50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA

Proc Natl Acad Sci U S A. 2022 Apr 5;119(14):e2120352119. doi: 10.1073/pnas.2120352119. Epub 2022 Mar 31.


Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2′-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144, which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.

Keywords: RNA modification; antibiotic resistance; methyltransferase; mycobacteria; ribosome.

MeSH terms

  • Bacterial Proteins* / chemistry
  • Catalytic Domain
  • Drug Resistance, Bacterial / genetics
  • Methyltransferases* / chemistry
  • Mycobacterium tuberculosis* / enzymology
  • Mycobacterium tuberculosis* / genetics
  • Protein Conformation, alpha-Helical
  • RNA, Ribosomal, 16S / chemistry
  • RNA, Ribosomal, 23S / chemistry
  • Ribosome Subunits, Large, Bacterial* / chemistry


  • Bacterial Proteins
  • RNA, Ribosomal, 16S
  • RNA, Ribosomal, 23S
  • TlyA protein, Mycobacterium tuberculosis
  • Methyltransferases
  • rRNA (adenosine-O-2'-)methyltransferase