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 (m(2)(2)G26) in tRNA. In the reaction, N2-guanine at position 26 (m(2)G26) 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 tRNA(Cys) has an m(2)(2)G26m(2)G27 or m(2)(2)G26m(2)(2)G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m(2)G27 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.

FIGURE 1.

Amino acid sequence alignment of Trm1 proteins. The alignment was generated by ClustalW 1.83 (59) and ESPript (60) programs. Amino acid sequences of Trm1 proteins experimentally identified are compared. Numbering of amino acid residues is based on the sequence of the A. aeolicus Trm1. Conserved and semi-conserved residues are boxed and highlighted, respectively. Secondary structure of P. horikoshii Trm1 is shown at the top of the alignment; arrows and coils represent β-sheets and helices, respectively. Asterisks and a reverse triangle show two Phe (Phe-27 and Phe-134) and Asp-132 residues, respectively.

FIGURE 2.

Purified A. aeolicus Trm1 protein. Three μg of purified A. aeolicus Trm1 was analyzed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. Mobility of standard markers is shown at the left.

FIGURE 3.

Western blotting analysis of Trm1 and AdoMet-dependent tRNA methyltransferase activity in A. aeolicus cell extract. A, cell extract (50 μg of proteins) of A. aeolicus was separated on a 15% SDS-polyacrylamide gel (left panel). The gel was stained with Coomassie Brilliant Blue. The Trm1 protein in the cell extract was detected by Western blotting analysis. The SDS-PAGE was performed under the same conditions as described in the left panel, and then electroblotting was performed. The primary antibody (anti-A. aeolicus Trm1 polyclonal antibody) was custom-made. The fluorescence derived from the secondary antibody was monitored on a Typhoon model 9410 system. The band corresponding to the Trm1 is shown by an arrow. B, E. coli tRNA mixture from [¹⁴C]AdoMet and A. aeolicus cell extract was incubated at 55 °C overnight, and ¹⁴C-methylated nucleotides were analyzed by two-dimensional TLC. Positions of standard markers (pA, pG, pC, and pU) are enclosed by dotted circles.

FIGURE 4.

Methyl transfer activity of A. aeolicus Trm1. A, nucleotide sequences of tested tRNA transcripts are compared. Positions from 8 to 48 are shown. We prepared full-length transcripts; sequences of the aminoacyl stem and T-arm are abbreviated by dotted lines. Yeast tRNA^Phe (G26U) is a mutant transcript, in which G26 is substituted with U. B, methyl group incorporation into tRNA transcripts and E. coli tRNA mixture at 15-min periods are shown by bars. This graph does not represent the initial velocities of the m²G26 formation, because m²₂G26, m²G27, and/or m²₂G27 formations are included in some cases (for example, A. aeolicus tRNA^Cys and tRNA^Tyr transcripts) as described in the text. Overnight incubation increases m²₂G via m²G in all methylated tRNAs.

FIGURE 5.

Mass spectrometry analysis of methylated A. aeolicus tRNA^Cys transcript. A, A. aeolicus tRNA^Cys transcript was methylated with nonradioisotope-labeled AdoMet, and then its nucleosides were analyzed by LC/MS. The eluted positions of m²G and m²₂G, which are determined by another control experiment, are shaded. B, methylated tRNA^Cys transcript was digested with RNase A, and its fragments were analyzed by LC/MS. The calculated m/z values are shown. C, fragments shown in B were analyzed by MS/MS. Nucleotide sequences determined by MS are shown in the panels.

FIGURE 6.

Purification and modified nucleotide analysis of native tRNA^Cys. A, nucleotide sequence of A. aeolicus tRNA^Cys is depicted as a cloverleaf structure. The 3′-biotin DNA probe region is illustrated. B, purified tRNA^Cys was analyzed by 10% PAGE (7 m urea). The gel was stained with toluidine blue. Lane 1, low molecular weight RNA fraction extracted from A. aeolicus cells (0.03 A₂₆₀ unit); lane 2, purified tRNA^Cys (0.009 A₂₆₀ unit). C, MS analysis of the RNase A-digested fragments of native tRNA^Cys. Two fragments derived from nucleotides from G26 to U33 were eluted, for which calculated m/z values and sequences are shown. D, nucleotide sequences of two fragments shown in C were determined by MS/MS analysis. C32 was found to be modified to Cm32 in addition to G26 and G27. E, modified nucleotides in A. aeolicus tRNA^Cys are shown in the cloverleaf structure.

FIGURE 7.

Kinetic study for tRNA^Tyr variants. A, nucleotide sequence of tRNA^Tyr is depicted as a cloverleaf structure. The mutation positions are highlighted in circles. B, kinetic parameters for the wild-type and mutant tRNA^Tyr transcripts are given. The parameters are average values of three independent experiments.

FIGURE 8.

Determination of the recognition sites by truncated tRNA transcripts. A, truncated tRNA transcripts are depicted as cloverleaf-like structures. As described in the text, we prepared two series of transcripts based on the tRNA^Tyr G26A,G27A mutant (B and C) and tRNA^Tyr A26G,A27G (original wild type; D and E). Transcript numbers 1–11 are based on the tRNA^Tyr G26A,G27A mutant, and numbers 12–22 are based on tRNA^Tyr A26G,A27G. B and D, methyl group incorporation was monitored by 10% PAGE (7 m urea). Left panels show the patterns of methylene blue staining, and right panels are autoradiograms of the same gels. The lane numbers correspond to the transcript numbers in A. C and E, initial velocities are compared. This is the average of three independent experiments.

FIGURE 9.

Schematic drawing of the target guanine recognition mechanisms of Trm1 proteins. A. aeolicus Trm1 recognizes the G26 and G27 bases from the T-arm region (A). In contrast, archaeal and eukaryotic enzymes recognize only the G26 base from the D-stem and variable region (B). A. aeolicus Trm1 recognizes the target sites distantly spaced as compared with archaeal and eukaryotic enzymes. This long distance target site recognition confers the multisite specificity of A. aeolicus Trm1.