Principal Investigator Uttam RajBhandary
We are working on developing methods for the incorporation of unnatural amino acids carrying fluorescent, photoaffinity or spectroscopic probes and heavy atoms, chemically reactive amino acids and phosphoamino acid analogues into proteins in vivo in eubacteria and in eukaryotic cells. Such proteins can be used for a variety of studies including the intracellular location of proteins, protein-protein interactions, signal transduction, analysis of structure, assembly and function of proteins and use of proteins as biosensors. A first step is the identification of:
(1) suppressor tRNAs that cannot be aminoacylated by any of the endogenous aminoacyl-tRNA synthetases, and
(2) aminoacyl-tRNA synthetases, which aminoacylate the suppressor tRNA but no other tRNA in the cell.
We have identified such orthogonal synthetase-tRNA pairs for use in E. coli, yeast and mammalian cells. We are now working on isolation of mutants in the aminoacyl-tRNA synthetase, which utilize the desired amino acid analogue instead of the normal amino acid. In collaboration with the group of Thomas Sakmar at Rockefeller University, we have recently described the incorporation of two chemically reactive keto amino acids at various sites of two G protein-coupled receptors, rhodopsin and CCR5.
We have also described a potentially more generally applicable system involving import into cells of suppressor tRNAs aminoacylated with the unnatural amino acid. The only requirement for this approach is that the suppressor tRNA that is imported should not be aminoacylated in the cells. We have identified such suppressor tRNAs and have imported amber (UAG) and ochre (UAA) suppressor tRNAs into mammalian cells. We have further shown that amber and ochre codons present in a reporter mRNA can be concomitantly suppressed by these tRNAs. These results open up the possibility of incorporating two different unnatural amino acids into the same protein in mammalian cells.