Recognition of nucleotide G745 in 23 S ribosomal RNA by the rrmA methyltransferase.
Methylation of the N1 position of nucleotide G745 in hairpin 35 of Escherichia coli 23 S ribosomal RNA (rRNA) is mediated by the methyltransferase enzyme RrmA. Lack of G745 methylation results in reduced rates of protein synthesis and growth. Addition of recombinant plasmid-encoded rrmA to an rrmA-deficient strain remedies these defects. Recombinant RrmA was purified and shown to retain its activity and specificity for 23 S rRNA in vitro. The recombinant enzyme was used to define the structures in the rRNA that are necessary for the methyltransferase reaction. Progressive truncation of the rRNA substrate shows that structures in stem-loops 33, 34 and 35 are required for methylation by RrmA. Multiple contacts between nucleotides in these stem-loops and RrmA were confirmed in footprinting experiments. No other RrmA contact was evident elsewhere in the rRNA. The RrmA contact sites on the rRNA are inaccessible in ribosomal particles and, consistent with this, 50 S subunits or 70 S ribosomes are not substrates for RrmA methylation. RrmA resembles the homologous methyltransferase TlrB (specific for nucleotide G748) as well as the Erm methyltransferases (nucleotide A2058), in that all these enzymes methylate their target nucleotides only in the free RNA. After assembly of the 50 S subunit, nucleotides G745, G748 and A2058 come to lie in close proximity lining the peptide exit channel at the site where macrolide, lincosamide and streptogramin B antibiotics bind.
Hansen
L H
LH
Department of Biochemistry and Molecular Biology, Odense University, Denmark.
Kirpekar
F
F
Douthwaite
S
S
D000900:Anti-Bacterial Agents / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D023282:Nuclease Protection Assays; D009690:Nucleic Acid Conformation; D021122:Protein Subunits; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013463:Sulfuric Acid Esters / Q000378:metabolism
D000900:Anti-Bacterial Agents; D021122:Protein Subunits; D012338:RNA, Ribosomal, 23S; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D013463:Sulfuric Acid Esters; D008780:Methyltransferases; C110074:23S rRNA m1G745 methyltransferase; C007482:dimethyl sulfate