The canonical genetic code can be expanded... it includes the incorporation of an unnatural amino acid into a target protein through assignment of a stop codon (e.g. the amber site UAG). Although this has been done (and in some sense has revolutionized biotechnology), there are major obstacles in the way of achieving a high level of incorporation especially in cases where there are more than one occurences of the unnatural codon in the coding sequence. This is in turn due to the competitive activity of the RF-1 protein which terminates translation upon recognition of the amber sites. Evidently, RF-1 is an essential gene and knocking it out is not an option. The authors in this paper come up with a decent idea for circumventing RF-1.
Genetic code expansion is achieved through introduction of an orthogonal tRNA and AA-tRNA synthetase; orthogonal in the sense that the natural AA-tRNA synthetases don't charge the introduced tRNA and the orthogonal AA-tRNA synthetase does not recognize any other tRNAs. In this paper, Wang et al. have taken this notion one step further: introducing an orthogonal ribosome which is unique for translating the target RNA and doesn't bind RF-1. They evolve such ribosome through mutagenizing 16S rRNA and then selecting for a clone that can efficiently read through an amber mutation in order to grow on chloramphenicole (see the figure below). In the rest of the paper, they attempt to validate the activity of this ribosome (which the call Ribo-X).
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