Update of the Pyrrolysyl-tRNA Synthetase/tRNA
            <sup>Pyl</sup>
            Pair and Derivatives for Genetic Code Expansion

Gong, Xuemei; Zhang, Haolin; Shen, Yue; Fu, Xian

doi:10.1128/jb.00385-22

Cited by 11 publications

(3 citation statements)

References 140 publications

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“…Amber stop codons are used often, because they are relatively rare in most organisms and thus minimize the adverse effect of unnatural‐amino‐acid incorporation at unintended sites. The Pyl‐based system is useful due to low cross‐reactivity with endogenous aminoacyl‐tRNA synthetases (aaRSs), and the broad‐substrate tolerance of the pyrrolysyl‐tRNA synthetase (PylRS) (Brabham & Fascione, 2017; Gong et al, 2023; Hancock et al, 2010; Wan et al, 2014). PylRS catalyzes ligation of Pyl and tRNA Pyl (Srinivasan et al, 2002; Wan et al, 2014).…”

Section: Resultsmentioning

confidence: 99%

Amber‐codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase

Avila‐Cobian,

Hoshino,

Horsman

et al. 2023

Protein Science

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The 11 lytic transglycosylases of Pseudomonas aeruginosa have overlapping activities in the turnover of the cell‐wall peptidoglycan. Rare lipoprotein A (RlpA) is distinct among the 11 by its use of only peptidoglycan lacking peptide stems. The spatial localization of RlpA and its interactome within P. aeruginosa are unknown. We employed suppression of introduced amber codons at sites in the rlpA gene for the introduction of the unnatural‐amino‐acids Νζ‐[(2‐azidoethoxy)carbonyl]‐l‐lysine (compound 1) and Nζ‐[[[3‐(3‐methyl‐3H‐diazirin‐3‐yl)propyl]amino]carbonyl]‐l‐lysine (compound 2). In live P. aeruginosa, full‐length RlpA incorporating compound 1 into its sequence was fluorescently tagged using strained‐promoted alkyne‐azide cycloaddition and examined by fluorescence microscopy. RlpA is present at low levels along the sidewall length of the bacterium, and at higher levels at the nascent septa of replicating bacteria. In intact P. aeruginosa, UV photolysis of full‐length RlpA having compound 2 within its sequence generated a transient reactive carbene, which engaged in photoaffinity capture of neighboring proteins. Thirteen proteins were identified. Three of these proteins—PBP1a, PBP5, and MreB—are members of the bacterial divisome. The use of the complementary methodologies of non‐canonical amino‐acid incorporation, photoaffinity proximity analysis, and fluorescent microscopy confirm a dominant septal location for the RlpA enzyme of P. aeruginosa, as a divisome‐associated activity. This accomplishment adds to the emerging recognition of the value of these methodologies for identification of the intracellular localization of bacterial proteins.

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Section: Resultsmentioning

confidence: 99%

Amber‐codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase

Avila‐Cobian,

Hoshino,

Horsman

et al. 2023

Protein Science

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“…This system has been extensively researched for its use in GCE because of the promiscuity of the PylRS binding pocket and the orthogonality of the archaeal PylRS:tRNA Pyl protein synthesis system in E. coli ( Tharp et al, 2017 ) and eukaryotic systems ( Chin, 2014 ; de la Torre and Chin, 2021 ). Significant research has focused on expanding the substrate capacity of PylRS and engineering PylRS:tRNA Pyl pairs to be mutually orthogonal ( Wan et al, 2014 ; Beattie et al, 2023 ; Gong et al, 2023 ). However, others have focused on optimizing tRNA Pyl for improved translational fidelity ( Wang J. et al, 2016 ).…”

Section: Acceptor Domain Engineering For Improved Ef-tu Interactionsmentioning

confidence: 99%

Tuning tRNAs for improved translation

Weiss,

Decker,

Bolano

et al. 2024

Front. Genet.

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Transfer RNAs have been extensively explored as the molecules that translate the genetic code into proteins. At this interface of genetics and biochemistry, tRNAs direct the efficiency of every major step of translation by interacting with a multitude of binding partners. However, due to the variability of tRNA sequences and the abundance of diverse post-transcriptional modifications, a guidebook linking tRNA sequences to specific translational outcomes has yet to be elucidated. Here, we review substantial efforts that have collectively uncovered tRNA engineering principles that can be used as a guide for the tuning of translation fidelity. These principles have allowed for the development of basic research, expansion of the genetic code with non-canonical amino acids, and tRNA therapeutics.

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“…In this review, we will focus on recent developments for phage-assisted identification of peptides that contain ncAAs and other noncanonical motifs, with a particular focus on consolidating methods to generate modifications on phage. Table summarizes recent reviews on additional topics that are relevant to this work, including overviews of phage display, ,, genetic code expansion, − platforms for genetically encoded libraries, , and peptide therapeutics. ,− This review begins with the application of genetic code expansion to phage-displayed libraries (Section ), followed by chemical post-translational modifications (Section ), and it concludes with a general overview of remaining challenges in displaying noncanonical motifs on phages (Section ).…”

Section: Introductionmentioning

confidence: 99%

Diversification of Phage-Displayed Peptide Libraries with Noncanonical Amino Acid Mutagenesis and Chemical Modification

Hampton,

Liu

2024

Chem. Rev.

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Sitting on the interface between biologics and small molecules, peptides represent an emerging class of therapeutics. Numerous techniques have been developed in the past 30 years to take advantage of biological methods to generate and screen peptide libraries for the identification of therapeutic compounds, with phage display being one of the most accessible techniques. Although traditional phage display can generate billions of peptides simultaneously, it is limited to expression of canonical amino acids. Recently, several groups have successfully undergone efforts to apply genetic code expansion to introduce noncanonical amino acids (ncAAs) with novel reactivities and chemistries into phage-displayed peptide libraries. In addition to biological methods, several different chemical approaches have also been used to install noncanonical motifs into phage libraries. This review focuses on these recent advances that have taken advantage of both biological and chemical means for diversification of phage libraries with ncAAs. CONTENTS 3.6. Outlook of Chemically Modified Phage Libraries 6070 4.

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Update of the Pyrrolysyl-tRNA Synthetase/tRNA ^Pyl Pair and Derivatives for Genetic Code Expansion

Cited by 11 publications

References 140 publications

Amber‐codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase

Amber‐codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase

Tuning tRNAs for improved translation

Diversification of Phage-Displayed Peptide Libraries with Noncanonical Amino Acid Mutagenesis and Chemical Modification

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Update of the Pyrrolysyl-tRNA Synthetase/tRNA Pyl Pair and Derivatives for Genetic Code Expansion

Cited by 11 publications

References 140 publications

Amber‐codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase

Amber‐codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase

Tuning tRNAs for improved translation

Diversification of Phage-Displayed Peptide Libraries with Noncanonical Amino Acid Mutagenesis and Chemical Modification

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Product

Resources

About

Update of the Pyrrolysyl-tRNA Synthetase/tRNA ^Pyl Pair and Derivatives for Genetic Code Expansion