Progress toward an expanded eukaryotic genetic code.
Expanding the eukaryotic genetic code to include unnatural amino acids with novel properties would provide powerful tools for manipulating protein function in eukaryotic cells. Toward this goal, a general approach with potential for isolating aminoacyl-tRNA synthetases that incorporate unnatural amino acids with high fidelity into proteins in Saccharomyces cerevisiae is described. The method is based on activation of GAL4-responsive HIS3, URA3, or lacZ reporter genes by suppression of amber codons in GAL4. The optimization of GAL4 reporters is described, and the positive and negative selection of active Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA(CUA) is demonstrated. Importantly, both selections can be performed on a single cell and with a range of stringencies. This method will facilitate the isolation of a range of aminoacyl-tRNA synthetase (aaRS)/tRNA(CUA) activities from large libraries of mutant synthetases.
Chin
Jason W
JW
Department of Chemistry, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Cropp
T Ashton
TA
Chu
Stephanie
S
Meggers
Eric
E
Schultz
Peter G
PG
D000596:Amino Acids / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism*; D017930:Genes, Reporter; D005815:Genetic Code; D005821:Genetic Techniques; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014158:Transcription, Genetic
D000596:Amino Acids; D011506:Proteins; D012346:RNA, Transfer, Amino Acyl; D000604:Amino Acyl-tRNA Synthetases