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Nucleic Acids Research, Vol 24, Issue 6 1059-1064, Copyright © 1996 by Oxford University Press

ARTICLES

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

X Gu, A Matsuda, KM Ivanetich and DV Santi
Department of Biochemistry, University of California, San Francisco 94143-0448 USA.

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.
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