Translational impacts of enzymes that modify ribosomal RNA around the peptidyl transferase centre

Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally-important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the “critical region” of the peptidyl transferase center (PTC) in 23S rRNA that exhibited only a minor growth defect at 37°C (although major at 20°C). However, our combined KO of modification enzymes RluC and RlmE resulted in conditional lethality (at 20°C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20 and 37°C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Rates of protein synthesis in vivo , as judged by the kinetics of β-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37°C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg ²⁺ . Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se , rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.