flavin-adenine-dinucleotide and ribothymidine

TrmFO is a N(5) , N(10) -methylenetetrahydrofolate (CH2 THF)-/FAD-dependent tRNA methyltransferase, which synthesizes 5-methyluridine at position 54 (m(5) U54) in tRNA. Thermus thermophilus is an extreme-thermophilic eubacterium, which grows in a wide range of temperatures (50-83 °C). In T. thermophilus, modified nucleosides in tRNA and modification enzymes form a network, in which one modification regulates the degrees of other modifications and controls the flexibility of tRNA. To clarify the role of m(5) U54 and TrmFO in the network, we constructed the trmFO gene disruptant (∆trmFO) strain of T. thermophilus. Although this strain did not show any growth retardation at 70 °C, it showed a slow-growth phenotype at 50 °C. Nucleoside analysis showed increase in 2'-O-methylguanosine at position 18 and decrease in N(1) -methyladenosine at position 58 in the tRNA mixture from the ∆trmFO strain at 50 °C. These in vivo results were reproduced by in vitro experiments with purified enzymes. Thus, we concluded that the m(5) U54 modification have effects on the other modifications in tRNA through the network at 50 °C. (35) S incorporations into proteins showed that the protein synthesis activity of ∆trmFO strain was inferior to the wild-type strain at 50 °C, suggesting that the growth delay at 50 °C was caused by the inferior protein synthesis activity.

Topics: Flavin-Adenine Dinucleotide; Folic Acid; Guanosine; Mutation; RNA, Transfer; Temperature; Thermus thermophilus; tRNA Methyltransferases; Uridine

The conserved U54 in tRNA is often modified to 5-methyluridine (m(5)U) and forms a reverse Hoogsteen base pair with A58 that stabilizes the L-shaped tRNA structure. In Gram-positive and some Gram-negative eubacteria, m(5)U54 is produced by folate/FAD-dependent tRNA (m(5)U54) methyltransferase (TrmFO). TrmFO utilizes N(5),N(10)-methylenetetrahydrofolate (CH(2)THF) as a methyl donor. We previously reported an in vitro TrmFO assay system, in which unstable [(14)C]CH(2)THF was supplied from [(14)C]serine and tetrahydrofolate by serine hydroxymethyltransferase. In the current study, we have improved the TrmFO assay system by optimization of enzyme and substrate concentrations and introduction of a filter assay system. Using this assay, we have focused on the tRNA recognition mechanism of TrmFO. 42 tRNA mutant variants were prepared, and experiments with truncated tRNA and microhelix RNAs revealed that the minimum requirement of TrmFO exists in the T-arm structure. The positive determinants for TrmFO were found to be the U54U55C56 sequence and G53-C61 base pair. The gel mobility shift assay and fluorescence quenching showed that the affinity of TrmFO for tRNA in the initial binding process is weak. The inhibition experiments showed that the methylated tRNA is released before the structural change process. Furthermore, we found that A38 prevents incorrect methylation of U32 in the anticodon loop. Moreover, the m(1)A58 modification clearly accelerates the TrmFO reaction, suggesting a synergistic effect of the m(5)U54, m(1)A58, and s(2)U54 modifications on m(5)s(2)U54 formation in Thermus thermophilus cells. The docking model of TrmFO and the T-arm showed that the G53-C61 base pair is not able to directly contact the enzyme.

Topics: Anticodon; Bacterial Proteins; Base Sequence; Electrophoretic Mobility Shift Assay; Enzyme Assays; Flavin-Adenine Dinucleotide; Folic Acid; Gene Expression Regulation, Bacterial; Glycine; Glycine Hydroxymethyltransferase; Kinetics; Models, Biological; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; RNA, Messenger; RNA, Transfer; RNA, Transfer, Phe; Serine; Substrate Specificity; Thermus thermophilus; tRNA Methyltransferases; Uridine

The methyl carbon of ribothymidine in Loop IV of the tRNA of Streptococcus faecalis, Bacillus subtilis, and some other microorganisms is derived directly from 5,10-methylenetetrahydrofolate, not S-adenosylmethionine. The pure enzyme from S. faecalis also requires FADH2. We have obtained evidence that tetrahydrofolate is a product of the reaction and demonstrated that label from [5-3H]5-deazaFMNH2 is incorporated into the methyl moiety of ribothymidine. These data indicate that the enzyme uses methylenetetrahydrofolate solely as a 1-carbon donor and employs FADH2 as a reducing agent in vitro according to the following reaction: tRNA(U psi C) + CH2 = THF + FADH2 leads to tRNA(T psi C) + THF + FAD.

Topics: Enterococcus faecalis; Flavin-Adenine Dinucleotide; Methylation; Methyltransferases; Oxidation-Reduction; Ribonucleosides; RNA, Transfer; Species Specificity; Tetrahydrofolates; Uridine