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Nucleic Acids Research, 2003, Vol. 31, No. 16 4738-4746
© 2003 Oxford University Press

Identifying the methyltransferases for m5U747 and m5U1939 in 23S rRNA using MALDI mass spectrometry

Christian Toft Madsen, Jonas Mengel-Jørgensen, Finn Kirpekar and Stephen Douthwaite*

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark

*To whom correspondence should be addressed. Tel: +45 6550 2395; Fax:+45 6550 2467; Email: srd{at}bmb.sdu.dk

There are three sites of m5U modification in Escherichia coli stable RNAs: one at the invariant tRNA position U54 and two in 23S rRNA at the phylogenetically conserved positions U747 and U1939. Each of these sites is modified by its own methyltransferase, and the tRNA methyltransferase, TrmA, is well-characterised. Two open reading frames, YbjF and YgcA, are approximately 30% identical to TrmA, and here we determine the functions of these candidate methyltransferases using MALDI mass spectrometry. A purified recombinant version of YgcA retains its activity and specificity, and methylates U1939 in an RNA transcript in vitro. We were unable to generate a recombinant version of YbjF that retained in vitro activity, so the function of this enzyme was defined in vivo by engineering a ybjF knockout strain. Comparison of the methylation patterns in 23S rRNAs from YbjF+ and YbjF– strains showed that the latter differed only in the lack of the m5U747 modification. With this report, the functions of all the E.coli m5U RNA methyltransferases are identified, and a more appropriate designation for YbjF would be RumB (RNA uridine methyltransferases B), in line with the recent nomenclature change for YgcA (now RumA).


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