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Expanding Genetic Code (Peter Schultz)

Expanding Genetic Code (Peter Schultz). DNA bases of a gene (A, G, C & T) mRNA (A, G, C & U) protein. tRNA : transfer RNA recognize one specific three base combination “codon” Genetic code : more codons than amino acid

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Expanding Genetic Code (Peter Schultz)

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  1. Expanding Genetic Code (Peter Schultz) DNA bases of a gene (A, G, C & T) mRNA (A, G, C & U) protein tRNA : transfer RNA recognize one specific three base combination “codon” Genetic code : more codons than amino acid 4 x 4 x 4 = 64 different possible ways transcription translation

  2. A method to encode unnatural amino acids with diverse physicochemical and biological properties genetically in bacteria, yeast and mammalian cells Three of them are non-sense codon Stop codon (UAA, UAG, UGA) they don’t code for any amino acid at all. A ribosome that is synthesizing a protein reaches a non-sense codon, the ribosome dissociated from the mRNA. In Schultz Lab. UAG Cell requires tRNA that recognizes UAG tRNA synthetase (loading enzyme that loaded the tRNA with an unusual A.A. site-specific insertion of the unnatural A.A. Functionally “orthogonal” pair (tRNA / synthatase pair) react with each other but not with endogenous yeast pairs. site-specific incorporation of novel A.A. into protein expressed by the yeast.

  3. Methodology Unnatural amino acid • Unique transfer-RNA : codon pair • corresponding aminoacyl-tRNA synthetase 1. Orthogonal tRNA for recognition amber nonsense codon (UAG) 2. Alterthe substrate specificity to recognize unnatural amino acid not Tyr or other endogenous amino acids generate large library

  4. An expanded genetic code • Chemically reactive group • photoreactive • post-translational modification • biophysical probe • redox-active • Metal chelator Unnatural amino acids that have been added to the genetic codes of prokaryotes and eukaryotes

  5. 3. Positive and negative selection A general positive and negative selection scheme for the development of synthetase variants that are specific for an unnatural amino acid in E. coli. Following the generation of a large library (~109 mutants) of, in this case, MjTyrRS active-site mutants, positive and negative selections were carried out. The positive selection was based on resistance to chloramphenicol, which was conferred in the presence of MjTyrRS and the unnatural amino acid (or any natural amino acid that the MjTyrRS could charge onto the orthogonal tRNA) by the suppression of an amber mutation (TAG) at a permissive site in the chloramphenicol acetyltransferase gene (labelled Cmr). The negative selection used the toxic barnase gene with amber mutations at permissive sites and was carried out in the absence of the unnatural amino acid. Only MjTyrRS variants that could acylate the orthogonal tRNATyr CUA with the unnatural amino acid and not with the endogenous amino acids could survive both selections.

  6. MjTyrRS library of mutants selection p-benzoyl-L-phenylalanine (pBpa) Photocrosslinking reactions with pBpa J.W. Chin et al., PNAS, 17, 11020-11024 (2002)

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