Characterizing the Role of 23S rRNA Peptidyltransferase Center Nucleotides in Translation

The ribosome, a 2.5MDa molecular machine that polymerizes α-amino acids into proteins, is the catalytic workhorse of the translation apparatus. The catalytic capacity of the translation machinery has attracted extensive efforts to repurpose it for novel functions. One key idea is that the natural translation machinery can be harnessed to synthesize polymers containing non-natural building blocks. Expanding the repertoire of ribosome substrates and functions is a difficult task, however, because the requirement of cell viability severely constrains the alterations that can be made to the ribosome, a catalyst that sustains the life of a cell. In practice, these constraints have made the natural ribosome nearly unevolvable and, so far, no generalizable approach for modifying the catalytic peptidyl transferase center (PTC) of the ribosome has been advanced. We propose to address this grand challenge by using cell-free systems that harness the biosynthetic potential of cellular machines without using intact cells, thus removing cell viability constraints. Here, we use our in vitro ribosome synthesis, assembly, and translation system (termed iSAT) to generate variant ribosomes with mutations in the 23S rRNA (especially those that are dominantly lethal in the PTC and exit tunnel) and inquire how these modifications affect protein synthesis. Using iSAT, we assembled more than 50 mutant ribosomes possessing single and some double base substitutions at 23S rRNA nucleotides in the PTC. By successfully quantifying full-length protein synthesis kinetics of iSAT-assembled WT and mutant ribosomes, we unexpectedly found many key PTC mutations, which were expected to abolish ribosome activity, still permitted full-length protein synthesis. We also assessed fidelity. Our work provides a comprehensive mapping of the systems effect of multiple ribosomal mutations on protein synthesis. The understanding gained facilitates efforts to engineer ribosomes for synthetic biology.