Expansion of the Genetic Codes

Expansion of the Genetic Codes 5.1.4.1 Amber and Other Stop Codons 

The second key step for the expansion of the biosynthesizing system to introduce nonnatural amino acids is the expansion of the genetic codes. Schultz [38] and Chamberlin [39] first assigned an amber (UAG) stop codon to a nonnatural amino acid (aa ). By adding an aa -tRNA with a CUA anticodon as a suppressor of the amber codon, they successfully introduced the nonnatural amino acid at that position. Since then, the amber suppression method has been employed by a number of researchers. This method is advantageous in that an unsuccessful decoding of the UAG codon automatically leads to 

It is not trivial that the amber suppression method can be used in living cells, because some of essential proteins may not be synthesized properly in the presence of a large amount of the aminoacylated suppressor tRNA. However, the amber suppression method has been reported to work successfully in Xenopus oocyte [40, 41], E. coli [23-25], and mammalian cells [28,42-44], 

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Selenoprotein synthesis An expansion of the genetic code. Trends Biochem. Sci. 16 463-467, 1991. 

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Ambrogelly A, Palioura S, Soil D. Natmal expansion of the genetic code. Nat. Chem. Biol. 2007 3 29-35. 

Bae JH, Rubini M, Jung G, Wiegand G, Seifert MHJ, Azim MK, Kim J-S, Zumbusch A, Holak TA, Moroder L, Huber R, Budisa N. Expansion of the genetic code enables design of a novel Gold Class of green fluorescent proteins. J. Mol. Biol. 2003 328 1071-1081. 

Aza-dC has been used to examine the role of methyltransferases/ where it was shown to be an inhibitor of Hhal DNA methyltransferase. Isonucleosides, e.g., isocytidine, can be used for new base-pair motifs. However, iso-dC is unstable to acidic conditions making ODN synthesis problematic. The substitution of iso-dC by 6-aza-iso-dC leads to more stable ODNs, though some duplex instability is introduced. Various pyrimidine analogues, such as (103) and (104), have been used as substrates of modified HIV-RT polymerase in DNA containing non-standard base pairs with them. Such new base pairs are of interest for expansion of the genetic code (see later). 

Ambrogelly et al (2007) Natural expansion of the genetic code. Nature Chem Biol 3 29-35 Bernstein MP, Dworkin JP, Sanford SA et al (2002) Racemic amino acids from the ultraviolet photolysis of interstellar ice analogues. Nature 416 401 —403 Blackmond DG, Klussmann M (2007) Spoilt for choice assessing phase behaviour models for the evolution of homochirality. Chem Commun 3990-3996 Breslow R (1959) The mechanism of the formose reaction. Tetrahedron Lett 21 22-26 Breslow R et al (2010) Initiating prebiotic homochirality on Earth. Orig Life Evol Biosph 40 11-26... 

T. Mitsui, T. Fujiwara, S. Yokoyama, A two-unnatural-base-pair system toward the expansion of the genetic code,/. Am. Chem. Soc. 2004, 126, 13298-13305. 

The trace element selenium is present in a number of proteins in the form of cotranslationally inserted selenocysteine (Sec). This insertion occurs when a unique selenocysteinyl-tRNA recognizes an internal UGA codon. Selenocysteine has therefore been called the 21st amino acid and its insertion at a nucleotide triplet that is normally a termination signal represents an expansion of the genetic code. 

Furter, R., Expansion of the genetic code site-directed p-fluoro-phenylalanine incorporation in Escherichia coli, Protein ScL, 7, 2, 419,1998. 

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