Engineering the elongation factor Tu for efficient selenoprotein synthesis

Haruna, Ken-ichi; Alkazemi, Muhammad H.; Liu, Yuchen; Söll, Dieter; Englert, Markus

doi:10.1093/nar/gku691

Cited by 54 publications

(54 citation statements)

References 28 publications

(47 reference statements)

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“…In redesigning EF-Tu to accommodate site-specific selenocysteine incorporation, certain EF-Tu variants that specifically recognized selenocysteinyl-tRNA in vitro were toxic to the cell when expressed in vivo (60). Similarly, EF-Tu mutants designed to carry a bulky fluorescent ncAA-tRNA did not release the ncAA-tRNA fast enough to support efficient translation (61).…”

Section: Discussionmentioning

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

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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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Section: Discussionmentioning

confidence: 99%

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

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

Self Cite

107

146

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“…The positively charged, highly conserved Arg181 and Arg236 in SelB contact the negatively charged selenol group (Se − , orange), whereas the aromatic ring of Tyr42 stacks onto the selenol group. The importance of these residues was demonstrated by mutational analysis 9,90 . The universally conserved Asp180, which is also important for Sec-tRNA Sec binding 9 , forms a secondary binding shell stabilizing Arg236.…”

mentioning

confidence: 99%

The pathway to GTPase activation of elongation factor SelB on the ribosome

Fischer

Neumann

Bock

et al. 2016

Nature

106

137

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“…tRNA engineering can also improve the efficiency of an oaaRS•tRNA pair by optimizing orthogonality of the tRNA, its binding to the o-aaRS or elongation factor Tu, or the decoding strength of the targeted codon [24–29] (Box 1). Also, the role of heterologous tRNA post-transcriptional modifications is emerging as a considerable factor in improving o-tRNA proficiency [30–32].…”

Section: Efficiency Of O-aars•trna Pairsmentioning

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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Engineering the elongation factor Tu for efficient selenoprotein synthesis

Cited by 54 publications

References 28 publications

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

The pathway to GTPase activation of elongation factor SelB on the ribosome

Upgrading aminoacyl-tRNA synthetases for genetic code expansion

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