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TrmH is a eubacterial tRNA methyltransferase responsible for formation of 2'-O-methylguaosine at position 18 (Gm18) in tRNA. In Escherichia coli cells, only 14 tRNA species possess the Gm18 modification. To investigate the substrate tRNA selection mechanism of E. coli TrmH, we performed biochemical and structural studies. Escherichia coli TrmH requires a high concentration of substrate tRNA for efficient methylation. Experiments using native tRNA SerCGA purified from a trmH gene disruptant strain showed that modified nucleosides do not affect the methylation. A gel mobility-shift assay reveals that TrmH captures tRNAs without distinguishing between relatively good and very poor substrates. Methylation assays using wild-type and mutant tRNA transcripts revealed that the location of G18 in the D-loop is very important for efficient methylation by E. coli TrmH. In the case of tRNASer, tRNATyrand tRNALeu, the D-loop structure formed by interaction with the long variable region is important. For tRNAGln, the short distance between G18 and A14 is important. Thus, our biochemical study explains all Gm18 modification patterns in E. coli tRNAs. The crystal structure of E. coli TrmH has also been solved, and the tRNA binding mode of E. coli TrmH is discussed based on the structure., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society.)

TrmFO catalyzes the formation of 5-methyluridine at position 54 in tRNA and uses N5, N10-methylenetetrahydrofolate (CH2THF) as the methyl group donor. We found that the trmFO gene-disruptant strain of Thermus thermophilus, an extremely thermophilic eubacterium, can grow faster than the wild-type strain in the synthetic medium at 70°C (optimal growth temperature). Nucleoside analysis revealed that the majority of modifications were appropriately introduced into tRNA, showing that the limited nutrients are preferentially consumed in the tRNA modification systems. CH2THF is consumed not only for tRNA methylation by TrmFO but also for dTMP synthesis by ThyX and methionine synthesis by multiple steps including MetF reaction. In vivo experiment revealed that methylene group derived from serine was rapidly incorporated into DNA in the absence of TrmFO. Furthermore, the addition of thymidine to the medium accelerated growth speed of the wild-type strain. Moreover, in vitro experiments showed that TrmFO interfered with ThyX through consumption of CH2THF. Addition of methionine to the medium accelerated growth speed of wild-type strain and the activity of TrmFO was disturbed by MetF. Thus, the consumption of CH2THF by TrmFO has a negative effect on dTMP and methionine syntheses and results in the slow growth under a nutrient-poor condition.

Dihydrouridine (D) is formed by tRNA dihydrouridine synthases (Dus). In mesophiles, multiple Dus enzymes bring about D modifications at several positions in tRNA. The extreme-thermophilic eubacterium Thermus thermophilus, in contrast, has only one dus gene in its genome and only two D modifications (D20 and D20a) in tRNA have been identified. Until now, an in vitro assay system for eubacterial Dus has not been reported. In this study, therefore, we constructed an in vitro assay system using purified Dus. Recombinant T. thermophilus Dus lacking bound tRNA was successfully purified. The in vitro assay revealed that no other factors in living cells were required for D formation. A dus gene disruptant (Δdus) strain of T. thermophilus verified that the two D20 and D20a modifications in tRNA were derived from one Dus protein. The Δdus strain did not show growth retardation at any temperature. The assay system showed that Dus modified tRNA(Phe) transcript at 60°C, demonstrating that other modifications in tRNA are not essential for Dus activity. However, a comparison of the formation of D in native tRNA(Phe) purified from the Δdus strain and tRNA(Phe) transcript revealed that other tRNA modifications are required for D formation at high temperatures., (© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)