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Updated: Jun 5, 2026
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Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
Published on: February 24, 2015
Minimal substrate features for Erm methyltransferases defined by using a combinatorial oligonucleotide library
Lykke H Hansen1, Sune Lobedanz, Stephen Douthwaite
1Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
Chembiochem : a European Journal of Chemical Biology
|January 26, 2011
Summary
Erm methyltransferases confer antibiotic resistance by modifying 23S ribosomal RNA. Researchers identified key RNA structural motifs essential for Erm enzyme activity, aiding in the design of novel resistance-blocking molecules.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Erm methyltransferases are crucial for bacterial resistance to macrolide, lincosamide, and streptogramin B antibiotics.
- These enzymes function by methylating the 23S ribosomal RNA at the A2058 nucleotide.
Purpose of the Study:
- To identify specific structural motifs within the rRNA substrate recognized by Erm methyltransferases.
- To understand the role of different RNA strands and modifications in the methylation process.
- To lay the groundwork for designing molecules that inhibit Erm activity.
Main Methods:
- Combinatorial synthesis of short RNA sequences to create substrate molecules.
- Incorporation of Locked Nucleic Acid (LNA) monomers to stabilize RNA structures.
- Functional assays to assess methylation efficiency with modified substrates.
Main Results:
- Erm recognizes specific structural features: an unpaired A2058 target following an irregular helix on the top strand.
- Stabilizing the top strand with LNA at G2056 enhanced methylation.
- The bottom strand is essential for methylation, primarily by maintaining the top strand's conformation, and can be substituted with DNA.
- Excessive LNA modification on the top strand hindered methylation, indicating a need for substrate flexibility.
Conclusions:
- The study elucidates the precise structural requirements of rRNA substrates for Erm-mediated methylation.
- Findings provide insights into the mechanism of antibiotic resistance conferred by Erm enzymes.
- This research facilitates the development of strategies to overcome Erm-mediated resistance by designing inhibitors.
