Towards synthesis of the E. coli ribosome: reconstitution of 5 modification reactions of 23S rRNA and crystal structure of one of the modification enzymes, RlmM

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Tyson Shepherd, Avinash S. Punekar, Josefine Liljeruhm, Maria Selmer, Anthony C. Forster

Uppsala University, Sweden

Synthesis of active E. coli ribosomes from in vitro-transcribed 23S ribosomal RNA (rRNA) is a key step towards several goals of synthetic biology, such as in vitro evolution of ribosomes, antibiotic discovery and synthesis of a minimal cell (1). Reconstitution analyses showed that active E. coli ribosomes could not be assembled using in vitro-transcribed rRNAs alone, but required one to six post-transcriptional chemical modifications in a 78-nucleotide critical region of the 23S rRNA comprising part of the peptidyl transferase center (2). Recently, five of the six enzymes responsible for these modifications were identified, namely methyltransferases RlmKL, RlmM and RlmN, and pseudouridine synthases RluC and RluE (3). Using a bottom-up approach, we have overexpressed these modification enzymes and reconstituted their reactions in a purified system. Tritium-labeling and primer-extension assays demonstrated that in vitro-transcribed 23S rRNA is a substrate for several of the enzymes, while combined modification reactions showed surprising cooperativity. In addition, we elucidated the crystal structure of modification enzyme RlmM in complex with its cofactor, S-adenysyl methionine (SAM) (4). RlmM is composed of a large N-terminal RNA recognition domain that presumably provides substrate specificity with a conserved, positively-charged patch. As expected, SAM was bound at the catalytic C-terminal methyltransferase domain at a Rossman fold. However, the SAM pocket was surprisingly shallow, suggesting that the site becomes deeper upon RNA binding. This new synthetic biology approach to rRNA modification reactions and ribosome reconstitution should illuminate the functions of these enigmatic modifications while moving us closer to synthesizing a minimal cell. 1. Forster AC, Church GM (2006) Mol Syst Biol 2:45. 2. Green R, Noller HF (1996) RNA 2:1011. 3. Purta E, O’Connor M, Bujnicki JM, Douthwaite S (2009) Mol Microbiol 72:1157. 4. Punekar AS, Shepherd TR, Liljeruhm J, Forster AC, Selmer M (2012) Nucleic Acids Res 40:10507.