The conformation of 23S rRNA nucleotide A2058 determines its recognition by the ErmE methyltransferase

The ErmE methyltransferase confers resistance to MLS antibiotics by specifically dimethylating adenine 2058 (A2058, Escherichia coli numbering) in bacterial 23S rRNA. To define nucleotides in the rRNA that are part of the motif recognized by ErmE, we investigated both in vivo and in vitro the effects of mutations around position A2058 on methylation. Mutagenizing A2058 (to G or U) completely abolishes methylation of 23S rRNA by ErmE. No methylation occurred at other sites in the rRNA, demonstrating the fidelity of ErmE for A2058. Breaking the neighboring G2057-C2611 Watson-Crick base pair by introducing either an A2057 or a U2611 mutation, greatly reduces the rate of methylation at A2058. Methylation remains impaired after these mutations have been combined to create a new A2057-U2611 Watson-Crick base interaction. The conformation of this region in 23S rRNA was probed with chemical reagents and it was shown that the A2057 and U2611 mutations alone and in combination alter the reactivity of A2058 and adjacent bases. However, mutagenizing position G-->A2032 in an adjacent loop, which has been implicated to interact with A2058, alters neither the ErmE methylation at A2058 nor the accessibility of this region to the chemical reagents. The data indicate that a less-exposed conformation at A2058 leads to reduction in methylation by ErmE. Nucleotide G2057 and its interaction with C2611 maintain the conformation at A2058, and are thus important in forming the structural motif that is recognized by the ErmE methyltransferase.