UC Berkeley

Orthogonal ribosomes are essential tools for expanding the genetic code, enabling the incorporation of non-canonical amino acids (ncAAs) into proteins, and facilitating the synthesis of novel biopolymers. However, current systems are limited by translation of endogenous mRNAs and cross-assembly with wild-type ribosomal subunits, preventing full orthogonality. This work presents multiple strategies to improve orthogonal ribosome function in Escherichia coli by addressing both of these limitations.First, we investigated the role of ribosomal protein bS1 in Shine-Dalgarno (SD) independent translation, hypothesizing that its exclusion from orthogonal 30S subunits might enhance specificity for orthogonal mRNAs. We engineered 16S rRNA variants with structural extensions in helix h26 to sterically block bS1 binding. While cryo-EM and biochemical assays confirmed successful bS1 exclusion, orthogonality was not significantly improved, indicating that additional mRNA features drive SD-independent translation.Second, we developed a novel strategy to disrupt cross-assembly by targeting inter-subunit rRNA bridges. Specifically, we mutated nucleotides in bridge B3 to prevent interaction between orthogonal 30S and wild-type 50S subunits. Cryo-EM analysis confirmed structural disruption at the interface, and we restored partial activity by rationally designing compensatory 23S rRNA mutations. To expand this approach, we screened a 23S rRNA library using a functional selection based on SecM stalling, followed by RNase H treatment to enrich for translationally competent complexes.Finally, we applied a parallel engineering strategy to bridge B7a, introducing phylogenetically guided mutations at 16S rRNA position A702 and pairing them with complementary mutations in 23S rRNA. Although less extensively characterized than the B3 bridge, this approach further supports the feasibility of engineering orthogonal ribosome pairs through targeted subunit interface design.Together, these findings provide a dual framework, modulating translation initiation and subunit specificity, for improving orthogonal ribosomes. This work lays the foundation for future efforts to construct fully orthogonal translation systems, enabling sophisticated genetic programs and expanded chemical capabilities in living cells.