Exploration of Methanomethylophilus alvus pyrrolysyl-tRNA synthetase activity in yeast

Archaeal pyrrolysyl-tRNA synthetases (PylRSs) have been used to genetically encode over 200 distinct noncanonical amino acids (ncAAs) in proteins in E. coli and mammalian cells. This vastly expands the range of chemical functionality accessible within proteins produced in these organisms. Despite these clear successes, explorations of PylRS function in yeast remains limited. In this work, we demonstrate that the Methanomethylophilus alvus PylRS (MaPylRS) and its cognate tRNACUA support the incorporation of ncAAs into proteins produced in S. cerevisiae using stop codon suppression methodologies. Additionally, we prepared three MaPylRS mutants originally engineered in E. coli and determined that all three were translationally active with one or more ncAAs, although with low efficiencies of ncAA incorporation in comparison to the parent MaPylRS. Alongside MaPylRS variants, we evaluated the translational activity of previously reported Methanosarcina mazei, Methanosarcina barkeri, and chimeric M. mazei and M. barkeri PylRSs. Using the yeast strain RJY100, and pairing these aaRSs with the M. barkeri tRNACUA, we did not observe any detectable stop codon suppression activity under the same conditions that produced moderately efficient ncAA incorporation with MaPylRS. The addition of MaPylRS to the orthogonal translation machinery toolkit in yeast potentially opens the door to hundreds of ncAAs that have not previously been genetically encodable using other aminoacyl-tRNA synthetase/tRNA pairs. Extending the scope of ncAA incorporation in yeast could powerfully advance chemical and biological research for applications ranging from basic biological discovery to enzyme engineering and therapeutic protein lead discovery.

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

10.1101/759662 ◽

2019 ◽

Cited By ~ 1

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.

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Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli

Genes ◽

10.3390/genes9100473 ◽

2018 ◽

Vol 9 (10) ◽

pp. 473 ◽

Cited By ~ 5

Author(s):

Takuya Umehara ◽

Saori Kosono ◽

Dieter Söll ◽

Koji Tamura

Keyword(s):

Escherichia Coli ◽

Trna Synthetase ◽

Synthetase Activity ◽

Lysine Acetylation ◽

Trna Synthetases ◽

E Coli ◽

Domains Of Life ◽

The Impact

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which Nε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.

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Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.18.8.4418 ◽

1998 ◽

Vol 18 (8) ◽

pp. 4418-4425 ◽

Cited By ~ 10

Author(s):

Ho-Jin Park ◽

Uttam L. RajBhandary

Keyword(s):

Mammalian Cells ◽

Trna Synthetase ◽

Nonsense Mutations ◽

Suppressor Trna ◽

E Coli ◽

Tetracycline Transactivator ◽

Hela Cell Lines ◽

Amber Codon ◽

Amber Suppressor ◽

Chloramphenicol Acetyltransferase Gene

ABSTRACT As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.

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Amber suppression in mammalian cells dependent upon expression of an Escherichia coli aminoacyl-tRNA synthetase gene.

Molecular and Cellular Biology ◽

10.1128/mcb.16.3.907 ◽

1996 ◽

Vol 16 (3) ◽

pp. 907-913 ◽

Cited By ~ 52

Author(s):

H J Drabkin ◽

H J Park ◽

U L RajBhandary

Keyword(s):

