Experimental challenges of sense codon reassignment: An innovative approach to genetic code expansion

AbstractThe genetic code is the universal cellular translation table to convert nucleotide into amino acid sequences. Changes to sense codons are expected to be highly detrimental. However, reassignments of single or multiple codons in mitochondria and nuclear genomes demonstrated that the code can evolve. Still, alterations of nuclear genetic codes are extremely rare leaving hypotheses to explain these variations, such as the ‘codon capture’, the ‘genome streamlining’ and the ‘ambiguous intermediate’ theory, in strong debate. Here, we report on a novel sense codon reassignment inPachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons we show that inPachysolenCUG codons are translated as alanine and not as the universal leucine. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by atRNA loss driven codon reassignmentmechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is outside the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects.

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Sense codon emancipation for proteome-wide incorporation of noncanonical amino acids: rare isoleucine codon AUA as a target for genetic code expansion

FEMS Microbiology Letters ◽

10.1111/1574-6968.12371 ◽

2014 ◽

Vol 351 (2) ◽

pp. 133-144 ◽

Cited By ~ 26

Author(s):

Nina Bohlke ◽

Nediljko Budisa

Keyword(s):

Amino Acids ◽

Genetic Code ◽

Noncanonical Amino Acids ◽

Genetic Code Expansion ◽

Sense Codon

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Directed Evolution of the Methanosarcina barkeri Pyrrolysyl tRNA/aminoacyl tRNA Synthetase Pair for Rapid Evaluation of Sense Codon Reassignment Potential

International Journal of Molecular Sciences ◽

10.3390/ijms22020895 ◽

2021 ◽

Vol 22 (2) ◽

pp. 895

Author(s):

David G. Schwark ◽

Margaret A. Schmitt ◽

John D. Fisk

Keyword(s):

Genetic Code ◽

Directed Evolution ◽

Trna Synthetase ◽

Methanosarcina Barkeri ◽

Rapid Evaluation ◽

Codon Reassignment ◽

Aminoacyl Trna Synthetase ◽

E Coli ◽

Orthogonal Pair ◽

Sense Codon

Genetic code expansion has largely focused on the reassignment of amber stop codons to insert single copies of non-canonical amino acids (ncAAs) into proteins. Increasing effort has been directed at employing the set of aminoacyl tRNA synthetase (aaRS) variants previously evolved for amber suppression to incorporate multiple copies of ncAAs in response to sense codons in Escherichia coli. Predicting which sense codons are most amenable to reassignment and which orthogonal translation machinery is best suited to each codon is challenging. This manuscript describes the directed evolution of a new, highly efficient variant of the Methanosarcina barkeri pyrrolysyl orthogonal tRNA/aaRS pair that activates and incorporates tyrosine. The evolved M. barkeri tRNA/aaRS pair reprograms the amber stop codon with 98.1 ± 3.6% efficiency in E. coli DH10B, rivaling the efficiency of the wild-type tyrosine-incorporating Methanocaldococcus jannaschii orthogonal pair. The new orthogonal pair is deployed for the rapid evaluation of sense codon reassignment potential using our previously developed fluorescence-based screen. Measurements of sense codon reassignment efficiencies with the evolved M. barkeri machinery are compared with related measurements employing the M. jannaschii orthogonal pair system. Importantly, we observe different patterns of sense codon reassignment efficiency for the M. jannaschii tyrosyl and M. barkeri pyrrolysyl systems, suggesting that particular codons will be better suited to reassignment by different orthogonal pairs. A broad evaluation of sense codon reassignment efficiencies to tyrosine with the M. barkeri system will highlight the most promising positions at which the M. barkeri orthogonal pair may infiltrate the E. coli genetic code.

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Faculty Opinions recommendation of A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1057894.512855 ◽

2007 ◽

Author(s):

Niles Lehman

Keyword(s):

Genetic Code ◽

Genetic Encoding ◽

Genetic Code Expansion

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Structural Basis for Genetic-Code Expansion with Various Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase

SSRN Electronic Journal ◽

10.2139/ssrn.3244022 ◽

2018 ◽

Cited By ~ 1

Author(s):

Tatsuo Yanagisawa ◽

Mitsuo Kuratani ◽

Eiko Seki ◽

Nobumasa Hino ◽

Kensaku Sakamoto ◽

...

Keyword(s):

Genetic Code ◽

Trna Synthetase ◽

Structural Basis ◽

Genetic Code Expansion

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Sense codon reassignment enables viral resistance and encoded polymer synthesis

Science ◽

10.1126/science.abg3029 ◽

2021 ◽

Vol 372 (6546) ◽

pp. 1057-1062

Author(s):

Wesley E. Robertson ◽

Louise F. H. Funke ◽

Daniel de la Torre ◽

Julius Fredens ◽

Thomas S. Elliott ◽

...

Keyword(s):

Stop Codon ◽

Synonymous Codon ◽

Viral Resistance ◽

Release Factor ◽

Codon Reassignment ◽

Efficient Synthesis ◽

Noncanonical Amino Acids ◽

Laboratory Evolution ◽

Transfer Rnas ◽

Sense Codon

It is widely hypothesized that removing cellular transfer RNAs (tRNAs)—making their cognate codons unreadable—might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.

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Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽

10.1007/s00726-020-02927-z ◽

2020 ◽

Author(s):

Thomas L. Williams ◽

Debra J. Iskandar ◽

Alexander R. Nödling ◽

Yurong Tan ◽

Louis Y. P. Luk ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Genetic Code Expansion ◽

Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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