Knocking out key modifications in E. coli 23S rRNA. (A) Secondary structure of domain V including the “critical region” of the 23S rRNA (boxed) around the PTC loop (adapted from Punekar et al. 2013). Shown are all domain V modifications (ho5C is partial), modification enzymes KO'ed in this study (inside colored boxes), and known modification enzymes remaining (black). (B) Direct evidence for reductions in modified nucleotides in 50S subunits due to two different combinations of KOs (ΔrluC/ΔrlmKL/ΔrlmN/ΔrlmM/ΔrluE is abbreviated to ΔCKLNMuE). rRNAs were digested into nucleosides that were separated by HPLC (Supplemental Fig. S2), and modification peak areas were compared with WT controls normalized to 1. Standard errors of two to three biological replicates are given with absent peaks indicated by absent bars. ΔrluC/ΔrlmE should delete 3/9 Ψs (RluC) and 1/1 Um (RlmE). ΔCKLNMuE should delete 4/9 Ψs (RluC and RluE), 1/1 m7G (RlmKL), 1/2 m2G (RlmKL), 1/1 m2A (RlmN), and 1/1 Cm (RlmM). The bars decreased by amounts close to those expected.
