Improved Amber and Opal Suppressor tRNAs for Incorporation of Unnatural Amino Acids In Vivo, Part 1. Minimizing Misacylation of Suppressor tRNAs
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Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: minimizing misacylation.
The incorporation of unnatural amino acids site-specifically is a valuable technique for structure-function studies, incorporation of biophysical probes, and determining protein-protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous am...
متن کاملImproved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 2: evaluating suppression efficiency.
The incorporation of unnatural amino acids into proteins is a valuable tool for addition of biophysical probes, bio-orthogonal functionalities, and photoreactive cross-linking agents, although these approaches often require quantities of protein that are difficult to access with chemically aminoacylated tRNAs. THG73 is an amber suppressor tRNA that has been used extensively, incorporating over ...
متن کاملComplete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells.
We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Acti...
متن کاملA general approach for the generation of orthogonal tRNAs.
BACKGROUND The addition of new amino acids to the genetic code of Escherichia coli requires an orthogonal suppressor tRNA that is uniquely acylated with a desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. A tRNA(Tyr)(CUA)-tyrosyl-tRNA synthetase pair imported from Methanococcus jannaschii can be used to generate such a pair. In vivo selections have been developed for sele...
متن کاملIn vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression.
Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for studying structure-function relationships, incorporating biophysical probes, and elucidating protein-protein interactions. In higher eukaryotic cells, the methodology is currently limited to incorporation of a single UAA in response to a stop codon, which is known as nonsense suppression. Frameshift...
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