A. aeolicus TrmD methylated the truncated RNA molecules efficiently and the D-stem structure of the tRNA inhibited the activity. In contrast, it has been reported that E. coli TrmD requires the T- and D-arm structures for the efficient methyl acceptance activity (Holmes et al. 1992; Redlak et al. 1997). These differences may be caused by the differences between the protein folds of A. aeolicus and E. coli TrmD proteins, because Liu et al. (2003) have reported the crystal structure of the A. aeolicus TrmD as a new fold, in which the trefoil knot is not presented.

Recently, we have reported the crystal structure of TrmH (Nureki et al. 2004), which is classified into the SPOUT protein group together with TrmD (Anantharaman et al. 2002). The catalytic domain of the SPOUT proteins is classified as class IV fold, which has a deep trefoil knot structure. We proposed a docking model of TrmH with tRNA complex (Nureki et al. 2004). Our model suggests that the interaction of TrmH and tRNA is quite different from that of TrmD and tRNA, even though the protein folds resemble each other. In order to understand the relationship of these two modification enzymes functionally, structurally and evolutionally, further studies will be necessary.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Materials

[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, USA. DE52 was a product of Whatman, UK. CM-Toyopearl 650M came from Tosoh, Japan. DNA oligomers were bought from Invitrogen, USA, and T7 RNA polymerase was from Toyobo, Japan. Other chemical reagents were of analytical grade.

Construction of TrmD expression system in E. coli

The trmD gene was amplified by PCR from A. aeolicus genomic DNA using the following primers: Nde1trmDF, 5'-CGG GCA TAT GAG TTC TAA TCC CCT CCG CTT CTT CG-3'; BamH1trmDR, 5'-CGG GGG ATC CTT AAA GAA GGT GCT TGT GC-3'. The underlined sequences show the sites of restriction enzymes, NdeI and BamHI, respectively. The amplified DNA fragment was cloned into the pET30a expression vector (Novagen, USA) utilizing NdeI and BamHI sites. In order to increase the yield of the expressed protein, the DNA sequences between SacI and XhoI sites were replaced by an artificial DNA sequence composed of codons optimized for translation in E. coli. Synthetic DNA oligomers (DC1–8) were phosphorylated, annealed, and ligated into the vector described above. DNA oligomers used to introduce this alteration were as follows: DC1, 5'-CCT GTC TGG ACA CCA CAA ACT GAT CGA ACT GTG GAA GCT GTG GCA CAG G-3'; DC2, 5'-ATC GAA AAC ACG GTA AAA AAG CGT CCG GAT CTG ATC CCG AAA GAC CTG A-3'; DC3, 5'-CAG AAC TGG AAA AAG ACA TTC TGA ATA GTA TCC TGT CTG GCA AGT CAT T-3'; DC4, 5'-CAA GGA ATG GCT GAA AGA GCA CAA GCA CCT TCT TTA ATA AC-3'; DC5, 5'-TCG AGT TAT TAA AGA AGG TGC TTG TGC TCT TTC AGC CAT TCC TTG AAT GAC TTG CC-3'; DC6, 5'-AGA CAG GAT ACT ATT CAG AAT GTC TTT TTC CAG TTC TGT CAG GTC TTT CGG-3'; DC7, 5'-GAT CAG ATC CGG ACG CTT TTT TAC CGT GTT TTC GAT CCT GTG CCA C-3'; DC8, 5'-AGC TTC CAC AGT TCG ATC AGT TTG TGG TGT CCA GAC AGG AGC T-3'. The resulting plasmid was named pET30trmD and introduced into E. coli BL21(DE3)-Codonplus-RIL strain (Stratagene, USA) for expression.

