UC Berkeley

Cells biosynthesize sequence-defined polymers of 20 canonical α-amino acids known as proteins, while chemists synthesize polymers of diverse monomer structures but with limited control over sequence and length. Expanding the monomer repertoire of the cellular translation system beyond α-amino acids could produce novel sequence-defined polymers with never-before-seen properties. Towards this end, various components of the translation apparatus require engineering to recognize non-native monomers for polymerization. In this dissertation, I describe my work to identify new aminoacyl-tRNA synthetase variants that acylate tRNAs with non-α-amino acids, the first step in cellular translation, and employ the variants to translate peptides containing new monomers. Screening known variants of pyrrolysyl-tRNA synthetase for in vitro acylation activity against a panel of non-α-amino acid monomers led to the discovery of the first α-thio acid, N-formyl-α-amino acid, and α-carboxyl acid synthetase substrates. The crystal structure of a pyrrolysyl-tRNA synthetase variant, MaFRSA, bound to an α-carboxyl acid substrate revealed a defined network of hydrogen bonds used to recognize the second substrate carboxyl group. The pyrrolysyl-tRNA synthetase variants supported translation of peptides bearing α-thio acid, N-formyl-α-amino acid, and α-carboxyl acid monomers at the N-terminus in vitro and translation of a model protein containing an α-hydroxy acid in vivo. To develop a T-box-based translation-independent selection strategy to evolve aminoacyl-tRNA synthetases for non-α-amino acids, I tested orthogonal tRNA recognition by several candidate T-boxes. Translation-dependent selection of pyrrolysyl-tRNA synthetase variants identified initial hits for acylation of several α-hydroxy acids and additional rounds of selection are ongoing.