Revisiting ribosomal catalysis

Bacterial protein synthesis is the major target of antibiotics but key requirements for the catalytic steps, peptide bond formation and release, are unclear. Both steps occur at an RNA active site within the largest ribosomal (r) RNA. In bacteria, this 23S rRNA is complexed with 5S rRNA and 32 r-proteins in the large (50S) ribosomal subunit.

Critical barriers to progress are the tricky E. coli model system and old unoptimized assays. This made determination of the minimal number of the 34 components needed for catalysis difficult.

Thus, one of the biggest questions in enzymology is still open: is rRNA an enzyme or are certain r-proteins required for catalysis? 50S Subunits from thermophilic bacteria are more stable and better experimental models.

In particular, conditions were identified to fold Thermus thermophilus unmodified 23S rRNA into a near-native structure (Lenz et al., 2017). So, we are exploiting this with new assays to finally settle the question above.

First, peptidyl transferase assays will be optimized by simultaneously saturating 50S with its P- and A-site substrates.

Second, our efficient, 30S-free, release assays will be used to test the role of 23S rRNA post-transcriptional modifications in release. Finally, T. thermophilus rRNAs alone will be assayed for catalytic activities.

If our preliminary activity is unverifiable, substrates will be cross-linked to rRNA and key recombinant r-proteins added to create the first synthetic ribosomal peptidyl transferase.