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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction of tRNA (m5U54)-methyltransferase (RUMT) with in vitro synthesized unmodified tRNA and a 17-base oligoribonucleotide analog of the T-arm of tRNA in the absence of AdoMet has been investigated. Binary complexes are formed which are isolable on nitrocellulose filters and are composed of noncovalent and covalent complexes in nearly equal amounts. The covalent RUMT-RNA complexes are stable to SDS-PAGE and migrate slower than free enzyme or RNA. Kinetic and thermodynamic constants involved in formation and disruption of noncovalent and covalent binary complexes have been determined and interpreted in the context of steady-state kinetic parameters of the enzyme-catalyzed methylation and 5-H exchange of substrate. The results show that the isolable covalent complex is kinetically incompetent as an intermediate for methylation. Isotope trapping experiments show that when AdoMet is added to preformed binary complex, all bound tRNA is converted to methylated product; thus, the covalent complexes are chemically competent to form products. We have concluded that, after a reversible binary complex is formed, the catalytic thiol adds to the 6-carbon of the U54 of tRNA. The initial adduct leaves the reaction pathway to protonation at carbon 5; the latter can deprotonate and re-enter the pathway to form methylated product. It is speculated that covalent binary RUMT-RNA adducts may serve as depots of enzyme-tRNA complexes primed for methylation, or in unknown roles with RNAs other than tRNA.
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PMID:Covalent adducts between tRNA (m5U54)-methyltransferase and RNA substrates. 142 Jan 48

16S rRNA, isolated from Escherichia coli or synthesized in vitro, is methylated by tRNA (m5U54)-methyltransferase (RUMT) and S-adenosyl-L-methionine to give ribothymidine (m5U). By methylation studies of 16S rRNA fragments, nearest-neighbor analysis, and nuclease protection experiments, the site of methylation was identified as U788. We have previously shown that the substrate consensus sequence for the T-arm of tRNA consists of a 2-5 base-pair stem and a 7-base loop, with certain constraints on base substitutions within the loop, and in the first two bases which close the loop [Gu, X., & Santi, D. V. (1991) Biochemistry 30, 2999-3002]. U788 of 16S rRNA is within a 9-base loop of a predicted stem-loop structure of 16S rRNA. If Ado substitution is allowed at the third and seventh positions of the loop and the first and ninth bases of the loop form an A-C base pair, the resulting stem-loop falls within the RUMT consensus sequence of the T-arm of tRNA. Individual mutants of the tRNA T-arm at these positions confirm that the substitutions are allowable, and expand the previous consensus sequence. Further, prevention of 7-base loop formation by requiring C-C base-pair formation at the loop closure abolishes substrate activity. RUMT forms a complex with Syn 16S rRNA which can be isolated on nitrocellulose filters or by SDS-PAGE electrophoresis. The enzyme also catalyzes exchange of tritium of [3H]Ura-16S rRNA for protons of water. By analogy with studies with tRNA [Gu, X., & Santi, D. V. (1991) Biochemistry 31, 10295-10302], the mechanism of methylation is proposed to involve formation of a covalent, albeit reversible, Michael adduct with the target U788 of 16S rRNA.
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PMID:In vitro methylation of Escherichia coli 16S rRNA by tRNA (m5U54)-methyltransferase. 811 82

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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PMID:Interaction of tRNA (uracil-5-)-methyltransferase with NO2Ura-tRNA. 860 39