Interaction of tRNA (Uracil-5-)-Methyltransferase with NO2Ura-tRNA

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-b...

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Bibliographic Details
Published in:Nucleic acids research 1996-03, Vol.24 (6), p.1059-1064
Main Authors: Gu, Xiangrong, Matsuda, Akira, Ivanetich, Kathryn M., Santi, Daniel V.
Format: Article
Language:English
Subjects:
Binding Sites
Binding, Competitive
Collodion
Escherichia coli - metabolism
Kinetics
Macromolecular Substances
RNA, Bacterial - chemistry
RNA, Bacterial - metabolism
RNA, Bacterial - pharmacology
RNA, Transfer - chemistry
RNA, Transfer - metabolism
RNA, Transfer - pharmacology
Substrate Specificity
tRNA Methyltransferases - antagonists & inhibitors
tRNA Methyltransferases - metabolism
Uracil - analogs & derivatives
Uracil - chemistry
Uracil - metabolism
Uracil - pharmacology
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Summary: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.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/24.6.1059