Distinct Modified Nucleosides in tRNA^Trp from the Hyperthermophilic Archaeon Thermococcus kodakarensis and Requirement of tRNA m²G10/m²₂G10 Methyltransferase (Archaeal Trm11) for Survival at High Temperatures

tRNA m²G10/m²₂G10 methyltransferase (archaeal Trm11) methylates the 2-amino group in guanosine at position 10 in tRNA and forms N²,N²-dimethylguanosine (m²₂G10) via N²-methylguanosine (m²G10). We determined the complete sequence of tRNA^Trp, one of the substrate tRNAs for archaeal Trm11 from Thermococcus kodakarensis, a hyperthermophilic archaeon. Liquid chromatography/mass spectrometry following enzymatic digestion of tRNA^Trp identified 15 types of modified nucleoside at 21 positions. Several modifications were found at novel positions in tRNA, including 2'-O-methylcytidine at position 6, 2-thiocytidine at position 17, 2'-O-methyluridine at position 20, 5,2'-O-dimethylcytidine at position 32, and 2'-O-methylguanosine at position 42. Furthermore, methylwyosine was found at position 37 in this tRNA^Trp, although 1-methylguanosine is generally found at this location in tRNA^Trp from other archaea. We constructed trm11 (Δtrm11) and some gene disruptant strains and compared their tRNA^Trp with that of the wild-type strain, which confirmed the absence of m²₂G10 and other corresponding modifications, respectively. The lack of 2-methylguanosine (m²G) at position 67 in the trm11 trm14 double disruptant strain suggested that this methylation is mediated by Trm14, which was previously identified as an m²G6 methyltransferase. The Δtrm11 strain grew poorly at 95°C, indicating that archaeal Trm11 is required for T. kodakarensis survival at high temperatures. The m²₂G10 modification might have effects on stabilization of tRNA and/or correct folding of tRNA at the high temperatures. Collectively, these results provide new clues to the function of modifications and the substrate specificities of modification enzymes in archaeal tRNA, enabling us to propose a strategy for tRNA stabilization of this archaeon at high temperatures.IMPORTANCEThermococcus kodakarensis is a hyperthermophilic archaeon that can grow at 60 to 100°C. The sequence of tRNA^Trp from this archaeon was determined by liquid chromatography/mass spectrometry. Fifteen types of modified nucleoside were observed at 21 positions, including 5 modifications at novel positions; in addition, methylwyosine at position 37 was newly observed in an archaeal tRNA^Trp The construction of trm11 (Δtrm11) and other gene disruptant strains confirmed the enzymes responsible for modifications in this tRNA. The lack of 2-methylguanosine (m²G) at position 67 in the trm11 trm14 double disruptant strain suggested that this position is methylated by Trm14, which was previously identified as an m²G6 methyltransferase. The Δtrm11 strain grew poorly at 95°C, indicating that archaeal Trm11 is required for T. kodakarensis survival at high temperatures.