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 the recently discovered methanogen aminoacylsynthetase like protein, O-phosphoseryl tRNA synthetase and its cognate tRNACys research will be initiated to develop a prokaryotic and a eukaryotic system for the cotranslational insertion of phosphoserine at pre determined positions in a polypeptide based on the nonsense codon UAG in a messenger RNA. Given the importance of phosphoserine in eukaryotic proteins essential for cell proliferation, cellular signaling, and cell death, a robust method of making phosphoserine containing proteins will have wide application and great significance. This project will provide interdisciplinary training for undergraduate and postdoctoral students.
From the simplest single celled bacterium to the most complex cells in the human brain, proteins are responsible for many of the chemical reactions and processes that allow cells to grow and thrive. In all the diversity of life, most proteins are made of a particular sequence of 20 amino acids that are linked together to form polypeptide chains also known as proteins. Most fascinatingly, after proteins are made, the individual amino acids can be later altered with particular chemical modifications of which many hundreds are now known. One of the most common and important types of these modified amino acids are phosphorylated amino acids. Phosphorylated amino acids play key roles in cellular signaling pathways, which allow cells to sense chemical signals and then use these signals to determine whether that particular cell should, for example, live or die. Mutations in certain proteins that make up these signaling pathways are found in up to 30% of all human cancers. Because these proteins can be phosphorylated at multiple sites, it has so far been impossible to study the different roles of particular phosphorylated amino acids in normal cellular signaling and in disease states such as cancer. While working with an unusual microbe that makes its living nearby deep sea hydrothermal vents, our lab discovered an enzyme that could be exploited to incorporate phosphorylated serine (phosphoserine or Sep) at any desired site in a protein. The enzyme (called phosphoseryl-tRNA synthetase or SepRS) chemically attaches Sep to a transfer RNA molecule (called Sep-tRNA) that can then be utilized to include Sep in protein synthesis. Since Sep is not a normal amino acid for protein synthesis, we found that the normal protein synthesis machinery of the cell would not accept this unusual amino acid. We applied a protein engineering scheme to alter a component of the protein synthesis machinery (called elongation factor Tu or EF-Tu), which is responsible for delivering Sep-tRNA to the ribosome. The ribosome is a large molecule, composed of RNA and protein, which conducts the synthesis of proteins. Our engineering effort led to the first successful incorporation of Sep into a growing polypeptide chain. To further test our system, we showed successful incorporation of Sep at a desired location in a protein that plays a major role in the human signaling pathways mentioned above. This technological achievement will allow for advances in our basic understating of how signaling pathways function and also for the design of specific inhibitors of this signaling pathway, which could ultimately lead to better treatment options and outcomes for patients suffering from a variety of malignances. The system we developed will also be useful for protein engineering endeavors that can now use an alphabet of 21 instead of 20 amino acids to develop proteins with novel properties. A number of undergraduate students, some from underrepresented groups, and also high school students and a high school teacher were involved in different stages of this funded research. By engaging young students in such exciting research, our project was able to broaden the impact of the research funding by attracting more young people to science specifically and by expanding scientific literacy among young students in general.
