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

• Main Authors: Alessandro De Simone, Carlos G. Acevedo-Rocha, Michael Georg Hoesl, Nediljko Budisa
• Format: Article
• Language: English
• Published: Croatian Chemical Society 2016-06-01
• Series: Croatica Chemica Acta
• Online Access: http://hrcak.srce.hr/file/248152
• ISSN: 0011-1643; 1334-417X

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. <i>Escherichia coli</i> methionyl-tRNA synthetase (MetRS) charges two tRNAs with Met: tRNA^fMet initiates protein synthesis (starting AUG codon), whereas elongator tRNA^Met participates in protein elongation (internal AUG codon(s)). Preliminary <i>in vitro</i> 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 / tRNA^Met pair from the archaeon <i>Sulfolobus acidocaldarius</i>, respectively. Here, we explored whether this configuration would allow a differential decoding during <i>in vivo</i> protein initiation and elongation. First, we eliminated the elongator tRNA^Met from a methionine auxotrophic <i>E. coli</i> 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 <i>in vivo</i> AUG codon reassignment is possible. To achieve full orthogonality during elongation, we imported the known orthogonal pair of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) / tRNA^Pyl and devised a genetic selection system based on the suppression of an amber stop codon in an important glycolytic gene, <i>pfkA</i>, 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. <br><a rel="license" href="http://creativecommons.org/licenses/by/4.0/"><img alt="Creative Commons License" style="border-width:0" src="https://i.creativecommons.org/l/by/4.0/80x15.png" /></a> This work is licensed under a <a rel="license" href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution 4.0 International License</a>.