Computationally-guided design and selection of high performing ribosomal active site mutants

Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g. library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing of combined ribosomal assembly and translation activity, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design and selection may serve as a powerful tool for rRNA engineering.

Automated summary

Understanding how modifications to the ribosome affect its function is important for various applications, such as studying ribosome biogenesis and repurposing ribosomes for synthetic biology. However, designing sequence-modified ribosomes has been challenging due to functional limitations caused by point mutations, especially in the catalytic active site. In this study, a computational rRNA design approach was developed to overcome these limitations and successfully engineer ribosomes with mutant active sites. The approach also identified new epistatic interactions and improved ribosomal phenotypes.