scholarly journals Aminoacyl-tRNA Synthetases and tRNAs for an Expanded Genetic Code: What Makes them Orthogonal?

2019 ◽  
Vol 20 (8) ◽  
pp. 1929 ◽  
Author(s):  
Sergey V. Melnikov ◽  
Dieter Söll
Keyword(s):  
Amino Acids ◽  
Synthetic Biology ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Trna Synthetases ◽  
Cross Reactions ◽  
The Past ◽  
Aminoacyl Trna Synthetases ◽  
Expanded Genetic Code ◽  
Fundamental Requirement

In the past two decades, tRNA molecules and their corresponding aminoacyl-tRNA synthetases (aaRS) have been extensively used in synthetic biology to genetically encode post-translationally modified and unnatural amino acids. In this review, we briefly examine one fundamental requirement for the successful application of tRNA/aaRS pairs for expanding the genetic code. This requirement is known as “orthogonality”—the ability of a tRNA and its corresponding aaRS to interact exclusively with each other and avoid cross-reactions with additional types of tRNAs and aaRSs in a given organism.

scholarly journals Measuring the tolerance of the genetic code to altered codon size

2021 ◽  
Author(s):  
E. DeBenedictis ◽  
D. Söll ◽  
K. Esvelt
Keyword(s):  
Genetic Code ◽  
Protein Translation ◽  
Single Amino Acid ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases ◽  
Triplet Codon ◽  
Orthogonal Set ◽  
Expanded Genetic Code ◽  
Primordial Earth

SummaryProtein translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet-codon, rather than quadruplet-codon, genetic code? Here, we investigate the tolerance of the E. coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all twenty canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs), many of which selectively incorporate a single amino acid in response to a specified four-base codon. However, efficient quadruplet codon translation often requires multiple tRNA mutations, potentially constraining evolution. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results illuminate factors that led to selection and maintenance of triplet codons in primordial Earth and provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.

scholarly journals The Role of Orthogonality in Genetic Code Expansion

Life ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 58 ◽  
Author(s):  
Pol Arranz-Gibert ◽  
Jaymin R. Patel ◽  
Farren J. Isaacs
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Genetic Code ◽  
Cross Reactivity ◽  
Elongation Factors ◽  
Translational Machinery ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Genetic Code Expansion

The genetic code defines how information in the genome is translated into protein. Aside from a handful of isolated exceptions, this code is universal. Researchers have developed techniques to artificially expand the genetic code, repurposing codons and translational machinery to incorporate nonstandard amino acids (nsAAs) into proteins. A key challenge for robust genetic code expansion is orthogonality; the engineered machinery used to introduce nsAAs into proteins must co-exist with native translation and gene expression without cross-reactivity or pleiotropy. The issue of orthogonality manifests at several levels, including those of codons, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and elongation factors. In this concept paper, we describe advances in genome recoding, translational engineering and associated challenges rooted in establishing orthogonality needed to expand the genetic code.

scholarly journals Protein evolution with an expanded genetic code

2008 ◽  
Vol 105 (46) ◽  
pp. 17688-17693 ◽  
Author(s):  
Chang C. Liu ◽  
Antha V. Mack ◽  
Meng-Lin Tsao ◽  
Jeremy H. Mills ◽  
Hyun Soo Lee ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Directed Evolution ◽  
Unnatural Amino Acids ◽  
Selective Advantage ◽  
Display System ◽  
Antibody Library ◽  
Evolution Of Proteins ◽  
Expanded Genetic Code ◽  
Selection Of

We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in VH CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded “synthetic” genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.

scholarly journals Oxidation of cellular amino acid pools leads to cytotoxic mistranslation of the genetic code

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Tammy J Bullwinkle ◽  
Noah M Reynolds ◽  
Medha Raina ◽  
Adil Moghal ◽  
Eleftheria Matsa ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Genetic Code ◽  
Trna Synthetase ◽  
Control Mechanisms ◽  
Cellular Toxicity ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
The Cost

Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress can lead to the accumulation of non-canonical amino acids that must be excluded from the proteome in order to maintain cellular viability.

On the Classes of Aminoacyl-tRNA Synthetases, Amino Acids and the Genetic Code

2004 ◽  
Vol 34 (4) ◽  
pp. 407-420 ◽  
Author(s):  
Andre R. O. Cavalcanti ◽  
Elisa Soares Leite ◽  
Benício B. Neto ◽  
Ricardo Ferreira
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases

scholarly journals Engineering aminoacyl-tRNA synthetases for use in synthetic biology

2020 ◽  
pp. 351-395
Author(s):  
Natalie Krahn ◽  
Jeffery M. Tharp ◽  
Ana Crnković ◽  
Dieter Söll
Keyword(s):  
Synthetic Biology ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases

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 Destruxin A Interacts with Aminoacyl tRNA Synthases in Bombyx mori

2021 ◽  
Vol 7 (8) ◽  
pp. 593
Author(s):  
Jingjing Wang ◽  
Alexander Berestetskiy ◽  
Qiongbo Hu
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Bombyx Mori ◽  
Metarhizium Anisopliae ◽  
Free Amino ◽  
Molecular Target ◽  
Trna Synthetases ◽  
Interaction Mode ◽  
Aminoacyl Trna Synthetases ◽  
Wide Range

Destruxin A (DA), a hexa-cyclodepsipeptidic mycotoxin produced by the entomopathogenic fungus Metarhizium anisopliae, exhibits insecticidal activities in a wide range of pests and is known as an innate immunity inhibitor. However, its mechanism of action requires further investigation. In this research, the interactions of DA with the six aminoacyl tRNA synthetases (ARSs) of Bombyx mori, BmAlaRS, BmCysRS, BmMetRS, BmValRS, BmIleRS, and BmGluProRS, were analyzed. The six ARSs were expressed and purified. The BLI (biolayer interferometry) results indicated that DA binds these ARSs with the affinity indices (KD) of 10−4 to 10−5 M. The molecular docking suggested a similar interaction mode of DA with ARSs, whereby DA settled into a pocket through hydrogen bonds with Asn, Arg, His, Lys, and Tyr of ARSs. Furthermore, DA treatments decreased the contents of soluble protein and free amino acids in Bm12 cells, which suggested that DA impedes protein synthesis. Lastly, the ARSs in Bm12 cells were all downregulated by DA stress. This study sheds light on exploring and answering the molecular target of DA against target insects.

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