Identifying the methyltransferases for m5U747 and m5U1939 in 23S rRNA using MALDI mass spectrometry
Open Access
- 15 August 2003
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 31 (16), 4738-4746
- https://doi.org/10.1093/nar/gkg657
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).Keywords
This publication has 53 references indexed in Scilit:
- Instruction of Translating Ribosome by Nascent PeptideScience, 2002
- The rlmB Gene Is Essential for Formation of Gm2251 in 23S rRNA but Not for Ribosome Maturation in Escherichia coliJournal of Bacteriology, 2001
- The Structural Basis of Ribosome Activity in Peptide Bond SynthesisScience, 2000
- The Complete Atomic Structure of the Large Ribosomal Subunit at 2.4 Å ResolutionScience, 2000
- The FtsJ/RrmJ Heat Shock Protein of Escherichia coli Is a 23 S Ribosomal RNA MethyltransferasePublished by Elsevier BV ,2000
- Identification of the 16S rRNA m5C967 Methyltransferase from Escherichia coliBiochemistry, 1999
- Nucleotides in 23S rRNA protected by the association of 30S and 50S ribosomal subunitsJournal of Molecular Biology, 1999
- Gapped BLAST and PSI-BLAST: a new generation of protein database search programsNucleic Acids Research, 1997
- Isolation of mutants of Escherichia coli lacking 5-methyluracil in transfer ribonucleic acid or 1-methylguanine in ribosomal RNAJournal of Molecular Biology, 1970
- Nucleotide Sequences from Specific Areas of the 16S and 23S Ribosomal RNAs of E. coliJBIC Journal of Biological Inorganic Chemistry, 1969