INTERACTION OF TRANSFER-RNA (URACIL-5-)-METHYLTRANSFERASE WITH NO(2)URA-TRANSFER-RNA

tRNA in which uracil is completely replaced by 5-nitro-uracil was prep ared 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 feat ure in their catalytic mechanism, When assayed shortly after mixing, t he tRNA analog, NO(2)Ura-tRNA, is a potent competitive inhibitor of tR NA-Ura methyl transferase (RUMT), Upon incubation, the analog causes a time-dependent inactivation of RUMT which could be reversed by diluti on into a large excess of tRNA substrate, Covalent RUMT-NO(2)Ura-tRNA complexes could be isolated on nitrocellulose filters or by SDS-PAGE, The interaction of RUMT and NO(2)Ura-tRNA was deduced to involve forma tion of a reversible complex, followed by formation of a reversible co valent complex in which Cys 324 of RUMT is linked to the 6-position of NO(2)Ura 54 in NO(2)Ura-tRNA.