Efficient Reassignment of a Frequent Serine Codon in Wild-Type <i>Escherichia coli</i>

Ho, Joanne M. L.; Reynolds, Noah M.; Rivera, Keith; Connolly, Morgan; Guo, Li; Ling, Jiqiang; Pappin, Darryl; Church, George M.; Söll, Dieter

doi:10.1021/acssynbio.5b00197

Cited by 41 publications

(36 citation statements)

References 47 publications

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“…[23] Recently, O'Donoghue et al showed that the machinery of selenocysteine can be modulated to recognize 58 of the 64 naturally occurring codons, in many cases completely outcompeting the endogenous tRNA, although other codons resulted in ambiguous translation. [24] Söll and co-workers showed that the serine sense codon AGU can be reassigned to 3-iodo-Lphenylalanine (3-I-Phe), [25] while Kwon and co-workers recently showed that it is possible to achieve ambiguous reading of the leucine UUG codon with 2-naphthylalanine. [26] In general, amino acids encoded by several codons (e.g., serine, leucine and arginine) offer more chances to be recoded since the wobble pairing at the 3 rd base could be outcompeted by a more energetically favorable WatsonCrick base pairing with the orthogonal tRNA.…”

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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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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“…reported the first example of breaking the degeneracy of the genetic code by incorporating ncAAs in response to one of two Phe codons (28). Recent reports have described the incorporation of ncAAs in response to reassigned rare arginine, isoleucine and serine codons (29–34). …”

Section: Introductionmentioning

confidence: 99%

Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment

2016

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Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon: tRNAOpt. We suspected a modification of the tRNAOptAUG anticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNAOptAUG is converted to inosine. We identified tRNAOptAUG anticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues to infiltrate the genetic codes of diverse organisms.

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“…That tRNA Pyl is a natural amber suppressor and that PylRS largely interacts with the G73, G1:C72, and unique core tertiary motifs of tRNA Pyl but not with its anticodon [61, 62] has made the PylRS•tRNA Pyl pair popular in GCE applications in both bacteria and eukaryotes [1, 63]. More importantly, the lack of tRNA anticodon recognition of PylRS has been exploited to not only incorporate ncAAs in response to stop codons but also in response to sense [64] and quadruplet codons [65]. …”

Section: Trna Dictates Orthogonality: Recognition By Aarssmentioning

confidence: 99%

The central role of tRNA in genetic code expansion

Reynolds

Vargas-Rodriguez

Söll

et al. 2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background The development of orthogonal translation systems (OTSs) for genetic code expansion (GCE) has allowed for the incorporation of a diverse array of non-canonical amino acids (ncAA) into proteins. Transfer RNA, the central molecule in the translation of the genetic message into proteins, plays a significant role in the efficiency of ncAA incorporation. Scope of Review Here we review the biochemical basis of OTSs for genetic code expansion. We focus on the role of tRNA and discuss strategies used to engineer tRNA for the improvement of ncAA incorporation into proteins. Major Conclusions The engineering of orthogonal tRNAs for GCE has significantly improved the incorporation of ncAAs. However, there are numerous unintended consequences of orthogonal tRNA engineering that cannot be predicted ab initio. General Significance Genetic code expansion has allowed for the incorporation of a great diversity of ncAAs and novel chemistries into proteins, making significant contributions to our understanding of biological molecules and interactions.

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Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli

Cited by 41 publications

References 47 publications

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

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

Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment

The central role of tRNA in genetic code expansion

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