(627e) Characterizing the Role of 23S rRNA a- and P-Site Mutations in Translation

Characterizing the role of 23S rRNA A- and P-site mutations in translation

Tasfia Azim, Anne d’Aquino, Michael C. Jewett

Northwestern University

The ribosome, a 2.5-MDa 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 ribosomal 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 in the A- and P-sites of the PTC and inquire how these modifications affect protein synthesis. There is currently no literature on the responses of A- and P-loops to single-base mutations, thus, these studies will not only provide insight into the basic biochemistry of these nucleotides in translation, but also provide the groundwork for engineering the catalytic center of the ribosome. Using iSAT, we have assembled mutant ribosomes possessing single-base substitutions on 23S rRNA nucleotides in the A- and P-loops. By successfully quantifying full-length protein synthesis kinetics of iSAT-assembled wild type and mutant ribosomes, we unexpectedly found many key PTC mutations, which were expected to abolish ribosomal activity, still permitted full-length protein synthesis. We also assessed translation fidelity. Our work provides a comprehensive mapping of the effects of multiple ribosomal mutations on protein synthesis. The understanding gained facilitates efforts to engineer and evolve ribosomes for synthetic biology.