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Updated: Aug 18, 2026

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues
12:07

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Published on: November 22, 2014

m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts

Y Liu1, D V Santi

  • 1Departments of Biochemistry and Biophysics, and Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0446, USA.

Proceedings of the National Academy of Sciences of the United States of America
|July 19, 2000
PubMed

Insights

Researchers identified a large family of RNA methyltransferases (MTases). Surprisingly, RNA m(5)C MTases utilize a distinct catalytic cysteine than DNA m(5)C MTases, challenging prior assumptions.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genomics

Background:

  • A novel family of over 55 RNA methyltransferases (MTases) has been identified through genomic database searches.
  • These RNA m(5)C MTases share homologous sequence motifs with DNA m(5)C MTases, including a conserved ProCys motif.

Purpose of the Study:

  • To investigate the catalytic mechanism of RNA m(5)C methyltransferases.
  • To determine the specific cysteine residue responsible for catalysis in RNA m(5)C MTases.

Main Methods:

  • Iterative genomic sequence database searches using Fmu (16S rRNA m(5)C 967 MTase) as a probe.
  • Site-directed mutagenesis of conserved cysteine residues within the ProCys motif and an additional conserved cysteine.
  • Assay of methyltransferase activity and formation of covalent complexes with modified RNA.

Main Results:

  • Mutagenesis of the cysteine in the ProCys motif did not abolish activity; instead, it formed a covalent complex with 5-fluorocytosine-containing RNA.
  • Mutagenesis of the other conserved cysteine resulted in complete loss of activity and complex formation.
  • RNA m(5)C MTases employ a different catalytic cysteine compared to DNA m(5)C MTases.

Conclusions:

  • The catalytic cysteine in methyltransferases is determined by the substrate type (DNA vs. RNA), not the target base.
  • RNA m(5)C MTases utilize a distinct catalytic cysteine residue than DNA m(5)C MTases.
  • The precise function of the conserved ProCys sequence in RNA m(5)C MTases remains to be elucidated.

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