Purification of transfer RNA (m5U54)-methyltransferase from Escherichia coli. Association with RNA.
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.
Ny
T
T
Department of Microbiology, University of Umeå, Sweden.
Lindström
H R
HR
Hagervall
T G
TG
Björk
G R
GR
D000596:Amino Acids / Q000032:analysis; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005784:Gene Amplification; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D010957:Plasmids; D012329:RNA, Bacterial / Q000302:isolation & purification*; D012359:tRNA Methyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D012329:RNA, Bacterial; D012359:tRNA Methyltransferases; C022769:tRNA(uracil-5)-methyltransferase