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 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.

scholarly journals Translational switching of Cry1 protein expression confers reversible control of circadian behavior in arrhythmic Cry-deficient mice

2018 ◽  
Vol 115 (52) ◽  
pp. E12388-E12397 ◽  
Author(s):  
Elizabeth S. Maywood ◽  
Thomas S. Elliott ◽  
Andrew P. Patton ◽  
Toke P. Krogager ◽  
Johanna E. Chesham ◽  
...  
Keyword(s):  
Stop Codon ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Adeno Associated Virus ◽  
Biologically Relevant ◽  
Negative Feedback Loops ◽  
Translational Readthrough ◽  
Genetic Code Expansion ◽  
And Behavior ◽  
Circadian Behavior

The suprachiasmatic nucleus (SCN) is the principal circadian clock of mammals, coordinating daily rhythms of physiology and behavior. Circadian timing pivots around self-sustaining transcriptional–translational negative feedback loops (TTFLs), whereby CLOCK and BMAL1 drive the expression of the negative regulators Period and Cryptochrome (Cry). Global deletion of Cry1 and Cry2 disables the TTFL, resulting in arrhythmicity in downstream behaviors. We used this highly tractable biology to further develop genetic code expansion (GCE) as a translational switch to achieve reversible control of a biologically relevant protein, Cry1, in the SCN. This employed an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair delivered to the SCN by adeno-associated virus (AAV) vectors, allowing incorporation of a noncanonical amino acid (ncAA) into AAV-encoded Cry1 protein carrying an ectopic amber stop codon. Thus, translational readthrough and Cry1 expression were conditional on the supply of ncAA via culture medium or drinking water and were restricted to neurons by synapsin-dependent expression of aminoacyl tRNA-synthetase. Activation of Cry1 translation by ncAA in neurons of arrhythmic Cry-null SCN slices immediately and dose-dependently initiated TTFL circadian rhythms, which dissipated rapidly after ncAA withdrawal. Moreover, genetic activation of the TTFL in SCN neurons rapidly and reversibly initiated circadian behavior in otherwise arrhythmic Cry-null mice, with rhythm amplitude being determined by the number of transduced SCN neurons. Thus, Cry1 does not specify the development of circadian circuitry and competence but is essential for its labile and rapidly reversible activation. This demonstrates reversible control of mammalian behavior using GCE-based translational switching, a method of potentially broad neurobiological interest.

scholarly journals Pyrrolysyl-tRNA Synthetase, an Aminoacyl-tRNA Synthetase for Genetic Code Expansion

2016 ◽  
Vol 89 (2) ◽  
Author(s):  
Ana Crnković ◽  
Tateki Suzuki ◽  
Dieter Söll ◽  
Noah M. Reynolds
Keyword(s):  
Genetic Code ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Genetic Code Expansion

scholarly journals An Expanded Genetic Code Enables Trimethylamine Metabolism in Human Gut Bacteria

mSystems ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Veronika Kivenson ◽  
Stephen J. Giovannoni
Keyword(s):  
Cardiovascular Disease ◽  
Genetic Code ◽  
Human Health ◽  
Cell Biology ◽  
Meta Analysis ◽  
Health Impact ◽  
Gut Bacteria ◽  
Human Gut ◽  
Demethylation Pathway ◽  
Genetic Code Expansion

ABSTRACT Cardiovascular disease (CVD) has been linked to animal-based diets, which are a major source of trimethylamine (TMA), a precursor of the proatherogenic compound trimethylamine-N-oxide (TMAO). Human gut bacteria in the genus Bilophila have genomic signatures for genetic code expansion that could enable them to metabolize both TMA and its precursors without production of TMAO. We uncovered evidence that the Bilophila demethylation pathway is actively transcribed in gut microbiomes and that animal-based diets cause Bilophila to rapidly increase in abundance. CVD occurrence and Bilophila abundance in humans were significantly negatively correlated. These data lead us to propose that Bilophila, which is commonly regarded as a pathobiont, may play a role in mitigating cardiovascular disease. Human gut microbiomes have been shown to affect the development of a myriad of disease states, but mechanistic connections between diet, health, and microbiota have been challenging to establish. The hypothesis that Bilophila reduces cardiovascular disease by circumventing TMAO production offers a clearly defined mechanism with a potential human health impact, but investigations of Bilophila cell biology and ecology will be needed to fully evaluate these ideas. IMPORTANCE Links between trimethylamine-N-oxide (TMAO) and cardiovascular disease (CVD) have focused attention on mechanisms by which animal-based diets have negative health consequences. In a meta-analysis of data from foundational gut microbiome studies, we found evidence that specialized bacteria have and express a metabolic pathway that circumvents TMAO production and is often misannotated because it relies on genetic code expansion. This naturally occurring mechanism for TMAO attenuation is negatively correlated with CVD. Ultimately, these findings point to new avenues of research that could increase microbiome-informed understanding of human health and hint at potential biomedical applications in which specialized bacteria are used to curtail CVD development.

scholarly journals An Expanded Genetic Code in Candida albicans To Study Protein-Protein Interactions In Vivo

2013 ◽  
Vol 12 (6) ◽  
pp. 816-827 ◽  
Author(s):  
Silke Palzer ◽  
Yannick Bantel ◽  
Franziska Kazenwadel ◽  
Michael Berg ◽  
Steffen Rupp ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Candida Albicans ◽  
Amino Acid ◽  
Genetic Code ◽  
Molecular Interactions ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Content Type ◽  
Suppressor Trna

ABSTRACT For novel insights into the pathogenicity of Candida albicans , studies on molecular interactions of central virulence factors are crucial. Since methods for the analysis of direct molecular interactions of proteins in vivo are scarce, we expanded the genetic code of C. albicans with the synthetic photo-cross-linking amino acid p -azido- l -phenylalanine (AzF). Interacting molecules in close proximity of this unnatural amino acid can be covalently linked by UV-induced photo-cross-link, which makes unknown interacting molecules available for downstream identification. Therefore, we applied an aminoacyl-tRNA synthetase and a suppressor tRNA pair ( Ec TyrtRNA CUA ) derived from Escherichia coli , which was previously reported to be orthogonal in Saccharomyces cerevisiae . We further optimized the aminoacyl-tRNA synthetase for AzF (AzF-RS) and Ec TyrtRNA CUA for C. albicans and identified one AzF-RS with highest charging efficiency. Accordingly, incorporation of AzF into selected model proteins such as Tsa1p or Tup1p could be considerably enhanced. Immunologic detection of C-terminally tagged Tsa1p and Tup1p upon UV irradiation in a strain background containing suppressor tRNA and optimized AzF-RS revealed not only the mutant monomeric forms of these proteins but also higher-molecular-weight complexes, strictly depending on the specific position of incorporated AzF and UV excitation. By Western blotting and tandem mass spectrometry, we could identify these higher-molecular-weight complexes as homodimers consisting of one mutant monomer and a differently tagged, wild-type version of Tsa1p or Tup1p, respectively, demonstrating that expanding the genetic code of C. albicans with the unnatural photo-cross-linker amino acid AzF and applying it for in vivo binary protein interaction analyses is feasible.

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”

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