ErmE site-directed mutants associated with an erythromycin-sensitive phenotype display RNA methylation defects in vitro. (A) SDS-PAGE analysis of wild-type ErmE and variants indicates that the proteins are reasonably pure and display the expected molecular weight. (B) Wild-type ErmE and variants produce similar circular dichroism spectra indicating there is no major change in protein structure in the variants. (C) Single-turnover kinetics assays were used to characterize RNA methylation by selected site-directed mutants from the basic patch region. 3H-S-adenosylmethionine (SAM) was used as the methyl donor to follow product formation, resulting in the transfer of a radio-isotopically labeled methyl group (red circle) from SAM to RNA. (D) Single-turnover kinetics assays were performed on site-directed mutants from the adenosine pocket and α4-cleft. A scatter plot of three replicates is shown along with a best-fit line derived by nonlinear regression.
