Towards synthesis of the E. coli ribosome: in vitro characterization of 23S rRNA methyltransferases RlmE, RlmJ and RlmM

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Josefine Liljerum, Tyson R. Shepherd, Jinfan Wang, Avinash S. Punekar, Maria Selmer, Anthony C. Forster

Uppsala University, Sweden

The biggest hurdle to synthesizing the E. coli ribosome in vitro is reconstituting several post-transcriptional modifications of the peptidyl transferase center (PTC) of 23S ribosomal RNA (1). The modification reaction whose knock out produces the most significant growth defect is methylation of the ribose 2’O of U2552 by RlmE (2). Two other modification reactions of the PTC are performed by the recently-discovered enzymes RlmJ (3) and RlmM (4) which methylate the base of A2030 and the ribose 2’O of C2498, respectively. These two nucleotides are adjacent in the ribosome due to a tertiary interaction between A2030 and C2498. All three enzymes use S-adenosyl-methionine as the methyl donor. In this study, all three enzymes were overexpressed in His-tagged form and their reactions assayed and optimized using reverse transcription and tritium-incorporation assays. RlmE required the full 50S ribosomal subunit as a substrate, as previously demonstrated (2). In contrast, RlmJ and RlmM were both found to specifically methylate 23S rRNA in unmodified form, facilitating substrate recognition studies. Domain V alone was sufficient for recognition by RlmM (5), while RlmJ only required a 26-nucleotide stem loop. This work should aid future ribosome reconstitution from transcripts for directed evolution of ribosomes and synthesis of a minimal cell. References 1. Forster, A C, Church, G M (2006) Mol Syst Biol, 2(45), 1. 2. Caldas T, Binet E, Bouloc P, Costa A, Desgres J, Richarme G (2000) J Biol Chem. 275(22), 16414. 3. Golovina A Y, Dzama M M, Osterman I A, Sergiev P V, Serebryakova M V, Bogdanov A A, Dontsova O A (2012) RNA, 18, 1725. 4. Purta E, O’Connor M, Bujnicki J M, Douthwaite S (2009) Molecular Microbiology 72(5), 1147. 5. Punekar A S, Shepherd T R, Liljeruhm J, Forster A C, Selmer M (2012), Nucleic Acids Res, 40, 10507.