Exploring the Substrate Range of Wild‐Type Aminoacyl‐tRNA Synthetases

Fan, Chenguang; Ho, Joanne M. L.; Chirathivat, Napon; Söll, Dieter; Wang, Yane‐Shih

doi:10.1002/cbic.201402083

Cited by 34 publications

(47 citation statements)

References 37 publications

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“…The sequences of specific antisense DNA oligos for those tRNA isoacceptors were based on previous reports [28], and were listed in the Methods section. We chose superfolder green fluorescent protein (sfGFP) as a reporter, which was engineered to have improved tolerance of circular permutation, greater resistance to chemical denaturants, and enhanced folding kinetics, thus being widely used in the field of genetic code expansion [32, 33]. And we selected phosphoserine (Sep) incorporation system [29], which recently has been used in CFPS systems to make phosphoprotein but with the contamination from near-cognate suppression [22].…”

Section: Resultsmentioning

confidence: 99%

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

Gan

Fan

2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. Methods We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. Results By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNALys, tRNATyr, and tRNAGln(CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Conclusions Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. General significance We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems.

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Section: Resultsmentioning

confidence: 99%

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

Gan

Fan

2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…We determined the substrate range of two AcKRS enzymes by screening with a chemically diverse library of 313 ncAAs (11,32), followed by rescreening with a selected subset of Phe and Lys derivatives (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

“…The o-AARS should be specific in ligating a desired ncAA to a stop codon decoding tRNA, and both the o-tRNA and o-AARS are assumed not to cross-react with endogenous AARSs or tRNAs. Although some AARSs evolved in nature to recognize certain ncAAs (10)(11)(12), many genetic code expansion systems require a mutated AARS active site. The active site of the o-AARS is usually redesigned via directed evolution (6), including positive and negative selective rounds, to produce an enzyme that is assumed to be specific for an ncAA and not active with the 20 canonical amino acids.…”

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confidence: 99%

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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107

146

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Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA Pyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N e -acetyl-Lys (AcK) onto tRNA Pyl . Here, we examine an N e -acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.aminoacyl-tRNA synthetase | genetic code | genetic selection | posttranslational modification | synthetic biology T he standard genetic code table relates the 64 nucleotide triplets to three stop signals and 20 canonical amino acids. Some organisms, including humans, naturally evolved expanded genetic codes that accommodate 21 amino acids (1), or possibly 22 amino acids in rare cases (2). Engineering translation system components, including tRNAs (3, 4), aminoacyl-tRNA synthetases (AARSs) (5, 6), elongation factors (7), and the ribosome itself (8), have produced organisms with artificially expanded genetic codes. Products of genetic code engineering include bacterial, yeast, and mammalian cells and animals that are able to synthesize proteins with sitespecifically inserted noncanonical amino acids (ncAAs) (9).Genetic code expansion systems rely on an orthogonal AARS/ tRNA pair (o-AARS, o-tRNA) (5, 6). The o-AARS should be specific in ligating a desired ncAA to a stop codon decoding tRNA, and both the o-tRNA and o-AARS are assumed not to cross-react with endogenous AARSs or tRNAs. Although some AARSs evolved in nature to recognize certain ncAAs (10-12), many genetic code expansion systems require a mutated AARS active site. The active site of the o-AARS is usually redesigned via directed evolution (6), including positive and negative selective rounds, to produce an enzyme that is assumed to be specific for an ncAA and not active with the 20 canonical amino acids. Genetic code expansion technology is rapidly evolving (13), and the ability to incorporate multiple ncAAs into a protein using quadruplet-codon decoding (14) or sense-codon recoding (15-19) is now becoming feasible. Protein synthesis with multiple ncAAs will require o-AARSs that are able to discriminate their ncAA substrate not only from canonical amino acids in the cell but from other ncAAs that are added to the cell.Probing the effects of amino acid analogs on bacterial cell...

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“…Many ncAAs may be substrates of host aaRSs, which can lead to proteome-wide incorporation of the ncAA [28]. Moreover, ncAAs may interfere with essential metabolic pathways or be metabolized into a different substrate [43].…”

Section: Figurementioning

confidence: 99%

Upgrading aminoacyl-tRNA synthetases for genetic code expansion

Vargas-Rodriguez

Sevostyanova

Söll

et al. 2018

Current Opinion in Chemical Biology

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106

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Synthesis of proteins with non-canonical amino acids via genetic code expansion is at the forefront of synthetic biology. Progress in this field has enabled site-specific incorporation of over 200 chemically and structurally diverse amino acids into proteins in an increasing number of organisms. This has been facilitated by our ability to repurpose aminoacyl-tRNA synthetases to attach non-canonical amino acids to engineered tRNAs. Current efforts in the field focus on overcoming existing limitations to the simultaneous incorporation of multiple non-canonical amino acids or amino acids that differ from the l-α-amino acid structure (e.g. d-amino acid or β-amino acid). Here, we summarize the progress and challenges in developing more selective and efficient aminoacyl-tRNA synthetases for genetic code expansion.

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Exploring the Substrate Range of Wild‐Type Aminoacyl‐tRNA Synthetases

Cited by 34 publications

References 37 publications

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Upgrading aminoacyl-tRNA synthetases for genetic code expansion

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