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

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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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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Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine

10.1101/631432 ◽

2019 ◽

Author(s):

Junetha Syed ◽

Saravanan Palani ◽

Scott T. Clarke ◽

Zainab Asad ◽

Andrew R. Bottrill ◽

...

Keyword(s):

Genetic Code ◽

Protein Function ◽

Fluorescent Protein ◽

Trna Synthetase ◽

Glutathione S Transferase ◽

Base Pairs ◽

Site Specific ◽

Green Fluorescent ◽

Natural Amino Acids ◽

Sense Codon

AbstractSite-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function [1-4]. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA [5, 6]. Here, we report expansion of the zebrafish genetic code to incorporate the UNAAs, Azido-lysine (AzK), bicyclononyne-lysine (BCNK), and Diazirine-lysine (AbK) into green fluorescent protein (GFP) and Glutathione-S-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of UNAA mutagenesis to investigate and engineer protein function in zebrafish.

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

FEBS Letters ◽

10.1016/j.febslet.2013.11.039 ◽

2013 ◽

Vol 588 (3) ◽

pp. 383-388 ◽

Cited By ~ 20

Author(s):

Radha Krishnakumar ◽

Jiqiang Ling

Keyword(s):

Genetic Code ◽

Innovative Approach ◽

Codon Reassignment ◽

Genetic Code Expansion ◽

Sense Codon

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Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine

International Journal of Molecular Sciences ◽

10.3390/ijms20102577 ◽

2019 ◽

Vol 20 (10) ◽

pp. 2577 ◽

Cited By ~ 1

Author(s):

Junetha Syed ◽

Saravanan Palani ◽

Scott T. Clarke ◽

Zainab Asad ◽

Andrew R. Bottrill ◽

...

Keyword(s):

Genetic Code ◽

Protein Function ◽

Fluorescent Protein ◽

Trna Synthetase ◽

Glutathione S Transferase ◽

Base Pairs ◽

Site Specific ◽

Green Fluorescent ◽

Natural Amino Acids ◽

Sense Codon

Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, using a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA. Here, we report an expansion of the zebrafish genetic code to incorporate the UNAAs, azido-lysine (AzK), bicyclononyne-lysine (BCNK), and diazirine-lysine (AbK) into green fluorescent protein (GFP) and glutathione-s-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of AzK, BCNK, and AbK introduction into proteins as a means to investigate and engineer their function in zebrafish.

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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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Context-specific action of macrolide antibiotics on the eukaryotic ribosome

Nature Communications ◽

10.1038/s41467-021-23068-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Maxim S. Svetlov ◽

Timm O. Koller ◽

Sezen Meydan ◽

Vaishnavi Shankar ◽

Dorota Klepacki ◽

...

Keyword(s):

Amino Acid Sequences ◽

Macrolide Antibiotics ◽

Single Mutation ◽

Sequence Motifs ◽

Eukaryotic Translation ◽

Eukaryotic Ribosome ◽

Cellular Translation ◽

Distinct Sequence ◽

Exit Tunnel ◽

Context Specific

AbstractMacrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

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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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Isolation, Identification, and Genomic Analysis of a Novel Reovirus from Healthy Grass Carp and Its Dynamic Proliferation In Vitro and In Vivo

Viruses ◽

10.3390/v13040690 ◽

2021 ◽

Vol 13 (4) ◽

pp. 690

Author(s):

Ke Zhang ◽

Wenzhi Liu ◽

Yiqun Li ◽

Yong Zhou ◽

Yan Meng ◽

...

Keyword(s):

Cell Lines ◽

Grass Carp ◽

Amino Acid Sequences ◽

Gibel Carp ◽

Fish Cell ◽

Fish Cell Lines ◽

Total Size ◽

Sequence Comparisons ◽

Grass Carp Reovirus

A new grass carp reovirus (GCRV), healthy grass carp reovirus (HGCRV), was isolated from grass carp in 2019. Its complete genome sequence was determined and contained 11 dsRNAs with a total size of 23,688 bp and 57.2 mol% G+C content, encoding 12 proteins. All segments had conserved 5' and 3' termini. Sequence comparisons showed that HGCRV was closely related to GCRV-873 (GCRV-I; 69.57–96.71% protein sequence identity) but shared only 22.65–45.85% and 23.37–43.39% identities with GCRV-HZ08 and Hubei grass carp disease reovirus (HGDRV), respectively. RNA-dependent RNA-polymerase (RdRp) protein-based phylogenetic analysis showed that HGCRV clustered with Aquareovirus-C (AqRV-C) prior to joining a branch common with other aquareoviruses. Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that HGCRV was in the same evolutionary cluster as GCRV-I. Thus, HGCRV could be a new GCRV isolate of GCRV-I but is distantly related to other known GCRVs. Grass carp infected with HGCRV did not exhibit signs of hemorrhage. Interestingly, the isolate induced a typical cytopathic effect in fish cell lines, such as infected cell shrank, apoptosis, and plague-like syncytia. Further analysis showed that HGCRV could proliferate in grass carp liver (L28824), gibel carp brain (GiCB), and other fish cell lines, reaching a titer of up to 7.5 × 104 copies/μL.

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