Recombinant thiopeptides containing noncanonical amino acids

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.

Download Full-text

Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0601167103 ◽

2006 ◽

Vol 103 (27) ◽

pp. 10180-10185 ◽

Cited By ~ 132

Author(s):

A. J. Link ◽

M. K. S. Vink ◽

N. J. Agard ◽

J. A. Prescher ◽

C. R. Bertozzi ◽

...

Keyword(s):

Amino Acids ◽

Cell Surface ◽

Surface Display ◽

Cell Surface Display ◽

Trna Synthetase ◽

Synthetase Activity ◽

Aminoacyl Trna Synthetase ◽

Noncanonical Amino Acids

Download Full-text

Extracurricular Functions of tRNA Modifications in Microorganisms

Genes ◽

10.3390/genes11080907 ◽

2020 ◽

Vol 11 (8) ◽

pp. 907

Author(s):

Ashley M. Edwards ◽

Maame A. Addo ◽

Patricia C. Dos Santos

Keyword(s):

Messenger Rna ◽

Chemical Reactivity ◽

Trna Synthetase ◽

Structure Stability ◽

Cellular Viability ◽

Aminoacyl Trna Synthetase ◽

Nutritional Factors ◽

Transfer Rnas ◽

Trna Modifications ◽

Dynamic Landscape

Transfer RNAs (tRNAs) are essential adaptors that mediate translation of the genetic code. These molecules undergo a variety of post-transcriptional modifications, which expand their chemical reactivity while influencing their structure, stability, and functionality. Chemical modifications to tRNA ensure translational competency and promote cellular viability. Hence, the placement and prevalence of tRNA modifications affects the efficiency of aminoacyl tRNA synthetase (aaRS) reactions, interactions with the ribosome, and transient pairing with messenger RNA (mRNA). The synthesis and abundance of tRNA modifications respond directly and indirectly to a range of environmental and nutritional factors involved in the maintenance of metabolic homeostasis. The dynamic landscape of the tRNA epitranscriptome suggests a role for tRNA modifications as markers of cellular status and regulators of translational capacity. This review discusses the non-canonical roles that tRNA modifications play in central metabolic processes and how their levels are modulated in response to a range of cellular demands.

Download Full-text

A New Functional Suppressor tRNA/Aminoacyl−tRNA Synthetase Pair for the in Vivo Incorporation of Unnatural Amino Acids into Proteins

Journal of the American Chemical Society ◽

10.1021/ja000595y ◽

2000 ◽

Vol 122 (20) ◽

pp. 5010-5011 ◽

Cited By ~ 84

Author(s):

Lei Wang ◽

Thomas J. Magliery ◽

David R. Liu ◽

Peter G. Schultz

Keyword(s):

Amino Acids ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Aminoacyl Trna Synthetase ◽

Suppressor Trna

Download Full-text

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

ChemBioChem ◽

10.1002/cbic.201700646 ◽

2017 ◽

Vol 19 (1) ◽

pp. 1-1 ◽

Cited By ~ 5

Author(s):

Vangmayee Sharma ◽

Yu Zeng ◽

W. Wesley Wang ◽

Yuchen Qiao ◽

Yadagiri Kurra ◽

...

Keyword(s):

Amino Acids ◽

Trna Synthetase ◽

Noncanonical Amino Acids ◽

Front Cover ◽

Terminal Domain

Download Full-text

Aminoacyl-tRNA synthetase activities specific to twenty amino acids in rat, rabbit and human myocardium

Journal of Molecular and Cellular Cardiology ◽

10.1016/0022-2828(73)90012-6 ◽

1973 ◽

Vol 5 (5) ◽

pp. 419-425 ◽

Cited By ~ 2

Author(s):

K Gibson

Keyword(s):

Amino Acids ◽

Trna Synthetase ◽

Human Myocardium ◽

Aminoacyl Trna Synthetase

Download Full-text

A Rationally Designed Aminoacyl-tRNA Synthetase for Genetically Encoded Fluorescent Amino Acids

10.1101/065227 ◽

2016 ◽

Cited By ~ 1

Author(s):

Ximena Steinberg ◽

Jason Galpin ◽

Gibran Nasir ◽

Jose Sepulveda-Ugarte ◽

Romina V. Sepúlveda ◽

...

Keyword(s):

Amino Acids ◽

Protein Structure ◽

Structure Function ◽

Trna Synthetase ◽

Aminoacyl Trna Synthetase ◽

E Coli ◽

Promising Strategy ◽

Fluorescent Amino Acids

AbstractThe incorporation of non-canonical amino acids into proteins has emerged as a promising strategy to manipulate and study protein structure-function relationships with superior precision in vitro and in vivo. To date, fluorescent non-canonical amino acids (f-ncAA) have been successfully incorporated in proteins expressed in bacterial systems, Xenopus oocytes, and HEK-293T cells. Here, we describe the rational generation of an orthogonal aminoacyltRNA synthetase based on the E. coli tyrosine synthetase that is capable of encoding the f-ncAA tyr-coumarin in HEK-293T cells.

Download Full-text

Did Amino Acid Side Chain Reactivity Dictate the Composition and Timing of Aminoacyl-tRNA Synthetase Evolution?

Genes ◽

10.3390/genes12030409 ◽

2021 ◽

Vol 12 (3) ◽

pp. 409

Author(s):

Tamara L. Hendrickson ◽

Whitney N. Wood ◽

Udumbara M. Rathnayake

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Chemical Reactivity ◽

Trna Synthetase ◽

Amino Acid Side Chain ◽

Standard Genetic Code ◽

Last Universal Common Ancestor ◽

Trna Synthetases ◽

Universal Common Ancestor

The twenty amino acids in the standard genetic code were fixed prior to the last universal common ancestor (LUCA). Factors that guided this selection included establishment of pathways for their metabolic synthesis and the concomitant fixation of substrate specificities in the emerging aminoacyl-tRNA synthetases (aaRSs). In this conceptual paper, we propose that the chemical reactivity of some amino acid side chains (e.g., lysine, cysteine, homocysteine, ornithine, homoserine, and selenocysteine) delayed or prohibited the emergence of the corresponding aaRSs and helped define the amino acids in the standard genetic code. We also consider the possibility that amino acid chemistry delayed the emergence of the glutaminyl- and asparaginyl-tRNA synthetases, neither of which are ubiquitous in extant organisms. We argue that fundamental chemical principles played critical roles in fixation of some aspects of the genetic code pre- and post-LUCA.

Download Full-text