Progress toward the evolution of an organism with an expanded genetic code.
Several significant steps have been completed toward a general method for the site-specific incorporation of unnatural amino acids into proteins in vivo. An "orthogonal" suppressor tRNA was derived from Saccharomyces cerevisiae tRNA2Gln. This yeast orthogonal tRNA is not a substrate in vitro or in vivo for any Escherichia coli aminoacyl-tRNA synthetase, including E. coli glutaminyl-tRNA synthetase (GlnRS), yet functions with the E. coli translational machinery. Importantly, S. cerevisiae GlnRS aminoacylates the yeast orthogonal tRNA in vitro and in E. coli, but does not charge E. coli tRNAGln. This yeast-derived suppressor tRNA together with yeast GlnRS thus represents a completely orthogonal tRNA/synthetase pair in E. coli suitable for the delivery of unnatural amino acids into proteins in vivo. A general method was developed to select for mutant aminoacyl-tRNA synthetases capable of charging any ribosomally accepted molecule onto an orthogonal suppressor tRNA. Finally, a rapid nonradioactive screen for unnatural amino acid uptake was developed and applied to a collection of 138 amino acids. The majority of glutamine and glutamic acid analogs under examination were found to be uptaken by E. coli. Implications of these results are discussed.
Liu
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DR
Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Schultz
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PG
D019943:Amino Acid Substitution; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005815:Genetic Code; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D012343:RNA, Transfer / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases