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. 2001 Mar 30;307(3):755-69.
doi: 10.1006/jmbi.2001.4518.

Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of "shifty" four-base codons with a library approach in Escherichia coli

T J Magliery  1 , J C Anderson, P G Schultz
Affiliations

Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of "shifty" four-base codons with a library approach in Escherichia coli

T J Magliery et al. J Mol Biol. .

Abstract

Naturally occurring tRNA mutants are known that suppress +1 frameshift mutations by means of an extended anticodon loop, and a few have been used in protein mutagenesis. In an effort to expand the number of possible ways to uniquely and efficiently encode unnatural amino acids, we have devised a general strategy to select tRNAs with the ability to suppress four-base codons from a library of tRNAs with randomized 8 or 9 nt anticodon loops. Our selectants included both known and novel suppressible four-base codons and resulted in a set of very efficient, non-cross-reactive tRNA/four-base codon pairs for AGGA, UAGA, CCCU and CUAG. The most efficient four-base codon suppressors had Watson-Crick complementary anticodons, and the sequences of the anticodon loops outside of the anticodons varied with the anticodon. Additionally, four-base codon reporter libraries were used to identify "shifty" sites at which +1 frameshifting is most favorable in the absence of suppressor tRNAs in Escherichia coli. We intend to use these tRNAs to explore the limits of unnatural polypeptide biosynthesis, both in vitro and eventually in vivo. In addition, this selection strategy is being extended to identify novel five- and six-base codon suppressors.

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Figures

Figure 1
Figure 1
Schematic of β-lactamase libraries and tRNASer libraries. Insertion of NNNN at Ser70 or Ser124 results in abortive translation unless the frameshift is suppressed.
Figure 2
Figure 2
Suppression efficiency of four-base codon/tRNA pairs measured by nitrocefin turnover. See Materials and Methods for definition of units. Readthrough of UAG, AGGA, CCCU, CUAG and UAGA was 0.8–1.7 units, while TOP10 cells alone exhibit about 0.9 unit. Suppression of four-base codons resulted in 17–240 units of activity, compared to 970 for suppression of UAG by supD. Results are the average of three to five independent trials per data point.
Figure 3
Figure 3
Nucleotide representation at anticodon loop sites in E. coli tRNAs and moderate and weak tRNA suppressors of AGGA with 8 nt and 9 nt anticodon loops. Suppression is at S124 and listed in ampicillin concentration of μg ml−1.
See this image and copyright information in PMC

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