Methylation kinetics and RNA affinity binding measurements are consistent with the position of E160 in the ErmE–RNA model. (A) Single-turnover kinetics assays of RNA methylation by ErmE variants were performed using 3H-S-adenosylmethionine (SAM) as the methyl donor. Assays were performed under conditions of limiting SAM or limiting RNA. An oligonucleotide mimicking 23S rRNA helix 73 was used as the substrate. A scatter plot of three replicates is shown along with a best-fit line determined by nonlinear regression. (B) Measurements of binding affinity of E160 variants for the helix 73 analogs were performed by fluorescence polarization using a fluorescein-labeled RNA oligonucleotide. A scatter plot of three replicates is shown for wt, E160A, and E160W. A single replicate of a titration of pepsin is shown as a negative control (Neg.) for RNA binding. E160W possesses reduced affinity for RNA, consistent with a steric clash with the RNA, but E160A does not.
