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https://doi.org/10.5562/cca2915

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

Alessandro De Simone   ORCID icon orcid.org/0000-0001-9858-9543 ; Department of Chemistry, Biocatalysis Group, Technical University Berlin/Berlin Institute of Technology, Müller-Breslau-Str. 10, Berlin DE-10623, Germany
Carlos G. Acevedo-Rocha ; Biosyntia ApS, Fruebjergvej 3, boks 54, DK-2100 Copenhagen Ø, Denmark
Michael Georg Hoesl ; Clariant Produkte (Deutschland) GmbH, Semmelweisstraße 1, DE-82152 Planegg
Nediljko Budisa   ORCID icon orcid.org/0000-0001-8437-7304 ; Department of Chemistry, Biocatalysis Group, Technical University Berlin/Berlin Institute of Technology, Müller-Breslau-Str. 10, Berlin DE-10623, Germany

Puni tekst: engleski, pdf (6 MB) str. 243-253 preuzimanja: 585* citiraj
APA 6th Edition
De Simone, A., Acevedo-Rocha, C.G., Georg Hoesl, M. i Budisa, N. (2016). Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation. Croatica Chemica Acta, 89 (2), 243-253. https://doi.org/10.5562/cca2915
MLA 8th Edition
De Simone, Alessandro, et al. "Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation." Croatica Chemica Acta, vol. 89, br. 2, 2016, str. 243-253. https://doi.org/10.5562/cca2915. Citirano 22.10.2019.
Chicago 17th Edition
De Simone, Alessandro, Carlos G. Acevedo-Rocha, Michael Georg Hoesl i Nediljko Budisa. "Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation." Croatica Chemica Acta 89, br. 2 (2016): 243-253. https://doi.org/10.5562/cca2915
Harvard
De Simone, A., et al. (2016). 'Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation', Croatica Chemica Acta, 89(2), str. 243-253. https://doi.org/10.5562/cca2915
Vancouver
De Simone A, Acevedo-Rocha CG, Georg Hoesl M, Budisa N. Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation. Croatica Chemica Acta [Internet]. 2016 [pristupljeno 22.10.2019.];89(2):243-253. https://doi.org/10.5562/cca2915
IEEE
A. De Simone, C.G. Acevedo-Rocha, M. Georg Hoesl i N. Budisa, "Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation", Croatica Chemica Acta, vol.89, br. 2, str. 243-253, 2016. [Online]. https://doi.org/10.5562/cca2915

Sažetak
Genetic encoding of noncanonical amino acids (ncAAs) through sense codon reassignment is an efficient tool for expanding the chemical functionality of proteins. Incorporation of multiple ncAAs, however, is particularly challenging. This work describes the first attempts to reassign the sense methionine (Met) codon AUG to two different ncAAs in bacterial protein translation. Escherichia coli methionyl-tRNA synthetase (MetRS) charges two tRNAs with Met: tRNAfMet initiates protein synthesis (starting AUG codon), whereas elongator tRNAMet participates in protein elongation (internal AUG codon(s)). Preliminary in vitro experiments show that these tRNAs can be charged with the Met analogues azidohomoalanine (Aha) and ethionine (Eth) by exploiting the different substrate specificities of EcMetRS and the heterologous MetRS / tRNAMet pair from the archaeon Sulfolobus acidocaldarius, respectively. Here, we explored whether this configuration would allow a differential decoding during in vivo protein initiation and elongation. First, we eliminated the elongator tRNAMet from a methionine auxotrophic E. coli strain, which was then equipped with a rescue plasmid harboring the heterologous pair. Although the imported pair was not fully orthogonal, it was possible to incorporate preferentially Eth at internal AUG codons in a model protein, suggesting that in vivo AUG codon reassignment is possible. To achieve full orthogonality during elongation, we imported the known orthogonal pair of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) / tRNAPyl and devised a genetic selection system based on the suppression of an amber stop codon in an important glycolytic gene, pfkA, which restores enzyme functionality and normal cellular growth. Using an evolved PylRS able to accept Met analogues, it should be possible to reassign the AUG codon to two different ncAAs by using directed evolution.

Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 International License.

Ključne riječi
genetic code; sense codon reassignment; methionyl-tRNA synthetase; genetic selection system; synthetic biology; unnatural amino acid

Hrčak ID: 168168

URI
https://hrcak.srce.hr/168168

Posjeta: 807 *