Identification of a novel gene encoding a flavin-dependent tRNA:m5U methyltransferase in bacteria--evolutionary implications

Abstract

Formation of 5-methyluridine (ribothymidine) at position 54 of the T-psi loop of tRNA is catalyzed by site-specific tRNA methyltransferases (tRNA:m5U-54 MTase). In all Eukarya and many Gram-negative Bacteria, the methyl donor for this reaction is S-adenosyl-l-methionine (S-AdoMet), while in several Gram-positive Bacteria, the source of carbon is N5, N10-methylenetetrahydrofolate (CH2H4folate). We have identified the gene for Bacillus subtilis tRNA:m5U-54 MTase. The encoded recombinant protein contains tightly bound flavin and is active in Escherichia coli mutant lacking m5U-54 in tRNAs and in vitro using T7 tRNA transcript as substrate. This gene is currently annotated gid in Genome Data Banks and it is here renamed trmFO. TrmFO (Gid) orthologs have also been identified in many other bacterial genomes and comparison of their amino acid sequences reveals that they are phylogenetically distinct from either ThyA or ThyX class of thymidylate synthases, which catalyze folate-dependent formation of deoxyribothymine monophosphate, the universal DNA precursor.

The tRNA Recognition Mechanism of Folate/FAD-dependent tRNA Methyltransferase (TrmFO)

“…The formation of a complex between E. coli TrmA and 16S rRNA is essential for cell viability (64). It has been reported that B. subtilis trmFO gene disruption strains can survive (46). However, the enzyme (at least B. subtilis TrmFO) has the potential to form a covalent bond with other RNA species as well as tRNA because the RNA recognition mechanism of TrmFO is relatively simple, as described in this paper.…”

“…The stability of the covalent complex would therefore seem to be quite different between B. subtilis and T. thermophilus TrmFO enzymes. The striking similarity of the amino acid sequences of both enzymes suggests that these TrmFO enzymes have the same catalytic mechanism (46). In previous work, we proposed the other cysteine (Cys-51 in T. thermophilus TrmFO, which corresponds to Cys-53 in B. subtilis TrmFO) to be the site of covalent bond formation based on the crystal structure (47).…”

“…Because this method is difficult for most biochemical researchers, TrmFO remained a relatively unstudied enzyme. Urbonavicius et al (46,60) detected TrmFO activity by 32 P-internally labeled tRNA. Although this system is convenient, measurement of velocities is not so easy.…”

“…However, for a long time, the gene encoding the enzyme remained unidentified until Urbonavicius et al (46) biochemically identified the gene, namely trmFO, from B. subtilis. Their study suggested that TrmFO is found widely among Gram-positive and some Gramnegative bacteria, including T. thermophilus (46). In our previous study, we devised an in vitro assay system for TrmFO activity and solved the crystal structures of free, tetrahydrofolate (THF)-bound, and glutathione-bound forms of T. thermophilus TrmFO (47).…”

RNA-methyltransferase TrmA is a dual-specific enzyme responsible forC5-methylation of uridine in both tmRNA and tRNA

“…Moreover, in B. subtilis, m 5 U at position 54 in tRNA is catalyzed by TrmFO, a methylene-tetrahydrofolate-dependent enzyme, while in E. coli it is the SAM-dependent TrmA enzyme that catalyzes the formation of the same m 5 U-54 in tRNA. [19][20][21] Very recently, Yamagami and collaborators have demonstrated that TrmFO, like TrmA, recognizes the T-arm structure of T. thermophilus tRNAs. 22 We can thus infer from this and our results that TrmFO most probably methylates the TLD T-loop in tmRNAs of Gram-positive bacteria (another dual-specific enzyme).…”