Escherichia Coli ◽

Mammalian Cells ◽

Trna Gene ◽

Trna Synthetase ◽

Developmentally Regulated ◽

Coding Sequence ◽

Suppressor Trna ◽

Trna Synthetases ◽

E Coli ◽

Nonsense Codons

As an approach to inducible suppression of nonsense mutations in mammalian and in higher eukaryotic cells, we have analyzed the expression of an Escherichia coli glutamine-inserting amber suppressor tRNA gene in COS-1 and CV-1 monkey kidney cells. The tRNA gene used has the suppressor tRNA coding sequence flanked by sequences derived from a human initiator methionine tRNA gene and has two changes in the coding sequence. This tRNA gene is transcribed, and the transcript is processed to yield the mature tRNA in COS-1 and CV-1 cells. We show that the tRNA is not aminoacylated in COS-1 cells by any of the endogenous aminoacyl-tRNA synthetases and is therefore not functional as a suppressor. Concomitant expression of the E. coli glutaminyl-tRNA synthetase gene results in aminoacylation of the suppressor tRNA and its functioning as a suppressor. These results open up the possibility of attempts at regulated suppression of nonsense codons in mammalian cells by regulating expression of the E. coli glutaminyl-tRNA synthetase gene in an inducible, cell-type specific, or developmentally regulated manner.

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Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene

10.1101/161984 ◽

2017 ◽

Author(s):

Noa Aloush ◽

Tomer Schvartz ◽

Andres I. König ◽

Sarit Cohen ◽

Eugene Brozgol ◽

...

Keyword(s):

Mammalian Cells ◽

Live Cell Imaging ◽

Cell Imaging ◽

Stop Codon ◽

Signal To Noise Ratio ◽

Trna Synthetase ◽

Live Cell ◽

Live Cells ◽

Copy Numbers ◽

Intracellular Proteins

ABSTRACTGenetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine synthetase/ pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNAPyl gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNA contributes to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNAPyl on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cells images by enhancing the signal-to-noise ratio and reducing an immobile tRNAPyl population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.

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A facile platform to engineer E. coli tyrosyl-tRNA synthetase adds new chemistries to the eukaryotic genetic code, including a phosphotyrosine mimic

10.1101/2021.11.28.470256 ◽

2021 ◽

Author(s):

Katherine T Grasso ◽

Soumya Jyoti Singha Roy ◽

Megan Jin Rae Yeo ◽

Chintan Soni ◽

Arianna O Osgood ◽

...

Keyword(s):

Mammalian Cells ◽

First Generation ◽

Dynamic Range ◽

Trna Synthetase ◽

Cross Reactivity ◽

Selection System ◽

Noncanonical Amino Acids ◽

E Coli ◽

Selection Systems ◽

Affinity Probes

The E. coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNAEcTyr pair offers an attractive platform to genetically encode new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed for its engineering, has impeded its success in the past. Recently, we showed that EcTyrRS can be engineered using a facile E. coli based selection system, in a strain where the endogenous tyrosyl pair has been substituted with an archaeal counterpart. However, a significant cross-reactivity between the UAG-suppressing tRNACUAEcTyr and the bacterial glutaminyl-tRNA synthetase limited the scope of this strategy, preventing the selection of moderately active EcTyrRS mutants. Here we report an engineered tRNACUAEcTyr that overcomes this cross-reactivity. Optimized selection systems using this tRNA enabled efficient enrichment of both strongly and weakly active ncAA-selective EcTyrRS mutants. We also developed a wide-dynamic range (WiDR) antibiotic selection to further enhance the activities of the weaker first-generation EcTyrRS mutants. We demonstrated the utility of our platform by developing several new EcTyrRS mutants that efficiently incorporate useful ncAAs in mammalian cells, including photo-affinity probes, bioconjugation handles, and a non-hydrolyzable mimic of phosphotyrosine.

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

10.1101/371757 ◽

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.

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Complementation of Human Immunodeficiency Virus Type 1 Replication by Intracellular Selection of Escherichia coli\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} Supplied in trans

Journal of Virology ◽

10.1128/jvi.00709-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9641-9650 ◽

Cited By ~ 2

Author(s):

Anna McCulley ◽

Casey D. Morrow

Keyword(s):

