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 Escheric hia coli 23 S ribosomal RNA (rRNA) is mediated by the methyltransferase enz yme RrmA. Lack of G745 methylation results in reduced rates of protein synt hesis and growth. Addition of recombinant plasmid-encoded rrmA to an rrmA-d eficient strain remedies these defects. Recombinant RrmA was purified and s hown to retain its activity and specificity for 23 S rRNA in vitro. The rec ombinant enzyme was used to define the structures in the rRNA that are nece ssary for the methyltransferase reaction. Progressive truncation of the rRN A substrate shows that structures in stem-loops 33, 34 and 35 are required for methylation by RrmA. Multiple contacts between nucleotides in these ste m-loops and RrmA were confirmed in footprinting experiments. No other RrmA contact was evident elsewhere in the rRNA. The RrmA contact sites on the rR NA are inaccessible in ribosomal particles and, consistent with this, 50 S subunits or 70 S ribosomes are not substrates for RrmA methylation. RrmA re sembles the homologous methyltransferase TlrB (specific for nucleotide G748 ) as well as the Erm methyltransferases (nucleotide A2058), in that all the se enzymes methylate their target nucleotides only in the free RNA. After a ssembly of the 50 S subunit, nucleotides G745, G748 and A2058 come to lie i n close proximity lining the peptide exit channel at the site where macroli de, lincosamide and streptogramin B antibiotics bind. (C) 2001 Academic Pre ss.