THE CONFORMATION OF 23S RIBOSOMAL-RNA NUCLEOTIDE A2058 DETERMINES ITSRECOGNITION BY THE ERME METHYLTRANSFERASE

The ErmE methyltransferase confers resistance to MLS antibiotics by sp ecifically dimethylating adenine 2058 (A2058, Escherichia coli numberi ng) 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 methylatio n. 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. Me thylation remains impaired after these mutations have been combined to create a new A2057-U2611 Watson-Crick base interaction. The conformat ion of this region in 23S rRNA was probed with chemical reagents and i t was shown that the A2057 and U2611 mutations alone and in combinatio n alter the reactivity of A2058 and adjacent bases. However, mutageniz ing position G --> A2032 in an adjacent loop, which has been implicate d to interact with A2058, alters neither the ErmE methylation at A2058 nor the accessibility of this region to the chemical reagents. The da ta indicate that a less-exposed conformation at A2058 leads to reducti on in methylation by ErmE. Nucleotide G2057 and its interaction with C 2611 maintain the conformation at A2058, and are thus important in for ming the structural motif that is recognized by the ErmE methyltransfe rase.