Within the broad field of synthetic biology, genetic code expansion (GCE) techniques enable creation of proteins with an expanded set of amino acids. This may be invaluable for applications in therapeutics, bioremediation, and biocatalysis. Central to GCE are aminoacyl-tRNA synthetases (aaRSs) as they link a non-canonical amino acid (ncAA) to their cognate tRNA, allowing ncAA incorporation into proteins on the ribosome. The ncAA-acylating aaRSs and their tRNAs should not cross-react with 20 natural aaRSs and tRNAs in the host, i.e., they need to function as an orthogonal translating system. All current orthogonal aaRS•tRNA pairs have been engineered from naturally occurring molecules to change the aaRS's amino acid specificity or assign the tRNA to a liberated codon of choice. Here we discuss the importance of orthogonality in GCE, laboratory techniques employed to create designer aaRSs and tRNAs, and provide an overview of orthogonal aaRS•tRNA pairs for GCE purposes.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8086897 | PMC |
| http://dx.doi.org/10.1016/bs.enz.2020.06.004 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Direct and quantitative analysis of tRNA acylation using intact tRNA liquid chromatography-mass spectrometry.
Nat Protoc
January 2025
Department of Chemistry, University of California, Berkeley, CA, USA.
Aminoacyl-tRNA synthetases (aaRSs) provide an essential functional link between an mRNA sequence and the protein it encodes. aaRS enzymes catalyze a two-step chemical reaction that acylates specific tRNAs with a cognate α-amino acid. In addition to their role in translation, acylated tRNAs contribute to non-ribosomal natural product biosynthesis and are implicated in multiple human diseases.
View Article and Find Full Text PDFThe mechanism of discriminative aminoacylation by isoleucyl-tRNA synthetase based on wobble nucleotide recognition.
Nat Commun
December 2024
State Key Laboratory of Anti-Infective Drug Discovery and Development, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China.
The faithful charging of amino acids to cognate tRNAs by aminoacyl-tRNA synthetases (AARSs) determines the fidelity of protein translation. Isoleucyl-tRNA synthetase (IleRS) distinguishes tRNA from tRNA solely based on the nucleotide at wobble position (N34), and a single substitution at N34 could exchange the aminoacylation specificity between two tRNAs. Here, we report the structural and biochemical mechanism of N34 recognition-based tRNA discrimination by Saccharomyces cerevisiae IleRS (ScIleRS).
View Article and Find Full Text PDFReaching New Heights in Genetic Code Manipulation with High Throughput Screening.
Chem Rev
November 2024
Chemical and Biological Engineering Department, Tufts University, Medford, Massachusetts 02155, United States.
The chemical and physical properties of proteins are limited by the 20 canonical amino acids. Genetic code manipulation allows for the incorporation of noncanonical amino acids (ncAAs) that enhance or alter protein functionality. This review explores advances in the three main strategies for introducing ncAAs into biosynthesized proteins, focusing on the role of high throughput screening in these advancements.
View Article and Find Full Text PDFGenetic code expansion (GCE) has become a critical tool in biology by enabling the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Central to GCE is the development of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs wherein engineered aaRSs recognize chosen ncAAs and charge them onto tRNAs that decode blank codons ( ., the amber stop codon).
View Article and Find Full Text PDFKinetic characterization of amino acid activation by aminoacyl-tRNA synthetases using radiolabelled γ-[P]ATP.
FEBS Open Bio
September 2024
Department of Chemistry, Faculty of Science, University of Zagreb, Zagreb, Croatia.
Aminoacyl-tRNA synthetases (AARSs) are fundamental enzymes that pair amino acids and tRNAs for protein synthesis. Aminoacylation occurs in two discrete steps. The amino acid is first activated by ATP, leading to an aminoacyl-adenylate intermediate and pyrophosphate (PP) formation.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!