Lost in Translation: Mapping Ribosomal Active Site Mutations in Vitro

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 with mutations in the active site, and inquire how these modifications impact protein synthesis. The active site of the ribosome is composed solely of ribosomal RNA (rRNA). This rRNA is assembled into three key loops, which facilitate translation: the peptidyl transferase loop, the A-loop, and the P-loop. The peptidyl transferase loop plays a key role in positioning substrates, is involved with peptide stalling, and is a key target for antibiotic binding. The A- and P-loops flank the peptidyl transferase loop within the ribosome’s active site. The A-loop is responsible for aminoacyl-tRNA interactions with the ribosome, and the P-loop is known to play a role in further facilitating peptide bond formation between amino acids. Because mutations to many of the nucleotides in these three active site loops confer lethal phenotypes, few to no studies exist in probing and fully characterizing them.

Understanding the effects of single-base mutations on these active site nucleotides will provide insight into the basic biochemistry of these bases in translation, but also provide the groundwork for engineering the catalytic center of the ribosome. Using iSAT, we have assembled 180 different variant ribosomes possessing single-base substitutions of 23S rRNA nucleotides in these active site 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 and ribosome assembly, as well as mapped mutant activity onto the ribosome’s crystal structure. Our work provides the first and only comprehensive mapping of the impacts of every mutation within the ribosome’s active site on protein synthesis. The understanding gained from these studies facilitates efforts to engineer and evolve ribosomes for synthetic biology.