The preparation of chemically-modified Cys-tRNACys and studies on its possible use for incorporating non-natural amino acids into proteins

grantor: University of TorontoBiosynthetic incorporation of fluorophores into proteins can be accomplished by supplying fluorescently-tagged aminoacyl-tRNA (aa-tRNA) to the cellular protein synthesis machinery. A key step in this process is the formation of a ternary complex consisting of an elongation factor (EF-Tu in bacteria), GTP, and the aminoacyl-tRNA. In an effort to explore the range of side-chain structure that can be tolerated by EF-Tu, we prepared Cys-tRNACys and reacted the cysteinyl side-chain with a structurally diverse set of fluorescent compounds. All modified aa-tRNA tested were found to bind to EF-Tu-GTP and binding was specific as demonstrated by the lack of an interaction with immobilized EF-Tu-GDP. These results are compared with the predictions of modelling based on the recently reported X-ray structure of the Cys-tRNACys-EF-Tu-GDPNP complex. It appears that, except perhaps for certain modifications very close to the backbone of the amino acid, a very wide range of side-chain structures can be tolerated by EF-Tu. We show that reverse-phase high performance liquid chromatography using a C8 column can be employed to purify fluorescently-labelled Cys-tRNA Cys from a crude mixture which also contains unaminoacylated tRNA Cys, unlabelled Cys-tRNACys, and disulphide-linked Cys-tRNACys. Towards expanding the range of methods available for site-specific protein labelling, we proposed a technique which is based on the use of chemically-modified cysteinyl-tRNACys. The synthetic tRNA is synthesized via a combination of enzymatic aminoacylation and chemical modification of the cysteinyl side-chain. When it is added to a rabbit reticulocyte lysate in vitro coupled transcription/translation system that has been programmed with the gene of interest, the non-standard amino acid should be incorporated into the target protein at positions that correspond to cysteine. We attempted to label firefly luciferase with fluorescein and a biotin derivative using fluorescein- and biotin-labelled Cys-tRNACys, respectively.Ph.D