PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine

Jiang, Ruisheng; Krzycki, Joseph A.

doi:10.1074/jbc.m112.396754

Cited by 59 publications

(81 citation statements)

References 40 publications

(60 reference statements)

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“…The N-terminal domain is highly insoluble and aggregates full-length PylRS. [26, 27] Using the truncated C-terminal catalytic core of PylRS from Methanosarcina mazei , apo-PylRS and PylRS complexes with different ligands were successfully determined, attributed mostly to contributions from Yokoyama, Söll, Steitz, and their coworkers. [28–32] The three dimensional organization of the PylRS catalytic core resembles that of other synthetases from the Class II AARS family.…”

Section: Pylrsmentioning

confidence: 99%

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

Wan¹,

Tharp²,

Liu³

2014

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

350

418

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The genetic incorporation of the 22nd proteinogenic amino acid, pyrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNAPyl. Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNAPyl. These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs.

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Section: Pylrsmentioning

confidence: 99%

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

Wan¹,

Tharp²,

Liu³

2014

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

350

418

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“…The mechanism by which A. arabaticum senses TMA and initiates the expression of the Pyl-decoding system remains unknown. Interestingly, despite encoding a functional PylRS 105,106 and tRNA Pyl pair 107 , Desulfitobacterium hafniense did not show detectable production of Pyl-tRNA Pyl or expression of MttB. Desulfitobacterium dehalogenans expressed transcripts for tRNA Pyl and PylRS, but no detectable transcription of the Pyl biosynthesis genes ( pylB , pylC and pylD ) was observed.…”

Section: Natural Genetic Code Expansionmentioning

confidence: 95%

Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology

2015

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The genetic code, initially thought to be universal and immutable, is now known to contain many variations, including biased codon usage, codon reassignment, ambiguous decoding and recoding. As a result of recent advances in the areas of genome sequencing, biochemistry, bioinformatics and structural biology, our understanding of genetic code flexibility has advanced substantially in the past decade. In this Review, we highlight the prevalence, evolution and mechanistic basis of genetic code variations in microorganisms, and we discuss how this flexibility of the genetic code affects microbial physiology.

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“…Each monomer of PylRS can be roughly divided into its N-terminal and C-terminal portions, which are encoded by a single gene in the Methanosarcinaceae species or by 2 separate genes in Desulfitobacterium hafniense ( pylSn and pylSc , respectively [6] ). Biochemical analysis of the N-terminal D. hafniense PylRS fragment demonstrate its role in tRNA binding [6] ; this is further supported by the lack of in vivo enzyme activity after truncation of the N-terminal PylRS domain [7] .…”

Section: The Pylrs•trnapyl Systemmentioning

confidence: 99%

“…Biochemical analysis of the N-terminal D. hafniense PylRS fragment demonstrate its role in tRNA binding [6] ; this is further supported by the lack of in vivo enzyme activity after truncation of the N-terminal PylRS domain [7] . A structural understanding of the role of the N-terminal domain of PylRS is lacking, as it does not have sequence homology with any known protein domain and its low solubility impedes production for crystallization purposes.…”

Section: The Pylrs•trnapyl Systemmentioning

confidence: 99%

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Pyrrolysyl-tRNA Synthetase, an Aminoacyl-tRNA Synthetase for Genetic Code Expansion

et al. 2016

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Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both in vitro and in vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In the Methanosarcinaceae the incorporation of the 22nd genetically encoded amino acid, pyrrolysine (Pyl), is facilitated by pyrrolysyl-tRNA synthetase (PylRS) and the cognate UAG-recognizing tRNAPyl. This unique aaRS•tRNA pair functions as an orthogonal translation system (OTS) in most model organisms. The facile directed evolution of the large PylRS active site to accommodate many ncAAs, and the enzyme’s anticodon-blind specific recognition of the cognate tRNAPyl make this system highly amenable for GCE purposes. The remarkable polyspecificity of PylRS has been exploited to incorporate >100 different ncAAs into proteins. Here we review the Pyl-OT system and selected GCE applications to examine the properties of an effective OTS.

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PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine

Cited by 59 publications

References 40 publications

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology

Pyrrolysyl-tRNA Synthetase, an Aminoacyl-tRNA Synthetase for Genetic Code Expansion

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