Front Cover: Evolving the N-Terminal Domain of Pyrrolysyl-tRNA Synthetase for Improved Incorporation of Noncanonical Amino Acids (ChemBioChem 1/2018)

Thiopeptides are a subclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with complex molecular architectures and an array of biological activities, including potent antimicrobial activity. Here we report the generation of thiopeptides containing noncanonical amino acids (ncAAs) by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin producer strain ofBacillus cereus. We demonstrate that thiopeptide variants containing ncAAs with bioorthogonal chemical reactivity can be further postbiosynthetically modified with biophysical probes, including fluorophores and photo-cross-linkers. This work allows the site-specific incorporation of ncAAs into thiopeptides to increase their structural diversity and probe their biological activity; similar approaches can likely be applied to other classes of RiPPs.

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Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

10.1101/2020.12.07.415521 ◽

2020 ◽

Author(s):

Jeffery M. Tharp ◽

Oscar Vargas-Rodriguez ◽

Alanna Schepartz ◽

Dieter Söll

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Trna Synthetase ◽

Dual Use ◽

Noncanonical Amino Acids ◽

Trna Synthetases ◽

Genetic Encoding ◽

Nonsense Codon ◽

N Terminus ◽

Nonsense Codons

ABSTRACTWe recently described an orthogonal initiator tRNA (itRNATy2) that can initiate protein synthesis with noncanonical amino acids (ncAAs) in response to the UAG nonsense codon. Here we report that a mutant of itRNATy2 (itRNATy2AUA) can efficiently initiate translation in response to the UAU tyrosine codon, giving rise to proteins with an ncAA at their N-terminus. We show that, in cells expressing itRNATy2AUA, UAU can function as a dual-use codon that selectively encodes ncAAs at the initiating position and tyrosine at elongating positions. Using itRNATy2AUA, in conjunction with its cognate tyrosyl-tRNA synthetase and two mutually orthogonal pyrrolysyl-tRNA synthetases, we demonstrate that UAU can be reassigned along with UAG or UAA to encode two distinct ncAAs in the same protein. Furthermore, by engineering the substrate specificity of one of the pyrrolysyl-tRNA synthetases, we developed a triply orthogonal system that enables simultaneous reassignment of UAU, UAG, and UAA to produce proteins containing three distinct ncAAs at precisely defined sites. To showcase the utility of this system, we produced proteins containing two or three ncAAs, with unique bioorthogonal functional groups, and demonstrate that these proteins can be separately modified with multiple fluorescent probes.TOC Image

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Orphan designation: 505 amino acid protein, corresponding to amino acids 2-506 of the wild-type human histidyl-tRNA synthetase, Treatment of limb-girdle muscular dystrophy

Case Medical Research ◽

10.31525/cmr-2935ce2 ◽

2020 ◽

Author(s):

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Muscular Dystrophy ◽

Trna Synthetase ◽

Limb Girdle Muscular Dystrophy ◽

Wild Type ◽

Orphan Designation ◽

Acid Protein ◽

Amino Acid Protein

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Noncanonical Amino Acids for Hypoxia-Responsive Peptide Self-Assembly and Fluorescence

Journal of the American Chemical Society ◽

10.1021/jacs.1c06435 ◽

2021 ◽

Author(s):

Binbin Hu ◽

Na Song ◽

Yawei Cao ◽

Mingming Li ◽

Xin Liu ◽

...

Keyword(s):

Amino Acids ◽

Self Assembly ◽

Noncanonical Amino Acids

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Exploring the Ability of LARS2 Carboxy-Terminal Domain in Rescuing the MELAS Phenotype

Life ◽

10.3390/life11070674 ◽

2021 ◽

Vol 11 (7) ◽

pp. 674

Author(s):

Francesco Capriglia ◽

Francesca Rizzo ◽

Giuseppe Petrosillo ◽

Veronica Morea ◽

Giulia d’Amati ◽

...

Keyword(s):

Protein Synthesis ◽

Molecular Mechanisms ◽

Mitochondrial Protein ◽

Trna Synthetase ◽

Mitochondrial Protein Synthesis ◽

Mitochondrial Translation ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Mitochondrial Functions ◽

Carboxy Terminal

The m.3243A>G mutation within the mitochondrial mt-tRNALeu(UUR) gene is the most prevalent variant linked to mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and a lack of post-transcriptional modification. In transmitochondrial cybrids, overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) has proven effective in rescuing the phenotype associated with m.3243A>G substitution. The rescuing activity resides in the carboxy-terminal domain (Cterm) of the enzyme; however, the precise molecular mechanisms underlying this process have not been fully elucidated. To deepen our knowledge on the rescuing mechanisms, we demonstrated the interactions of the Cterm with mutated mt-tRNALeu(UUR) and its precursor in MELAS cybrids. Further, the effect of Cterm expression on mitochondrial functions was evaluated. We found that Cterm ameliorates de novo mitochondrial protein synthesis, whilst it has no effect on mt-tRNALeu(UUR) steady-state levels and aminoacylation. Despite the complete recovery of cell viability and the increase in mitochondrial translation, Cterm-overexpressing cybrids were not able to recover bioenergetic competence. These data suggest that, in our MELAS cell model, the beneficial effect of Cterm may be mediated by factors that are independent of the mitochondrial bioenergetics.

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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 ◽

10.1007/s00726-020-02927-z ◽

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.

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Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

ACS Chemical Biology ◽

10.1021/acschembio.1c00120 ◽

2021 ◽

Vol 16 (4) ◽

pp. 766-774

Author(s):

Jeffery M. Tharp ◽

Oscar Vargas-Rodriguez ◽

Alanna Schepartz ◽

Dieter Söll

Keyword(s):

Amino Acids ◽

Noncanonical Amino Acids ◽

Genetic Encoding ◽

Nonsense Codons

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