Core sequence in the RNA motif recognized by the ErmE methyltransferase revealed by relaxing the fidelity of the enzyme for its target

Under physiological conditions, the ErmE methyltransferase specifically mod ifies a single adenosine within ribosomal RNA (rRNA), and thereby confers r esistance to multiple antibiotics. The adenosine (A2058 in Escherichia coli 23S rRNA) lies within a highly conserved structure, and is methylated effi ciently, and with equally high fidelity, in rRNAs from phylogenetically div erse bacteria. However, the fidelity of ErmE is reduced when magnesium is r emoved, and over twenty new sites of ErmE methylation appear in E. coli 16S and 23S rRNAs. These sites show widely different degrees of reactivity to ErmE. The canonical A2058 site is largely unaffected by magnesium depletion and remains the most reactive site in the rRNA. This suggests that methyla tion at the new sites results from changes in the RNA substrate rather than the methyltransferase. Chemical probing confirms that the rRNA structure o pens upon magnesium depletion, exposing potential new interaction sites to the enzyme. The new ErmE sites show homology with the canonical A2058 site, and have the consensus sequence aNNNcgGAHAg (ErmE methylation occurs exclu sively at adenosines (underlined); these are preceded by a guanosine, equiv alent to G2057; there is a high preference for the adenosine equivalent to A2060; H is any nucleotide except G; N is any nucleotide; and there are sli ght preferences for the nucleotides shown in lower case). This consensus is believed to represent the core of the motif that Erm methyltransferases re cognize at their canonical A2058 site. The data also reveal constraints on the higher order structure of the motif that affect methyltransferase recog nition.