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Expanding Genetic Code (Peter Schultz)

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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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slide1

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

slide2

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.

slide3

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

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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

slide6

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.

slide7

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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