The N-terminal basic patch and motif X contribute to rRNA methylation, and the basic patch contributes to rRNA binding. (A) Methylation reactions under single-turnover conditions were conducted using site-directed mutants in the N-terminal basic region or motif X of ErmE. (B) Affinity binding of ErmE site-directed mutants to an analog of helix 73 of 23S rRNA was measured by fluorescence polarization. Three independent binding reactions were performed for each variant, and replicate measurements are shown as a scatter plot. A cartoon depicting the assays is given. The fluorescein label on the 48 nt rRNA analog is indicated by an orange circle. (C) Dissociation constant (Kd) values of ErmE variants for RNA. Parentheses indicate 95% confidence interval. (****) P ≤ 0.001.
