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 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 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 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 Pyrrolysyl-tRNA Synthetase with a Unique Architecture Enhances the Availability of Lysine Derivatives in Synthetic Genetic Codes

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2460 ◽  
Author(s):  
Atsushi Yamaguchi ◽  
Fumie Iraha ◽  
Kazumasa Ohtake ◽  
Kensaku Sakamoto
Keyword(s):  
Amino Acid ◽  
Binding Pocket ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Genetic Codes ◽  
Genetic Code Expansion ◽  
Amino Acid Binding ◽  
Derivatives Of ◽  
Additional Nucleotide ◽  
Incorporation Efficiency

Genetic code expansion has largely relied on two types of the tRNA—aminoacyl-tRNA synthetase pairs. One involves pyrrolysyl-tRNA synthetase (PylRS), which is used to incorporate various lysine derivatives into proteins. The widely used PylRS from Methanosarcinaceae comprises two distinct domains while the bacterial molecules consist of two separate polypeptides. The recently identified PylRS from Candidatus Methanomethylophilus alvus (CMaPylRS) is a single-domain, one-polypeptide enzyme that belongs to a third category. In the present study, we showed that the PylRS—tRNAPyl pair from C. M. alvus can incorporate lysine derivatives much more efficiently (up to 14-times) than Methanosarcinaceae PylRSs in Escherichia coli cell-based and cell-free systems. Then we investigated the tRNA and amino-acid recognition by CMaPylRS. The cognate tRNAPyl has two structural idiosyncrasies: no connecting nucleotide between the acceptor and D stems and an additional nucleotide in the anticodon stem and it was found that these features are hardly recognized by CMaPylRS. Lastly, the Tyr126Ala and Met129Leu substitutions at the amino-acid binding pocket were shown to allow CMaPylRS to recognize various derivatives of the bulky Nε-benzyloxycarbonyl-l-lysine (ZLys). With the high incorporation efficiency and the amenability to engineering, CMaPylRS would enhance the availability of lysine derivatives in expanded codes.

scholarly journals Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine

2019 ◽  
Vol 20 (9) ◽  
pp. 2343 ◽  
Author(s):  
Tobias Baumann ◽  
Matthias Hauf ◽  
Florian Richter ◽  
Suki Albers ◽  
Andreas Möglich ◽  
...  
Keyword(s):  
Stop Codon ◽  
Trna Synthetase ◽  
Targeted Mutagenesis ◽  
General Strategy ◽  
Library Design ◽  
Active Site Residues ◽  
Enzyme Design ◽  
Computational Enzyme Design ◽  
Genetic Code Expansion

Engineering aminoacyl-tRNA synthetases (aaRSs) provides access to the ribosomal incorporation of noncanonical amino acids via genetic code expansion. Conventional targeted mutagenesis libraries with 5–7 positions randomized cover only marginal fractions of the vast sequence space formed by up to 30 active site residues. This frequently results in selection of weakly active enzymes. To overcome this limitation, we use computational enzyme design to generate a focused library of aaRS variants. For aaRS enzyme redesign, photocaged ortho-nitrobenzyl tyrosine (ONBY) was chosen as substrate due to commercial availability and its diverse applications. Diversifying 17 first- and second-shell sites and performing conventional aaRS positive and negative selection resulted in a high-activity aaRS. This MjTyrRS variant carries ten mutations and outperforms previously reported ONBY-specific aaRS variants isolated from traditional libraries. In response to a single in-frame amber stop codon, it mediates the in vivo incorporation of ONBY with an efficiency matching that of the wild type MjTyrRS enzyme acylating cognate tyrosine. These results exemplify an improved general strategy for aaRS library design and engineering.

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.

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