PMID:12907714
| Citation |
Madsen, CT, Mengel-Jørgensen, J, Kirpekar, F and Douthwaite, S (2003) Identifying the methyltransferases for m(5)U747 and m(5)U1939 in 23S rRNA using MALDI mass spectrometry. Nucleic Acids Res. 31:4738-46 |
|---|---|
| Abstract |
There are three sites of m(5)U 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 m(5)U747 modification. With this report, the functions of all the E.coli m(5)U 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). |
| Links | |
| Keywords |
Base Sequence; Cloning, Molecular; Escherichia/enzymology; Escherichia/genetics; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Methyltransferases/genetics; Methyltransferases/isolation & purification; Methyltransferases/metabolism; Molecular Sequence Data; Nucleic Acid Conformation; RNA, Ribosomal, 23S/chemistry; RNA, Ribosomal, 23S/genetics; RNA, Ribosomal, 23S/metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods; Uridine/metabolism; tRNA Methyltransferases/genetics; tRNA Methyltransferases/metabolism |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.