Expression and purification of TrmD

The expression of TrmD in E. coli was induced by addition of IPTG to a final concentration of 1 mM. After 4 h, the cells were collected by centrifugation at 6500 x g for 20 min, frozen in liquid nitrogen, and stored at –80 °C until required. The cells (5 g) were suspended in 25 mL of the buffer A (50 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 6 mM 2-mercaptoethanol, 50 mM KCl), and disrupted with an ultrasonic disruptor model UD-200 (Tomy, Japan). The cell debris was removed by centrifugation at 8000 g for 20 min. The supernatant fraction was then heated at 70 °C for 30 min. The denatured proteins were removed by centrifugation at 8000 g for 20 min. The supernatant fraction was applied on to a DE52 column (column volume, 10 mL). The flow-through fractions were collected and dialyzed against buffer B (50 mM HEPES-KOH (pH 6.8), 6 mM 2-mercaptoethanol). The dialyzed sample was loaded on to a CM-Toyoperal 650M column (column volume, 10 mL) and TrmD protein eluted with buffer B containing 200 mM KCl. The eluted sample was dialyzed against the buffer A and concentrated with Centriprep YM-10 centrifugal filter devices (Millipore). Glycerol was added to the purified protein to a final concentration of 50% v/v and stored at –30 °C. Quantification of protein was performed with a Bio-Rad protein assay kit using bovine serum albumin as the standard.

Preparation of tRNA transcripts

The yeast tRNAPhe, E. coli tRNALeu (CAG), tRNASer (UGA) and A. aeolcius tRNA transcripts were prepared as described previously (Hori et al. 2002, 2003). Briefly, yeast tRNAPhe, E. coli tRNALeu (CAG) and tRNASer (UGA) genes with T7 RNA polymerase promoter were amplified from plasmids by PCR and then run-off transcriptions were carried out. The template DNAs for A. aeolcius tRNA transcripts were prepared by annealing of two primers and in vitro DNA polymerase reaction: one primer contains the T7 RNA polymerase promoter sequence. For example, in the case of A. aeolicus tRNALeu (CAG), the following primers were used: AALeu (CAG) F, 5'-TAA TAC GAC TCA CTA TAG CGG GTG TGG CGG AAC TGG CAG ACG CGC CGG ACT CAG GAT CCG-3'; AALeu (CAG) R, 5'-GG TGC GGG TGG CGG GAG TCG AAC CCG CAC GCC CTT GCG GGC ACC GGA TCC TGA GTC CGG-3'. The template DNA was purified by phenol-chloroform extraction, recovered by ethanol precipitation and used for the run-off transcription. The primers for yeast tRNAAsp, tRNAPhe (A36U mutant), and H. volcanii tRNA transcripts were newly synthesized and used for in vitro transcription. All transcripts were purified by 10% polyacrylamide gel (7 M urea) electrophoresis.

Measurements of the enzymatic activities

The standard assay used during the enzyme purification measured the transfer of the methyl group from [methyl-14C]-AdoMet to A. aeolicus tRNAPro(GGG) transcript: 300 ng of the protein, 13.5 µM transcript, and 38 µM [methyl-14C]-AdoMet in 30 µL of buffer C (50 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 6 mM 2-mercaptoethanol, 50 mM KCl) were incubated for 5 min at 60 °C. An aliquot (20 µL) was then used for the conventional filter assay. RNA transcripts were prepared as reported previously (Hori et al. 2003). In order to visualize the methyl-transfer reaction, we used 10% polyacrylamide gel electrophoresis and an imaging analyzer system. Briefly, tRNA (0.3 A260 units) was incubated with 300 ng (5 pmol of dimer enzyme) of the purified TrmD and 38 µM [methyl-14C]-AdoMet for 10 min at 60 °C in 30 µL of buffer C, extracted with phenol, collected by ethanol precipitation, and then loaded on to a 10% polyacrylamide gel (7 M urea). The gel was stained with methylene blue and dried. The 14C-methyl incorporation into tRNAs was monitored with a Fuji Photo Film BAS2000 imaging analyzer. Apparent kinetic parameters, Km and Vmax, were determined by a Lineweaver–Burk plot of the methyl-transfer reaction as measured by the [methyl-3H]-AdoMet by the filter assay: 1 µg of the protein, 38 µM AdoMet, various concentration of the transcript in 50 µL of buffer C (50 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 6 mM 2-mercaptoethanol, 50 mM KCl) were incubated for 5 min at 55 °C. RNA concentrations in the assay were changed to the methyl acceptance activity of the transcript. RNA concentrations in the first measurement were 0.5, 1, 2, 3, 5, 8, and 12 µM. Based on this first measurement, we changed the RNA concentrations. When the estimated Km value was relatively small (< 2 µM), RNA concentrations were changed to be 0.2, 0.3, 0.5, 1, 2, 3, 5, 8, 12, and 15 µM. When the estimated Km value was relatively large (> 2 µM), RNA concentrations were changed to be 0.5, 1, 2, 3, 5, 10, 15, 20 and 30 µM.

