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

ABSTRACT

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 centre (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 (not RluE) 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°C and 37°C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Elongation 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 Mg²⁺. 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.

Graphical Abstract

KEYWORDS:

• Modification enzymes
• ribosomal RNA
• peptidyl transferase centre
• E. coli,translation

Acknowledgments

We are grateful to Nicola Freyer for pilot fragment reactions, Raymond Fowler for purified translation factors and technical assistance, and Leif Kirsebom and Michael Pavlov for discussions. This work was supported by the Carl Tryggers Foundation, the Tore Nilsons Foundation and the Åke Wibergs Foundation (CTS22:1886, 2022-004 and M22-0037 to G.B.), the Estonian Ministry of Education and Research (PUT PRG1179 to J.R.) and the Swedish Research Council (NT project grant 2017-04148 to A.C.F.).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

LB, JR & ACF conceptualized the study; LB, JL, RCB and GB obtained the data; all authors analysed the data; LB & AF wrote the manuscript and all authors edited it.

Data availability statement

The data supporting the study are presented in the figures and tables in the main text and the supplementary material and are available in more detail upon request from LB and AF.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15476286.2024.2368305

Additional information

Funding

The work was supported by the Carl Tryggers Foundation [CTS22:1886]; Tore Nilsons Foundation [2022-004]; Swedish Research Council [2017-04148]; Estonian Ministry of Education and Research [PUT PRG1179]; Åke Wibergs Foundation [M22-0037].