Genetic encoding of Post-translational modifications

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Post-translational modifications (PTMs) are covalent modifications on side chains or termini of proteins installed by specific enzymes after ribosomal protein synthesis in cells. PTMs play an important role in modulating several biological processes and is one of the methods used by nature to increase the repertoire of chemical functional groups in proteins. Methylation of arginine is an important PTM that modulates the activity of numerous proteins involved in RNA processing, transcriptional regulation, signal transduction and DNA repair. Tyrosine phosphorylation, another PTM, that plays an important role in cell-cycle progression, cell proliferation, cellular homeostasis, neural activity, transcriptional activation and ageing. Many diseases, including cancer and neurological disorders, are associated with abnormal levels of PTMs. Despite the importance of PTMs in biology, it is often difficult to obtain a homogeneous sample of protein with specific PTMs. This is mainly because cells have competing mechanisms to install and remove PTMs, and samples of post-translationally modified proteins also contain unmodified proteins. It is often difficult to separate post-translationally modified proteins from their unmodified analogues. The problem is further compounded if the protein has multiple sites for PTMs. In addition, proteomics analyses have revealed numerous previously unidentified sites of PTMs on many proteins. Often enzymes that install these PTMs are not known, thus further limiting the access to these post-translationally modified proteins. To address these challenges, here we propose to develop methods for genetic site-specific incorporation of methylarginines and phosphotyrosine. This work would provide facile access to several important post-translationally modified proteins. Proteins are synthesized in all organisms by a biological machine called the ribosome. The ribosome uses messenger RNA (mRNA) as a template and transfer RNA (tRNA) as an adaptor molecule to catalyse protein synthesis using a set of rules defined by the genetic code. For each naturally occurring amino acid there are, 1) a unique set of triplet bases in the DNA or mRNA, called codons, which correspond to the amino acid, and 2) a unique set of aminoacyl-tRNA synthetases (aaRS)/ tRNA pairs. aaRSs catalyze the charging of their corresponding tRNAs with their corresponding amino acids. During protein synthesis, the charged tRNA is taken to the ribosome where it binds to its cognate codon on the mRNA in the small subunit of the ribosome, and concurrently an amino acid is added to the growing peptide chain in the large subunit. Genetic incorporation of unnatural amino acids in cells has been achieved by assigning an amber stop codon or quadruplet codons to unnatural amino acids and supplying the cells with orthogonal aaRS/ tRNA pairs. Orthogonal aaRS/ tRNA pairs do not interfere with the tRNA synthetases and the tRNAs of the host cell, and specifically recognize the unnatural amino acid. Using this approach, numerous unnatural amino acids have been genetically encoded in different organisms. However, only a small number of PTMs have been genetically encoded. In this proposal, we explore different approaches to identify new orthogonal aaRS/ tRNA pairs in E. coli to enable genetic encoding of methylarginines and phosphotyrosine.