Interaction of tRNA (uracil-5-)-methyltransferase with NO2Ura-tRNA

Abstract

tRNA in which uracil is completely replaced by 5-nitro-uracil was prepared by substituting 5-nitro-UTP for UTP in an in vitro transcription reaction. The rationale was that the 5-nitro substituent activates the 6-carbon of the Ura heterocycle towards nucleophiles, and hence could provide mechanism-based inhibitors of enzymes which utilize this feature in their catalytic mechanism. When assayed shortly after mixing, the tRNA analog, NO2Ura-tRNA, is a potent competitive inhibitor of tRNA-Ura methyl transferase (RUMT). Upon incubation, the analog causes a time-dependent inactivation of RUMT which could be reversed by dilution into a large excess of tRNA substrate. Covalent RUMT-NO2Ura-tRNA complexes could be isolated on nitrocellulose filters or by SDS-PAGE. The interaction of RUMT and NO2Ura-tRNA was deduced to involve formation of a reversible complex, followed by formation of a reversible covalent complex in which Cys 324 of RUMT is linked to the 6-position of NO2Ura 54 in NO2Ura-tRNA.

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

“…Therefore, this enzyme catalyzes in vitro methyl transfer to part of 16S rRNA (24). In the reaction, TrmA forms a covalent bond complex with substrate RNA (25,26), and methyl transfer takes place by a single displacement mechanism (27).…”

Chemistry of Fluorinated Pyrimidines in the Era of Personalized Medicine

“…Furthermore, Ψ is a highly conserved modification at other sites in tRNA and in multiple RNA species. Substitution of 5-nitrouridine in tRNA was shown to trap tRNA-Ura methyl transferase (RUMT; [ 76 ]) by the formation of a reversible covalent complex in which Cys324 of RUMT forms a Michael adduct with NO 2 Ura54 in tRNA [ 77 ]. The role of TRMT2A, the mammalian tRNA methyltransferase 2 homolog A protein, for mammalian cell function remains unclear; however, a recent study demonstrated that TRMT2A-deficient cells displayed increased growth consistent with a role for TRMT2A in cell cycle regulation [ 78 ].…”