Suppressor tRNAs

Enzymatic Synthesis.—The total synthesis by chemical and enzymatic methods of the structural gene for the precursor of a tyrosine suppressor tRNA from E. coli, a DNA duplex that is 126 nucleotides long, has been described. This, Khorana s Meisterwerk to date, represents the highest state of the art, and proceeds by methods which are already famous, and the touchstone of those who wish to follow.135... 

A mutant amber suppressor tRNA library based on the... 

Initial attempts at introducing unnatural amino acids into proteins in mammalian cells involved the transfection of an amber suppressor tRNA that is chemically acylated with an unnatural amino acid in vitro This approach limits the amount of overall protein that can be produced since the acylated suppressor tRNA is consumed stoichiometrically and cannot be regenerated inside cells. It is also technically demanding to prepare the chemically acylated tRNA. 

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.

Escherichia coli tRNA -Derived Suppressor tRNA. 86... 

Addition of the modified gene to a cell-free system containing the appropriate RNA polymerase and semi-synthetic suppressor tRNA. 

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 suppressor tRNA developed by the Chamberlin lab for use in a rabbit reticulocyte lysate is based on an E. coli glycyl tRNA, which was initially chosen because glycyl-tRNA synthetases do not rely on a double-sieve editing mechanism for enzymatic hydrolysis of misacylated tRNAs [26]. Two base pair changes were made to the acceptor stem to allow incorporation of the optimal T7 RNA polymerase promoter into the DNA template for tRNA y-Con [27,28],... 

Fig. 4. Comparison of E. coli tRNA and semi-synthetic suppressor tRNA y. The residues that have been changed are highlighted...

The Lester and Dougherty labs, which have collaborated to extend the suppression mutagenesis technique to Xenopus oocytes with remarkable success [30, 31], began with a suppressor tRNA ( MN3 ) designed for in vivo use and demonstrated that it functioned more effectively in the oocyte system than a yeast tRNA -derived suppressor tRNA. They have since developed an alternative suppressor based on tRNA " from Tetrahymena thermophila that has proven to be considerably more versatile, efficient and accurate in the oocyte system [32], as well as showing good suppression efficiency in E. coli transcription-translation reactions [33]. 

Table 1. Comparison of suppressor tRNAs in the Xenopus oo cyte system. See text for details...

UAG Position in nAChR Suppressor tRNA Efficiency ratio... 

Escherichia coii tRNA "-Derived Suppressor tRNA... 

The ability of both suppressor tRNAs to incorporate the nonpolar amino acid valine as well as the polar noncoded homoglutamate into two proteins was tested in E. coli cell-free transcription-translation systems [35]. The proteins T4... 

When the DNA templates were added to M. luteus transcription-translation reactions containing the corresponding charged suppressor tRNAs, full-length... 

At this stage, it is clear that ribosomal protein synthesis will not allow the incorporation of D-amino acids or those that are too bulky, and that only a-ami-no and a-hydroxy acids can be introduced those with extended backbones such as y-amino acids cannot be introduced in this manner. Also, in spite of the development of improved suppressor tRNAs, the incorporation of small, highly polar amino acids remains difficult. 

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