A transfer RNA methyltransferase with an unusual domain composition catalyzes 2'-O-methylation at position 6 in tRNA

Abstract

Thermococcus kodakarensis tRNATrp contains 2'-O-methylcytidine at position 6 (Cm6). However, the tRNA methyltransferase responsible for the modification has not been identified. Using comparative genomics, we predicted TK1257 as a candidate gene for the modification enzyme. Biochemical and mass spectrometry studies of purified recombinant TK1257 gene product demonstrated that it possesses a tRNA methyltransferase activity for Cm6 formation. This protein has a highly unusual composition of domains, containing N-terminal ferredoxin-like, SPOUT catalytic, and THUMP domains. Previous to this study, all known THUMP-related tRNA methyltransferases were shown to contain a Rossmann fold catalytic domain and the nucleosides they produced were N2-methylguanosine and/or N2, N2-dimethylguanosine. Therefore, our findings extend the knowledge of architecture of tRNA methyltransferases. We named the TK1257 gene product TrmTS and showed that it can synthesize Am6 and Um6 as well as Cm6. A trmTS gene deletion strain showed slight growth retardation at high temperatures. Site-directed mutagenesis studies revealed catalytically and structurally important amino acid residues in TrmTS and identified a TrmTS-specific linker that is structurally essential. We revealed that TrmTS recognizes the 3'-CCA terminus of tRNA but does not require the three-dimensional structure of tRNA for its activity. Finally, we constructed a model of the binding between TrmTS and tRNA.

Graphical Abstract

Figure 1.

Nucleotide sequence of Thermococcus kodakarensis tRNA^Trp and scheme of comparative genomics. (A) The secondary structure of T. kodakarensis tRNA^Trp is depicted as a cloverleaf structure. The Cm6 is highlighted in red. (B) The responsible gene for the Cm6 modification in tRNA exists in T. kodakarensis and P. furiosus genomes was predicted by comparative genomics. H. volcanii, S. acidocaldarius and T. acidophilum do not possess the Cm6 modification in tRNA. The 570 genes were common in T. kodakarensis and P. furiosus. (C) Scheme of comparative genomics is illustrated. An explanation of details is described in the main text.

Figure 2.

TK1257 gene product possesses a tRNA methyltransferase activity for 2′-O-methylation at position 6. (A) Purified TK1257 gene product (3 μg) was analyzed by 15% SDS–PAGE. The gel was stained with Coomassie Brilliant Blue. (B) The methyl-transfer activity of TK1257 gene product was tested at 75°C using tRNA^Trp transcript and ³H-labeled SAM. This experiment was independently replicated three times (n = 3). The error bars show the standard deviations. (C) Three mutant tRNA^Trp transcripts, in which the C6-G67 base pair was replaced by A6-U67, U6-A67, or G6-C67 base pair, were prepared. The mutation site is highlighted in red. (D) The methylation speeds for the wild-type and mutant tRNA^Trp transcripts were compared. The methylation for the wild-type tRNA^Trp is expressed as 1.00. This experiment was independently replicated three times (n = 3). The error bars show the standard deviations. (E) ¹⁴C-methylated nucleotides were analyzed by two-dimensional thin-layer chromatography. When the wild-type tRNA^Trp transcript (WT) was used, ¹⁴C-pCm was detected. In contrast, when the mutant tRNA^Trp A6-U67 and U6-A67 transcripts were used, ¹⁴C-pAm and ¹⁴C-pUm were detected, respectively. Furthermore, when the mutant tRNA^Trp G6-C67 transcript was used, ¹⁴C-labeled nucleotide was not detected, consistent with the result in panel (D). (F) Apparent kinetic parameters of TK1257 gene product for the wild-type and mutant tRNA^Trp A6-U67 and U6-A67 transcripts were determined. These transcripts had comparable methyl group acceptance activities. The experiment was independently replicated three times (n = 3). The errors show the standard deviations. (G) Kinetic parameters of TK1257 gene product for SAM were determined.

Figure 3.

TrmTS is expressed in T. kodakarensis wild-type strain, and the trmTS gene deletion (ΔtrmTS) strain showed a slight growth retardation at 93°C. (A) The cell extracts from wild-type and ΔtrmTS strains were analyzed by western blotting. Purified TrmTS (right lane) was used as a positive control. The gel was stained with Coomassie Brilliant Blue. Because only 20 ng of TrmTS was used, the band of TrmTS was not visible by Coomassie Brilliant Blue staining. (B) Native tRNA^Trp were purified from the wild-type and ΔtrmTS strains. The gel was stained with methylene blue. (C) The Cm modification levels in purified tRNA^Trp from the wild-type (upper) and ΔtrmTS (lower) strains were analyzed by LC/MS. The abundance of Cm modification was normalized to the extracted ion chromatogram intensity of guanosine. The Cm levels from the wild-type strain set as 100%. (D) Growth curves of the wild-type (circle) and ΔtrmTS (square) strains are compared at 85°C and 93°C. At 93°C, a slight growth retardation of ΔtrmTS strain was observed. When TrmTS was expressed in the ΔtrmTS strain (triangle), the growth speed recovered. This experiment was independently replicated three times (n = 3). The error bars show the standard deviations.

