PROJECT SUMMARY Proteins are typically synthesized with 20 amino acids, yet over 300 amino acids are found in proteins as a result of posttranslational modifications (PTMs). These natural noncanonical amino acids (ncAAs) modulate protein function and control fundamental cellular processes. Satisfactory genetic encoding of ncAAs requires the development of efficient and accurate aminoacyl-tRNA formation and delivery to the ribosome by design of tRNAs, tRNA synthetases, and elongation factors that constitute orthogonal translation systems (OTSs). While some ncAAs have been genetically encoded (e.g., N-acetyllysine, phosphoserine (Sep)), OTSs have not been established for a number of critical PTMs. The overall goal of this proposal is to rewire translation by developing OTSs for facile and precise production of natural and engineered proteins containing naturally occurring and synthetic ncAAs. These general goals will be realized in three specific areas of the proposed work. (1) Selenium, in the form of selenocysteine (Sec), is an essential trace element for human health, exhibiting many advantageous chemical properties with its misincorporation implicated in many disease states. We will engineer efficient site-directed insertion of Sec and investigate the effects its insertion along with its precursor Sep into several enzymes of industrial and medical interest. (2) While the genetic code was once thought to be universal, natural codon reassignments in nature are now known to be widespread. We will couple bioinformatic analysis with our knowledge of tRNA identity elements to both reveal novel genetic codes and better characterize the role of this variability in nature. Additionally, we will use long-term evolution to produce an organism with a new genetic code utilizing synthetic amino acids. (3) We plan to create aminoacyl-tRNA synthetases for efficient synthesis of ncAA- tRNA for a series of phosphoamino acids and chemically reactive synthetic amino acids. Given the critical role of phosphorylation in cell signaling and the success of kinase inhibitors against cancer cells, and based on our success establishing an OTS for phosphoserine, we propose to establish OTSs for additional phosphoamino acids and their non-hydrolyzable analogs. Incorporation of chemically reactive amino acids will provide a robust tool to introduce PTMs, biophysical probes, or other valuable residues into a protein of interest. The proposed work is significant because the ability to produce, purify, biochemically and structurally char- acterize proteins containing ncAAs at defined sites is essential for elucidation of fundamental cellular processes and for construction of new tools for protein design. The innovation of the proposed work is to genetically encode these biologically relevant ncAAs, and provide efficient OTSs for biochemical and biomedical researchers to help unravel the complex network of PTMs and their role in biotechnology and human health.