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 Structural basis of nonnatural amino acid recognition by an engineered aminoacyl-tRNA synthetase for genetic code expansion

2005 ◽  
Vol 102 (5) ◽  
pp. 1366-1371 ◽  
Author(s):  
T. Kobayashi ◽  
K. Sakamoto ◽  
T. Takimura ◽  
R. Sekine ◽  
V. P. Kelly ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genetic Code ◽  
Trna Synthetase ◽  
Structural Basis ◽  
Aminoacyl Trna Synthetase ◽  
Genetic Code Expansion ◽  
Nonnatural Amino Acid ◽  
Amino Acid Recognition

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.

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 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 Aminoacyl-tRNA synthetase evolution and sectoring of the genetic code

Transcription ◽  
2018 ◽  
pp. 1-15 ◽  
Author(s):  
Daewoo Pak ◽  
Yunsoo Kim ◽  
Zachary F. Burton
Keyword(s):  
Genetic Code ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase

scholarly journals RECOGNITION OF THE AMINOACYL-tRNA-SYNTHETASE BY THE COGNATE U SHAPED tRNA AND ITS RELATION TO THE GENETIC CODE

1982 ◽  
Vol 11 (10) ◽  
pp. 1561-1564 ◽  
Author(s):  
Mikio Shimizu
Keyword(s):  
Genetic Code ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase
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