Mapping the Plasticity of theEscherichia coliGenetic Code with Orthogonal Pair-Directed Sense Codon Reassignment

Biochemistry ◽  
2018 ◽  
Vol 57 (19) ◽  
pp. 2762-2774 ◽  
Author(s):  
Margaret A. Schmitt ◽  
Wil Biddle ◽  
John D. Fisk
Keyword(s):  
Codon Reassignment ◽  
Orthogonal Pair ◽  
Sense Codon

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

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.

scholarly journals Sense codon reassignment enables viral resistance and encoded polymer synthesis

Science ◽  
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.

scholarly journals Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the Methanocaldococcus jannaschii Orthogonal Pair

Genes ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 546 ◽  
Author(s):  
David Schwark ◽  
Margaret Schmitt ◽  
John Fisk
Keyword(s):  
Stop Codon ◽  
Release Factor ◽  
Codon Reassignment ◽  
Methanocaldococcus Jannaschii ◽  
E Coli ◽  
Amber Stop Codon ◽  
Orthogonal Pair ◽  
Amber Codon ◽  
Quantitative Contribution ◽  
Stop Codon Reassignment

Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the Methanocaldococcus jannaschii orthogonal pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded Escherichia coli strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in E. coli DH10B and 76 to 104% in the RF1 deleted E. coli C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1.

scholarly journals Evaluating Sense Codon Reassignment with a Simple Fluorescence Screen

Biochemistry ◽  
2015 ◽  
Vol 54 (50) ◽  
pp. 7355-7364 ◽  
Author(s):  
Wil Biddle ◽  
Margaret A. Schmitt ◽  
John D. Fisk
Keyword(s):  
Codon Reassignment ◽  
Sense Codon

scholarly journals A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes

2016 ◽  
Author(s):  
Stefanie Mühlhausen ◽  
Peggy Findeisen ◽  
Uwe Plessmann ◽  
Henning Urlaub ◽  
Martin Kollmar
Keyword(s):  
Genetic Code ◽  
Trna Synthetase ◽  
Amino Acid Sequences ◽  
Codon Reassignment ◽  
Proteomics Data ◽  
Pachysolen Tannophilus ◽  
Sequence Comparisons ◽  
Codon Capture ◽  
Cellular Translation ◽  
Sense Codon

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.

scholarly journals The Influence of Competing tRNA Abundance on Translation: Quantifying the Efficiency of Sense Codon Reassignment at Rarely Used Codons

ChemBioChem ◽  
2020 ◽  
Vol 21 (16) ◽  
pp. 2274-2286 ◽  
Author(s):  
David G. Schwark ◽  
Margaret A. Schmitt ◽  
Wil Biddle ◽  
John D. Fisk
Keyword(s):  
Codon Reassignment ◽  
Sense Codon

scholarly journals Oligonucleotide-mediated tRNA sequestration enables one-pot sense codon reassignment in vitro

2018 ◽  
Vol 46 (12) ◽  
pp. 6387-6400 ◽  
Author(s):  
Zhenling Cui ◽  
Yue Wu ◽  
Sergey Mureev ◽  
Kirill Alexandrov
Keyword(s):  
Codon Reassignment ◽  
One Pot ◽  
Sense Codon

scholarly journals Analysis of Stop Codons within Prokaryotic Protein-Coding Genes Suggests Frequent Readthrough Events

2021 ◽  
Vol 22 (4) ◽  
pp. 1876
Author(s):  
Frida Belinky ◽  
Ishan Ganguly ◽  
Eugenia Poliakov ◽  
Vyacheslav Yurchenko ◽  
Igor B. Rogozin
Keyword(s):  
Stop Codon ◽  
Purifying Selection ◽  
Protein Product ◽  
Intermediate Step ◽  
Protein Coding ◽  
Stop Codons ◽  
Protein Coding Genes ◽  
Synonymous Sites ◽  
Prokaryotic Protein ◽  
Sense Codon

Nonsense mutations turn a coding (sense) codon into an in-frame stop codon that is assumed to result in a truncated protein product. Thus, nonsense substitutions are the hallmark of pseudogenes and are used to identify them. Here we show that in-frame stop codons within bacterial protein-coding genes are widespread. Their evolutionary conservation suggests that many of them are not pseudogenes, since they maintain dN/dS values (ratios of substitution rates at non-synonymous and synonymous sites) significantly lower than 1 (this is a signature of purifying selection in protein-coding regions). We also found that double substitutions in codons—where an intermediate step is a nonsense substitution—show a higher rate of evolution compared to null models, indicating that a stop codon was introduced and then changed back to sense via positive selection. This further supports the notion that nonsense substitutions in bacteria are relatively common and do not necessarily cause pseudogenization. In-frame stop codons may be an important mechanism of regulation: Such codons are likely to cause a substantial decrease of protein expression levels.

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