Publications

• Identification of the 16S rRNA m5C967 methyltransferase from Escherichia coli.

  Gu X.R., Gustafsson C., Ku J., Yu M., Santi D.V.

  The fmu gene product has been proposed to be an RNA methyltransferase [Koonin, E. V. (1994) Nucleic Acids Res. 22, 2476-2478]. Fmu has been cloned and expressed, and the encoded 47 kDa protein has been purified and characterized. The enzyme catalyzed specific methylation of C967 of unmodified 16S ... >> More

  The fmu gene product has been proposed to be an RNA methyltransferase [Koonin, E. V. (1994) Nucleic Acids Res. 22, 2476-2478]. Fmu has been cloned and expressed, and the encoded 47 kDa protein has been purified and characterized. The enzyme catalyzed specific methylation of C967 of unmodified 16S rRNA transcripts. A 16mer stem-loop structure containing C967 (nt 960-975) was also a good substrate for the enzyme in vitro. Methylation of C967 was confirmed by several methods including analysis of RNase T1 digests and nearest-neighbor analysis. Fmu did not catalyze methylation of transcripts of 23S rRNA. E. coli cells that contained kanr-disrupted fmu produced 16S rRNA that could be specifically methylated by Fmu in vitro at C967 but not C1407. Further, fmu disruption did not significantly alter the growth rate of E. coli in rich or minimal media. We propose renaming this ORF "rrmB" and the enzyme "RrmB" for rRNA methyltransferase. << Less

  Biochemistry 38:4053-4057(1999) [PubMed] [EuropePMC]

• Purification, cloning, and characterization of the 16S RNA m5C967 methyltransferase from Escherichia coli.

  Tscherne J.S., Nurse K., Popienick P., Michel H., Sochacki M., Ofengand J.

  The methyltransferase that forms m5C967 in Escherichia coli small subunit ribosomal RNA has been purified, cloned, and characterized. The gene was identified from the N-terminal sequence of the purified enzyme. The gene is a fusion of two open reading frames, fmu and fmv, previously believed to be ... >> More

  The methyltransferase that forms m5C967 in Escherichia coli small subunit ribosomal RNA has been purified, cloned, and characterized. The gene was identified from the N-terminal sequence of the purified enzyme. The gene is a fusion of two open reading frames, fmu and fmv, previously believed to be distinct due to a DNA sequencing error. The gene, here named rsmB, encodes a 429-amino acid protein that has a number of homologues in prokaryotes, Archaea, and eukaryotes. C-Terminal sequencing of the overexpressed and affinity-purified protein by mass spectrometry methods verified the sequence expected for the gene product. The recombinant protein exhibited the same specificity as the previously described native enzyme; that is, it formed only m5C and only at position 967. C1407, which is also m5C in natural 16S RNA, was not methylated. In vitro, the enzyme only recognized free 16S RNA. 30S ribosomal subunits were not a substrate. There was no requirement for added magnesium, suggesting that extensive secondary or tertiary structure in the RNA substrate may not be a requirement for recognition. << Less

  Biochemistry 38:1884-1892(1999) [PubMed] [EuropePMC]