Amber suppression

Figure 6 A general method for expressing prokaryotic tRNAs in mammalian cells, (a) A type-3 Pol III promoter, the H1 promoter, was combined with other gene elements in different ways to express the Escherichia coii amber suppressor tRNA . The GFP gene containing the TAG mutation at a permissive site serves as a fluorescence reporter for amber suppression. Translation of full-length GFP generates green fluorescence and indicates the expression of functional Escherichia coii amber suppressor tRNA in cells, (b) Total fluorescence intensity of cells transfected with constructs shown in (a). The combination in tRNA4 yields the most efficient tRNA expression.

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],... 

Fig.1. The in vitro site-directed suppression mutagenesis system. Utilizing site-directed mutagenesis a specific codon within a gene under the control of an inducible promoter is converted to an amber termination codon. The gene is added to a cell-free expression system and transcription is induced in the presence of an aminoacyl suppressor tRNA, yielding protein containing the noncoded amino acid at the site corresponding to the termination codon...

The principal drawbacks to the technique are its labor-intensive nature and low protein yields, which in the best cases reach only a milligram or less. Competition with release factors at the amber stop codon often results in truncated protein as the primary product [16, 17]. Suppression efficiency rates are also affected by the character of the amino acid, which determines whether it is a good substrate for the ribosome and protein elongation factors. In addition, context effects variously ascribed to the influence of neighboring mRNA... 

The amber suppressor tRNA (tRNASuP) is introduced to overcome the limitation associated with uniform incorporation of the noncoded amino acid into the target protein. The amino acid-loaded tRNASup incorporates the nonnatural amino acid at a specific site in the protein, corresponding to the nonsense suppression site in the genel144 Phe(4-Tmd)-tRNASup is used to study the interactions of translocating preprolactin with lipids and proteins at the endoplasmic reticulum membranei134 145 ... 

SCHEME 19.7 Amber codon suppression strategy for unnatural azido amino acid incorporation in proteins. 

Interrupted-Proton-Decoupling Experiments. Interrupted-pro-ton-decoupling experiments were carried out with a 50- xs interruption. This technique selects quaternary carbons (those lacking an attached proton) and rapidly moving methyl carbons. Resonances from methylene and methine carbons are suppressed unless, for some reason, their motion within the solid occurs at a rate similar to that of methyl groups. In essence, the interrupted-proton-decoupling experiment, which has not been previously reported for amber samples, provides an alternative fingerprint for the samples. 

Noren CJ, Anthony-Cahill SJ, Suich DJ, Noren KA, Griffith MC, Schultz PG. In vitro suppression of an amber mutation by a... 

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