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Purification of transfer RNA (m5U54)-methyltransferase from Escherichia coli. Association with RNA.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
1988 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 177, no 3, 467-75 p.Article in journal (Refereed) Published
Abstract [en]

tRNA (m5U54)-methyltransferase (EC 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.

Place, publisher, year, edition, pages
1988. Vol. 177, no 3, 467-75 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:umu:diva-59880PubMedID: 2461858OAI: diva2:556980
Available from: 2012-09-26 Created: 2012-09-26 Last updated: 2012-09-26

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