Mammalian Cells ◽

Infectious Virus ◽

Trna Synthetase ◽

E Coli ◽

Primer Selection ◽

Yeast Trna ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Selection Of

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) exclusively selects tRNA 3 Lys as the primer for the initiation of reverse transcription, even though both tRNA 3 Lys and tRNA 1,2 Lys are found in HIV-1 virions. Alteration of the HIV-1 primer-binding site (PBS) to be complementary to alternate tRNAs results in the use of those tRNAs for replication, indicating that primer complementarity with the PBS is an important determinant of primer selection. In previous studies, we have exploited this fact to develop a system in which yeast (Saccharomyces cerevisiae) tRNAPhe is provided in trans to complement the replication of HIV-1 with a PBS complementary to yeast tRNAPhe. Recent studies have demonstrated that the presence of lysyl-tRNA synthetase in HIV-1 virions might account for the preference for the selection of tRNA 3 Lys in HIV-1 replication. To establish a complementation system more reflective of HIV-1 primer selection, we have altered the HIV-1 PBS to be complementary to the Escherichia coli tRNA 3 Lys , which shares near identity with mammalian tRNA 3 Lys except in the 3′-terminal 18-nucleotide sequence that binds to the PBS. E. coli tRNA 3 Lys expressed from a plasmid was aminoacylated in mammalian cells. Cotransfection of cells with a plasmid that encodes E. coli tRNA 3 Lys and a plasmid encoding an HIV-1 provirus with a PBS complementary to E. coli tRNA 3 Lys resulted in the production of infectious virus. A comparison of the two complementation systems revealed that higher levels of intracellular E. coli tRNA 3 Lys than of yeast tRNAPhe were needed to achieve equal levels of infectious virus, indicating that there was no preferential selection of E. coli tRNA 3 Lys . To examine the specificity of tRNALys selection, E. coli tRNA 3 Lys was modified to tRNA 1,2 Lys . This tRNA was also aminoacylated when expressed in mammalian cells and complemented the infectivity of HIV-1 at levels similar to those seen for E. coli tRNA 3 Lys . Additional mutations in the anticodon of E. coli tRNA 3 Lys were constructed; these mutations did not significantly correlate with the capacity of the tRNA primer to complement infectivity of HIV-1, even though they had a drastic effect on the aminoacylation of the tRNAs. The results of these studies demonstrate that E. coli tRNA 3 Lys provided in trans can complement HIV-1 genomes with the PBS altered to E. coli tRNA 3 Lys . However, the capacity of tRNA 3 Lys to interact with lysyl-tRNA synthetase does not entirely explain the enhanced preference for selection of tRNA 3 Lys for the replication of HIV-1.

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Site-Specific Incorporation of Citrulline into Proteins in Mammalian Cells

10.1101/2020.06.06.137885 ◽

2020 ◽

Author(s):

Santanu Mondal ◽

Shu Wang ◽

Yunan Zheng ◽

Sudeshna Sen ◽

Abhishek Chatterjee ◽

...

Keyword(s):

Mammalian Cells ◽

Trna Synthetase ◽

Arginine Deiminase ◽

Neutrophil Extracellular Trap ◽

Post Translational Modification ◽

Site Specific ◽

E Coli ◽

Physiological Processes ◽

Nonsense Codon

AbstractCitrullination is a post-translational modification (PTM) of arginine that is crucial for several physiological processes, including gene regulation and neutrophil extracellular trap formation. Despite recent advances, studies of protein citrullination remain challenging due to the difficulty of accessing proteins homogeneously citrullinated at a specific site. Herein, we report a novel technology that enables the site-specific incorporation of citrulline (Cit) into proteins in mammalian cells. This approach exploits an E. coli-derived engineered leucyl tRNA synthetase-tRNA pair that incorporates a photocaged-citrulline (SM60) into proteins in response to a nonsense codon. Subsequently, SM60 is readily converted to Cit with light in vitro and in living cells. To demonstrate the utility of the method, we biochemically characterized the effect of incorporating Cit at two known autocitrullination sites in Protein Arginine Deiminase 4 (PAD4, R372 and R374) and showed that the R372Cit and R374Cit mutants are 181- and 9-fold less active than the wild-type enzyme. This powerful technology possesses immense potential to decipher the biology of citrullination.

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