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

Xiangrong Gu | Zendy; Akira Matsuda | Zendy; Kathryn M. Ivanetich | Zendy; Daniel V. Santi | Zendy

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

Author(s) -

Xiangrong Gu,

Akira Matsuda,

Kathryn M. Ivanetich,

Daniel V. Santi

Publication year - 1996

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/24.6.1059

Subject(s) - transfer rna , uracil , biology , methyltransferase , biochemistry , covalent bond , enzyme , stereochemistry , rna , methylation , chemistry , dna , gene , organic chemistry

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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