PMID:2461858
| Citation |
Ny, T, Lindström, HR, Hagervall, TG and Björk, GR (1988) Purification of transfer RNA (m5U54)-methyltransferase from Escherichia coli. Association with RNA. Eur. J. Biochem. 177:467-75 |
|---|---|
| Abstract |
tRNA (m5U54)-methyltransferase (EC 2.1.1.35) catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to transfer ribonucleic acid (tRNA) and thereby forming 5-methyluridine (m5U, ribosylthymine) in position 54 of tRNA. This enzyme, which is involved in the biosynthesis of all tRNA chains in Escherichia coli, was purified 5800-fold. A hybrid plasmid carrying trmA, the structural gene for tRNA (m5U54)-methyltransferase was used to amplify genetically the production of this enzyme 40-fold. The purest fraction contained three polypeptides of 42 kDa, 41 kDa and 32 kDa and a heterogeneous 48-57-kDa RNA-protein complex. All the polypeptides seem to be related to the 42/41-kDa polypeptides previously identified as the tRNA (m5U54)-methyltransferase. RNA comprises about 50% (by mass) of the complex. The RNA seems not to be essential for the methylation activity, but may increase the activity of the enzyme. The amino acid composition is presented and the N-terminal sequence of the 42-kDa polypeptide was found to be: Met-Thr-Pro-Glu-His-Leu-Pro-Thr-Glu-Gln-Tyr-Glu-Ala-Gln-Leu-Ala-Glu-Lys- . The tRNA (m5U54)-methyltransferase has a pI of 4.7 and a pH optimum of 8.0. The enzyme does not require added cations but is stimulated by Mg2+. The apparent Km for tRNA and S-adenosyl-L-methionine are 80 nM and 17 microM, respectively. |
| Links | |
| Keywords |
Amino Acids/analysis; Chromatography, DEAE-Cellulose; Chromatography, Gel; Escherichia coli/enzymology; Escherichia coli/genetics; Gene Amplification; Genes; Genes, Bacterial; Kinetics; Plasmids; RNA, Bacterial/isolation & purification; tRNA Methyltransferases/genetics; tRNA Methyltransferases/isolation & purification; tRNA Methyltransferases/metabolism |
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