Reassignment of a rare sense codon to a non-canonical amino acid in<i>Escherichia coli</i>

Mukai, Takahito; Yamaguchi, Atsushi; Ohtake, Kazumasa; Takahashi, Mihoko; Hayashi, Akiko; Iraha, Fumie; Kira, Satoshi; Yanagisawa, Takatoshi; Yokoyama, Shigeyuki; Hoshi, Hiroko; Kobayashi, T.; Sakamoto, Kensaku

doi:10.1093/nar/gkv787

Cited by 74 publications

(99 citation statements)

References 57 publications

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“…S7 and Dataset S5). Consistent with the findings of Mukai et al (25) and Baba et al (31), argW (tRNA Arg CCU ; decodes AGG only) was dispensable in C123a because it can be complemented by argU (tRNA Arg UCU ; decodes both AGG and AGA). However, argU is the only E. coli tRNA that can decode AGA and remains essential in C123a, probably because it is required to translate the AGR codons for the rest of the proteome (23).…”

Section: C-terminal Overlap (Mutation)supporting

confidence: 87%

“…Although the translation function of the AGG codon has been shown to permit efficient suppression with nonstandard amino acids (24)(25)(26), AGG necessarily remains translated as Arg in each of these studies. No study has yet demonstrated that all instances of any sense codon can be removed from essential genes.…”

Section: Significancementioning

confidence: 99%

“…Furthermore, their sparse use (123 instances in the essential genes of E. coli MG1655 and 4,228 instances in the entire genome) ( Table 1 and Dataset S1) made replacing all AGR instances in essential genes a tractable goal, with essential genes serving as a stringent test set for identifying any fitness impact from codon replacement (31). Additionally, recent work has shown the difficulty of directly mutating some AGR codons to other synonymous codons (25), although the authors do not explain the mechanism of failure or report successful implementation of alternative designs. We attempted to remove all 123 instances of AGR codons from essential genes by replacing them with the synonymous CGU codon.…”

Section: Significancementioning

confidence: 99%

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Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

Napolitano

Landon

Gregg

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 "recalcitrant" AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional "safe replacement zone" (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.codon choice | genome editing | recoded genomes

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Section: C-terminal Overlap (Mutation)supporting

confidence: 87%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 1 more Smart Citation

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

Napolitano

Landon

Gregg

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Recently, the rarest E. coli sense codon, AGG, was recoded from arginine (Arg) to L-homoarginine (LHR) using various tricks: all the AGG codons in essential genes were replaced with synonymous ones, the cellular levels of competing tRNAs Arg were eliminated/reduced, and a heterologous PylRS / tRNA Pyl CCU mutant specific for LHR was expressed. [21] In another example, it was possible to liberate the rare isoleucine codon AUA from its natural decoding mechanism. This was achieved by eliminating the enzyme catalyzing the tRNA modification C-to-L (2-lysyl-cytidine) at the wobble position of tRNA Ile CAU, which is important for codon recognition, and by rescuing the engineered cells with an heterologous IleRS / tRNA Ile UAU pair.…”

Section: "Rewiring" the Genetic Code: Sense Codons Reassignmentmentioning

confidence: 99%

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

Simone¹,

Acevedo‐Rocha²,

Hoesl³

et al. 2016

Croat. Chem. Acta

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Genetic encoding of noncanonical amino acids (ncAAs) through sense codon reassignment is an efficient tool for expanding the chemical functionality of proteins. Incorporation of multiple ncAAs, however, is particularly challenging. This work describes the first attempts to reassign the sense methionine (Met) codon AUG to two different ncAAs in bacterial protein translation. Escherichia coli methionyl-tRNA synthetase (MetRS) charges two tRNAs with Met: tRNA fMet initiates protein synthesis (starting AUG codon), whereas elongator tRNA Met participates in protein elongation (internal AUG codon(s)). Preliminary in vitro experiments show that these tRNAs can be charged with the Met analogues azidohomoalanine (Aha) and ethionine (Eth) by exploiting the different substrate specificities of EcMetRS and the heterologous MetRS / tRNA Met pair from the archaeon Sulfolobus acidocaldarius, respectively. Here, we explored whether this configuration would allow a differential decoding during in vivo protein initiation and elongation. First, we eliminated the elongator tRNA Met from a methionine auxotrophic E. coli strain, which was then equipped with a rescue plasmid harboring the heterologous pair. Although the imported pair was not fully orthogonal, it was possible to incorporate preferentially Eth at internal AUG codons in a model protein, suggesting that in vivo AUG codon reassignment is possible. To achieve full orthogonality during elongation, we imported the known orthogonal pair of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) / tRNA Pyl and devised a genetic selection system based on the suppression of an amber stop codon in an important glycolytic gene, pfkA, which restores enzyme functionality and normal cellular growth. Using an evolved PylRS able to accept Met analogues, it should be possible to reassign the AUG codon to two different ncAAs by using directed evolution.

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“…Several groups have deleted RF1 in an attempt to reassign UAG to a sense codon in E. coli by subsequently introducing an orthogonal aaRS–tRNA 33–38 pair to incorporate a non-canonical amino acid. Notably, when the native UAG codons are present, deleting RF1 and reassigning UAG as a sense codon causes severe growth defects 34,35 .…”

Section: Codon Reassignmentmentioning

confidence: 99%

Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology

2015

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The genetic code, initially thought to be universal and immutable, is now known to contain many variations, including biased codon usage, codon reassignment, ambiguous decoding and recoding. As a result of recent advances in the areas of genome sequencing, biochemistry, bioinformatics and structural biology, our understanding of genetic code flexibility has advanced substantially in the past decade. In this Review, we highlight the prevalence, evolution and mechanistic basis of genetic code variations in microorganisms, and we discuss how this flexibility of the genetic code affects microbial physiology.

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Reassignment of a rare sense codon to a non-canonical amino acid inEscherichia coli

Cited by 74 publications

References 57 publications

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology

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