Expanding the Genetic Code with Phosphothreonine and Phosphotyrosine

Yale University, New Haven CT

Investigators

Abstract

Synthesis of proteins containing amino acids outside of the genetic code has recently resurfaced as a topic of much interest and presents a great challenge intellectually and technically. Reprogramming the genetic code requires two key events: (i) The creation of a new efficient 'orthogonal' aminoacyl tRNA synthetase:tRNA pair able to generate solely a non-canonical aminoacyl tRNA. (ii) The ability to specify the position in which this amino acid will be inserted in the protein by recoding a particular mRNA codon. Based on the natural existence of two archaeal aminoacyl-tRNA synthetase-like proteins (O-phosphoseryl tRNA synthetase with its cognate tRNACys and pyrrolysyl-tRNA synthetase with its cognate tRNAPyl) research will be initiated to develop an Escherichia coli system for the in vivo co translational insertion of phosphothreonine and phosphotyrosine at pre-determined positions in a polypeptide based on the nonsense codons UGA and UAG in a messenger RNA. Given the importance of phosphothreonine and phosphotyrosine in eukaryotic proteins essential for cell proliferation, cellular signaling, and cell death, a robust method of making proteins containing phospho-amino acids will have wide application and great significance. The broader impacts resulting from this project are: (i) The experimental tools to be developed will be widely employed in the future by other researchers in the life sciences. (ii) Development of a viable patent will contribute to commercial technology. (iii) This project will provide interdisciplinary training for undergraduate and postdoctoral students.