Figure 4.

Amino acid sequence alignment of TrmTS-like proteins and SPOUT tRNA 2′-O-methyltranserases and location of conserved amino acid residues. (A) The amino acid sequence alignment of TrmTS-like proteins is depicted. NFLD, THUMP, and SPOUT domains, and linker region are highlighted in gray, pale blue and purple, and green, respectively. Fifteen conserved amino acid residues were substituted by alanine and/or other amino acid residues. (B) Subunit structural model of TrmTS was constructed by AlphaFold 3. A SAM molecule was modeled on the structure where the crystal structure of T. thermophilus TrmH in complexed with SAM (PDB: 1V2X) was aligned with the TrmTS structure. The TrmH structure was then removed. SAM is colored red. The domains and linker region are colored in the same as in panel (A). (C) The mutation sites are mapped onto the SPOUT catalytic domain and linker region.

Figure 5.

Purities of TrmTS mutant proteins and their activities. (A) The wild-type and mutant TrmTS proteins (2 μg each) were analyzed by 15% SDS–PAGE. The gel was stained with Coomassie Brilliant Blue. (B) Relative methylation speed of the wild-type and mutant TrmTS proteins were compared using tRNA^Trp transcript and ³H-SAM as substrates. The methylation speed of the wild-type TrmTS is expressed as 1.00. This experiment was independently replicated three times (n = 3). The error bars show the standard deviations.

Figure 6.

CD spectra and apparent kinetic parameters of TrmTS mutant proteins. (A) CD spectra were measured at 25°C, 50°C, and 75°C. (B) Apparent kinetic parameters of mutant TrmTS proteins for SAM and tRNA^Trp transcripts were measured. (C) Apparent kinetic parameters of the wild-type and mutant proteins are summarized. “n. d.” means that the activity was not detectable. The experiment was independently replicated three times (n = 3). The errors show the standard deviations.

Figure 7.

Two conserved lysine residues (Lys155 and Lys160) in the linker region. (A) The linker from one monomer contacts the SPOUT domain from the other monomer. Asp270 residue of the catalytic domain in one subunit is able to contact with Lys160 residue of the linker region in another subunit. (B and C) Apparent kinetic parameters of TrmTS Lys155Ala mutant protein for (B) SAM and (C) tRNA^Trp transcript were determined. The experiment was independently replicated three times (n = 3) The errors show the standard deviations.

Figure 8.

Transfer RNA recognition by TrmTS. (A) Eight mutant tRNA^Trp transcripts were prepared. Transcript 1 is the wild-type tRNA^Trp transcript. Secondary structure of each transcript except for transcript 5 were predicted using RNAfold. (B) The features of mutant tRNA^Trp transcripts are summarized. (C) The mutant tRNA^Trp transcripts were treated with TrmTS and ¹⁴C-SAM and then analyzed by 10% PAGE (7 M urea). The gel was stained with methylene blue (left). The autoradiogram of the same gel was obtained (right). The lane numbers correspond to the transcript numbers in panels (A) and (B). (D) Two mutant tRNA^Trp transcripts (-ACCA and G6-C67), which are not methylated by TrmTS, were used for the inhibition experiments. Each mutant tRNA^Trp transcript (0, 10, 20, 30, 40, 50, 60, 70, and 80 μM) was titrated to the TrmTS reaction in the presence of 35 μM wild-type tRNA^Trp. The reaction was performed at 75°C for 10 min. The experiments were replicated three times (n = 3). The error bars show the standard deviations.

Figure 9.

Hypothetical binding model between TrmTS and tRNA. (A) Positive (blue) and negative (red) charged areas are mapped onto the surface of TrmTS dimer structure where ±5 kBT/e values were used for the visualization of the electrostatic potential map. (B) Conserved and nonconserved amino acid regions are colored in magenta and dark green, respectively. (C) A binding model between TrmTS and tRNA^Trp was predicted using AlphaFold3. TrmTS recognizes the CCA terminus of substrate tRNA and the positively charged area of the SPOUT domain interact with the three-dimensional core of tRNA. The key amino acid residues for TrmTS activities are labeled.