Mass spectrometry

Yeast tRNAPhe (50 µg) was methylated with 20 µg enzyme and cold AdoMet for 4 h at 60 °C in 100 µL of buffer C. The RNA was extracted with phenol, recovered by ethanol precipitation, and loaded on to a 10% polyacrylamide gel (7 M urea) electrophoresis. 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. An LCQ ion-trap (IT) mass spectrometer (ThermoFinnigan) equipped with an electrospray ionization (ESI) source and a HP1100 liquid chromatography system (Agilent) was used to analyze the total nucleosides and RNase T1-digested RNA. For nucleoside analysis, the sample (5 µg) was completely digested with nuclease P1 (3 µg) and bacterial alkaline phosphatase (0.1 unit) in 20 µL of reaction mixture containing 50 mM HEPES-KOH (pH 7.5) at 37 °C for 3 h. The hydrolysates were subjected to mass spectrometric analysis as previously described (Hori et al. 2002; Kaneko et al. 2003; Okamoto et al. 2004). For fragment analysis, methylated or unmethylated control tRNA (5 µg each) was digested with RNase T1 (2.5 unit) in 20 µL of reaction mixture containing 25 mM NH4OAc (pH 5.3) at 37 °C for 1 h and subjected to mass spectrometric analysis. Oligo-nucleotides produced by RNase T1 digestion were analyzed in negative ion form by LC/MS as previously described (Hori et al. 2002; Kaneko et al. 2003; Okamoto et al. 2004).

Amino acid sequence alignment and phylogenetic analyses

The amino acid sequences of eubacterial trmD proteins were retrieved from GENBANK. The amino acid sequences were aligned with CLUSTAL X (Thompson et al. 1997) using the default settings. The well aligned regions (163 sites) were selected with GBLOCKS (Castresana 2000).

For Maximum likelihood (ML) analysis (no site-by-site rate variations), the ML pair-wise distances of all combinations were estimated using the PROTML routine in MOLPHY 2.3b (Adachi & Hasegawa 1996), with the D (pair-wise distance calculation) option. A Neighbor-Joining (NJ) tree was then constructed using the NJDIST routine in MOLPHY. The topology of the NJ tree was then used as the initial tree for the ML tree search by the NNI search routine of PROTML.

For reconstruction of the NJ tree (Saitou & Nei 1987), the alpha parameter of gamma distribution of site-by-site variation of evolutionary rate was estimated with Tree-Puzzle 5.0 (Schmidt et al. 2002) with JTT model. The pair-wise distances of all combinations were estimated with PROTDIST in PHYLIP 3.6 (Felsenstein 1997) using the site-by-site variation model. The NJ tree was then reconstructed with NEIGHBOR in PHYLIP 3.6. For bootstrap analysis, the 1000 resamplings were performed with SEQBOOT in PHYLIP 3.6. The PROTDIST was used for estimation of pair-wise distances, and NEIGHBOR was used for reconstruction of NJ trees of resamplings. The bootstrap probability of each node was calculated with CONSENSE of PHYLIP 3.6.

For Maximum parsimony (MP) analysis, the PAUP 4.01b10 was used. The MP tree was estimated by heuristic search under the default condition except use of the option ó the random step-wise addition (10 replicates) of taxon. For bootstrap analysis, 100 resamplings were used.


   Acknowledgements
 
We thank Mr Yutaka Fukumoto (Ehime University) for help with cloning of A. aeolicus trmD. This work was partly supported by a Grant-in-aid (18048032) for Science Research on Priority Areas, and a Grant-in-aid (17613003) for Science Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Grant for Basic Science Research Projects (050137) from the Sumitomo Foundation.


   Footnotes
 
Communicated by: Yoshikazu Nakamura

aPresent address: Institute of Environmental Microbiology, Kyowa Kako Co. Ltd, Tadao 2-15-5, Machida 194-0035, Japan Back

* Correspondence: E-mail: hori{at}eng.ehime-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 25 March 2006
Accepted: 27 August 2006




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