Four-base Codons

Figure 10 Alteration of the genetic code for incorporation of non-natural amino acids, (a) In nonsense suppression, the stop codon UAG is decoded by a non-natural tRNA with the anticodon CUA. In vivo decoding of the UAG codon by this tRNA is in competition with termination of protein synthesis by release factor 1 (RFl). Purified in vitro translation systems allow omission of RF1 from the reaction mixture, (b) A new codon-anticodon pair can be created using four-base codons such as GGGU. Crystal structures of these codon-anticodon complexes in the ribosomal decoding center revealed that the C in the third anticodon position interacts with both the third and fourth codon position (purple line) while the extra A in the anticodon loop does not contact the codon.(c) Non-natural base pairs also allow creation of new codon-anticodon pairs. Shown here is the interaction of the base Y with either base X or (hydrogen bonds are indicated by red dashes).

Hohsaka T, Ashizuka Y, Taira H, Murakami H, Sisido M. Incorporation of normatural amino acids into proteins by using various four-base codons in an escherichia coU in vitro translation system. Biochemistry 2001 40 11060-11064. 

It is argued that the extension of the lengths of codons and anticodons might cause steric overcrowding between the tRNAs in the ribosomal A site and P site. The overcrowding in ribosome, however, has been avoided by a bend of mRNA chain at the junction between the A and P sites [52]. Because of this bend, the main bodies of the two tRNAs are well separated, while the two anticodons as well as the amino acid and the peptide C-terminal are close to each other. Indeed, even five-base codons [53] and a tandem four-base codon [54] have been reported to be successful. 

Similar to the amber codon method, four-base codon method has been shown to work in living cells [55]. 

K. Taira, M. Sisido, Position-specific incorporation of a fluorophore-quencher pair into a single streptavidin through orthogonal four-base codon/anticodon pairs, J. Am. Chem. Soc. 2002, 124, 14586-14589. 

M. Sisido, Four-base codon-mediated incorporation of nonnatural amino acids into proteins in a eukaryotic cell-free translation system, J. Biosci. Bioeng. 2005, 99, 473-476. 

R. Abe, H. Murakami, M. Sisido, Position-specific incorporation of dansylated nonnatural amino acids into streptavidin by using a four-base codon, FEBS Lett. 2004, 560, 173-177. 

Maghery, XJ., Anderson, J.C., and Schultz, RG., Expanding the genetic code selection of efficient suppressors of four- base codons and identification of shifiy four-base codons with a library approach in Escherichia coli, J. Mol. Biol, 307, 3, 755, 2001. 

---