Context effects of genetic code expansion by stop codon suppression

2018 ◽  
Vol 46 ◽  
pp. 146-155 ◽  
Author(s):  
Yonatan Chemla ◽  
Eden Ozer ◽  
Itay Algov ◽  
Lital Alfonta
Keyword(s):  
Genetic Code ◽  
Context Effects ◽  
Stop Codon ◽  
Genetic Code Expansion

scholarly journals Unique Characteristics of the Pyrrolysine System in the 7th Order of Methanogens: Implications for the Evolution of a Genetic Code Expansion Cassette

Archaea ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Guillaume Borrel ◽  
Nadia Gaci ◽  
Pierre Peyret ◽  
Paul W. O'Toole ◽  
Simonetta Gribaldo ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Genetic Code ◽  
Stop Codon ◽  
Trna Synthetase ◽  
Coding System ◽  
Genomic Features ◽  
Close Relationship ◽  
Seventh Order ◽  
Genetic Code Expansion

Pyrrolysine (Pyl), the 22nd proteogenic amino acid, was restricted until recently to few organisms. Its translational use necessitates the presence of enzymes for synthesizing it from lysine, a dedicated amber stop codon suppressor tRNA, and a specific amino-acyl tRNA synthetase. The three genomes of the recently proposed Thermoplasmata-related 7th order of methanogens contain the complete genetic set for Pyl synthesis and its translational use. Here, we have analyzed the genomic features of the Pyl-coding system in these three genomes with those previously known fromBacteriaandArchaeaand analyzed the phylogeny of each component. This shows unique peculiarities, notably anamber  tRNAPylwith an imperfect anticodon stem and a shortenedtRNAPylsynthetase. Phylogenetic analysis indicates that a Pyl-coding system was present in the ancestor of the seventh order of methanogens and appears more closely related to Bacteria than to Methanosarcinaceae, suggesting the involvement of lateral gene transfer in the spreading of pyrrolysine between the two prokaryotic domains. We propose that the Pyl-coding system likely emerged once in Archaea, in a hydrogenotrophic and methanol-H2-dependent methylotrophic methanogen. The close relationship between methanogenesis and the Pyl system provides a possible example of expansion of a still evolving genetic code, shaped by metabolic requirements.Corrigendum to “Unique Characteristics of the Pyrrolysine System in the 7th Order of Methanogens: Implications for the Evolution of a Genetic Code Expansion Cassette”

scholarly journals Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells

2018 ◽  
Author(s):  
Birthe Meineke ◽  
Johannes Heimgärtner ◽  
Lorenzo Lafranchi ◽  
Simon J Elsässer
Keyword(s):  
Genetic Code ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Human Gut ◽  
Methanosarcina Mazei ◽  
Genetic Code Expansion

ABSTRACTGenetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded non-canonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei (Mma PylRS/PylT) being the most active and versatile to date. We found a previously uncharacterized pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that the new PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its non-cognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

scholarly journals Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

2021 ◽  
Author(s):  
Devon A. Stork ◽  
Georgia R. Squyres ◽  
Erkin Kuru ◽  
Katarzyna A. Gromek ◽  
Jonathan Rittichier ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Genetic Code ◽  
Cell Biology ◽  
Stop Codon ◽  
Positive Bacterium ◽  
E Coli ◽  
Binding Interface ◽  
Genetic Code Expansion ◽  
And Control

AbstractBacillus subtilis is a model Gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, we demonstrate broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

scholarly journals Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Devon A. Stork ◽  
Georgia R. Squyres ◽  
Erkin Kuru ◽  
Katarzyna A. Gromek ◽  
Jonathan Rittichier ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Genetic Code ◽  
Cell Biology ◽  
Stop Codon ◽  
Positive Bacterium ◽  
E Coli ◽  
Binding Interface ◽  
Genetic Code Expansion ◽  
And Control

AbstractBacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, here we report broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

scholarly journals Genetic Code Expansion System for Tight Control of Gene Expression in Bombyx mori Cell Lines

