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Abstract 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. 

We demonstrate in vitro incorporation of cyclic β-amino acids into peptides by the ribosome through genetic code reprogramming. Further, we show that incorporation efficiency can be increased through the addition of elongation factor P. 

Abstract Ribosome-mediated polymerization of backbone-extended monomers into polypeptides is challenging due to their poor compatibility with the translation apparatus, which evolved to use α-L-amino acids. Moreover, mechanisms to acylate (or charge) these monomers to transfer RNAs (tRNAs) to make aminoacyl-tRNA substrates is a bottleneck. Here, we rationally design non-canonical amino acid analogs with extended carbon chains (γ-, δ-, ε-, and ζ-) or cyclic structures (cyclobutane, cyclopentane, and cyclohexane) to improve tRNA charging. We then demonstrate site-specific incorporation of these non-canonical, backbone-extended monomers at the N- and C- terminus of peptides using wild-type and engineered ribosomes. This work expands the scope of ribosome-mediated polymerization, setting the stage for new medicines and materials. 

Abstract Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ~1.7 × 10⁷member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ~4 × 10³rRNA variants. We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties. 

Abstract Ribo-T is a ribosome with covalently tethered subunits where core 16S and 23S ribosomal RNAs form a single chimeric molecule. Ribo-T makes possible a functionally orthogonal ribosome–mRNA system in cells. Unfortunately, use of Ribo-T has been limited because of low activity of its original version. Here, to overcome this limitation, we use an evolutionary approach to select new tether designs that are capable of supporting faster cell growth and increased protein expression. Further, we evolve new orthogonal Ribo-T/mRNA pairs that function in parallel with, but independent of, natural ribosomes and mRNAs, increasing the efficiency of orthogonal protein expression. The Ribo-T with optimized designs is able to synthesize a diverse set of proteins, and can also incorporate multiple non-canonical amino acids into synthesized polypeptides. The enhanced Ribo-T designs should be useful for exploring poorly understood functions of the ribosome and engineering ribosomes with altered catalytic properties.