Translation of UAG as Pyrrolysine

Krzycki, Joseph A.

doi:10.1007/978-0-387-89382-2_3

Cited by 7 publications

(11 citation statements)

References 77 publications

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“…In anti-strep immunoblots we only detected a single band at 71 kDa, indicating that translation was terminated at the first UAG codon. Kryzcki and coworkers noted that in highly expressed monomethylamine methyltransferase genes, the +1 and +2 nucleotides after the pyrrolysine-coding UAG codon are often GG [80]. Others have observed that the efficiency of pyl incorporation at UAG codons in heterologous systems can vary with the gene context [81, 82].…”

Section: Discussionmentioning

confidence: 99%

pNEB193-derived suicide plasmids for gene deletion and protein expression in the methane-producing archaeon, Methanosarcina acetivorans

Shea

Walter

Duszenko

et al. 2016

Plasmid

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Gene deletion and protein expression are cornerstone procedures for studying metabolism in any organism, including methane-producing archaea (methanogens). Methanogens produce coenzymes and cofactors not found in most bacteria, therefore it is sometimes necessary to express and purify methanogen proteins from the natural host. Protein expression in the native organism is also useful when studying post-translational modifications and their effect on gene expression or enzyme activity. We have created several new suicide plasmids to complement existing genetic tools for use in the methanogen, Methanosarcina acetivorans. The new plasmids are derived from the commercially available E. coli plasmid, pNEB193, and cannot replicate autonomously in methanogens. The designed plasmids facilitate markerless gene deletion, gene transcription, protein expression, and purification of proteins with cleavable affinity tags from the methanogen, Methanosarcina acetivorans.

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Section: Discussionmentioning

confidence: 99%

pNEB193-derived suicide plasmids for gene deletion and protein expression in the methane-producing archaeon, Methanosarcina acetivorans

Shea

Walter

Duszenko

et al. 2016

Plasmid

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“…While amber codons have been identified in Thg1 and transposase genes of one or two Methanosarcina spp., these are likely to be mutations stable in the context of tRNA Pyl and PylS, as homologs of these genes without amber codons exist in other Methanosarcinacea [21,28,29]. In sharp contrast, each of the organisms possessing the five pyl genes also possess one or more homologs of mtmB , mtbB , and/or mttB with conserved in-frame amber codons, as well as the genes for associated proteins such as the corrinoid proteins and RamA (Fig.…”

Section: Sequenced Genomes With Pyl Genesmentioning

confidence: 99%

“…The Desulfobacterium autotrophicum genome is interesting in this regard, as pylSn , pylT , and pylB , are present, but not pylSc , pylC , or pylD . A transposase gene homolog within the remaining pylTSnB cluster suggests the pyl gene cluster was disrupted [21]. Accordingly, although many mttB homologs are found in this genome, all lack an in frame amber codon.…”

Section: Sequenced Genomes With Pyl Genesmentioning

confidence: 99%

“…Incorporation of pyrrolysine into protein under the direction of amber codons does not require specific signals in the gene, as in-frame amber codons inserted into the E. coli uidA gene for β-glucuronidase are translated as pyrrolysine at 20–30% efficiency in M. acetivorans [20,21]. Translation of UAG as pyrrolysine even in foreign genes with an introduced amber codon suggests this level of translation occurs by a mechanism analogous to that underlying amber suppression, with tRNA Pyl acting as a suppressor tRNA.…”

Section: Introductionmentioning

confidence: 99%

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Functional context, biosynthesis, and genetic encoding of pyrrolysine

Gaston

Jiang

Krzycki

2011

Current Opinion in Microbiology

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Summary In Methanosarcina spp., amber codons in methylamine methyltransferase genes are translated as the 22nd amino acid, pyrrolysine. The responsible pyl genes plus amber-codon containing methyltransferase genes have been identified in four archaeal and five bacterial genera, including one human pathogen. In E. coli, the recombinant pylBCD gene products biosynthesize pyrrolysine from two lysine and the pylTS gene products direct pyrrolysine incorporation into protein. In the proposed biosynthetic pathway, PylB forms methylornithine from lysine, which is joined to another lysine by PylC, and oxidized to pyrrolysine by PylD. Structures of the catalytic domain of pyrrolysyl-tRNA synthetase (archaeal PylS or bacterial PylSc) revealed binding sites for tRNAPyl and pyrrolysine. PylS and tRNAPyl are now being exploited as an orthogonal pair in recombinant systems for introduction of useful modified amino acids into proteins.

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“…In 2002, the internal UAG (amber) codon present in the monomethylamine methyltransferase gene of Methanosarcina barkeri [1], a methanogenic archaeon, was shown to encode pyrrolysine ( 1 , Pyl, Figure 1) [2,3], establishing it as the 22 nd proteinogenic amino acid [4,5]. Apart from the engagement of UAG which is usually a nonsense (stop) codon, pyrrolysine behaves like a typical canonical amino acid and is charged directly into its cognate amber suppressor tRNA (tRNA Pyl ) by its own pyrrolysyl-tRNA synthetase (PylRS) [6,7].…”

Section: Introductionmentioning

confidence: 99%

The pyrrolysine translational machinery as a genetic-code expansion tool

Fekner

Chan

2011

Current Opinion in Chemical Biology

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The discovery of pyrrolysine not only expanded the set of the known proteinogenic amino acids but also revealed unusual features of its encoding mechanism. The engagement of a canonical stop codon and a unique aminoacyl-tRNA synthetase–tRNA pair that can be used to accommodate a broad range of unnatural amino acids while maintaining strict orthogonality in a variety of prokaryotic and eukaryotic expression systems has proven an invaluable combination. Within a few years since its unique properties were elucidated, the pyrrolysine translational machinery has become a popular choice for the synthesis of recombinant proteins bearing a wide variety of otherwise hard-to-introduce functional groups. It is also central to the development of new synthetic strategies that rely on stop-codon suppression.

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Translation of UAG as Pyrrolysine

Cited by 7 publications

References 77 publications

pNEB193-derived suicide plasmids for gene deletion and protein expression in the methane-producing archaeon, Methanosarcina acetivorans

pNEB193-derived suicide plasmids for gene deletion and protein expression in the methane-producing archaeon, Methanosarcina acetivorans

Functional context, biosynthesis, and genetic encoding of pyrrolysine

The pyrrolysine translational machinery as a genetic-code expansion tool

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