Insects ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1081
Author(s):  
Wei Lu ◽  
Ruolin Wang ◽  
Pan Wang ◽  
Sanyuan Ma ◽  
Qingyou Xia
Keyword(s):  
Gene Expression ◽  
Genetic Code ◽  
Bombyx Mori ◽  
Cell Lines ◽  
Stop Codon ◽  
Protein Markers ◽  
Control Of Gene Expression ◽  
New Genes ◽  
Expansion System ◽  
Genetic Code Expansion

Inducible gene expression systems are important tools for studying gene function and to control protein synthesis. With the completion of the detailed map of the silkworm (Bombyx mori) genome, the study of Bombyx mori has entered the post-genome era. While the functions of many genes have been described in detail, many coding genes remain unidentified. Except for the available tetracycline induction system, there is currently a dearth of other effective induction systems for B. mori. A genetic code expansion system can be used for protein labeling and to regulate gene expression. Here, we have established a genetic code expansion system for B. mori based on the well-researched tRNAPyl/PylRS pair from Methanosarcina mazei. We used H-Lys(Boc)-OH, which is a lysine derivative to efficiently and tightly control the expression of the reporter gene DsRed[TAG]EGFP (D[TAG]G), which encoded a H-Lys(Boc)-OH-bearing protein fused with DsRed and EGFP (here regarded as D[Boc]G) in B. mori cell lines BmE and BmNs. In D[TAG]G, the amber stop codon is recognized as the orthogonal tRNAPyl. Successful application of genetic code expansion system in silkworm cell lines will support the research into the function of silkworm genes and paves the way for the identification of new genes and protein markers in silkworm.

scholarly journals Generation of Recombinant Mammalian Selenoproteins through Genetic Code Expansion with Photocaged Selenocysteine

2019 ◽  
Author(s):  
Jennifer C. Peeler ◽  
Rachel E. Kelemen ◽  
Masahiro Abo ◽  
Laura C. Edinger ◽  
Jingjia Chen ◽  
...  
Keyword(s):  
Genetic Code ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Biological Evaluation ◽  
Trna Synthetase ◽  
Secis Element ◽  
E Coli ◽  
Domains Of Life ◽  
Translation Mechanism ◽  
Genetic Code Expansion

ABSTRACTSelenoproteins contain the amino acid selenocysteine and are found in all domains of life. The functions of many selenoproteins are poorly understood, partly due to difficulties in producing recombinant selenoproteins for cell-biological evaluation. Endogenous mammalian selenoproteins are produced through a non-canonical translation mechanism requiring suppression of the UGA stop codon, and a selenocysteine insertion sequence (SECIS) element in the 3’ untranslated region of the mRNA. Here, recombinant selenoproteins are generated in mammalian cells through genetic code expansion, circumventing the requirement for the SECIS element, and selenium availability. An engineered orthogonal E. coli leucyl-tRNA synthetase/tRNA pair is used to incorporate a photocaged selenocysteine (DMNB-Sec) at the UAG amber stop codon. Recombinantly expressed selenoproteins can be photoactivated in living cells with spatial and temporal control. Using this approach, the native selenoprotein methionine-R-sulfoxide reductase 1 is generated and activated in mammalian cells. The ability to site-specifically introduce selenocysteine directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.

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

2018 ◽  
Author(s):  
Tatsuo Yanagisawa ◽  
Mitsuo Kuratani ◽  
Eiko Seki ◽  
Nobumasa Hino ◽  
Kensaku Sakamoto ◽  
...  
Keyword(s):  
Genetic Code ◽  
Trna Synthetase ◽  
Structural Basis ◽  
Genetic Code Expansion

scholarly journals 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 ◽  
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.

scholarly journals ASIC Activation Mechanisms Delineated through Genetic Code Expansion

2021 ◽  
Vol 120 (3) ◽  
pp. 338a
Author(s):  
Matthew L. Rook ◽  
Tyler A. Couch ◽  
Jackson Hernandez ◽  
Alison J. Frontier ◽  
David M. MacLean
Keyword(s):  
Genetic Code ◽  
Activation Mechanisms ◽  
Genetic Code Expansion
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