Rapid and Inexpensive Evaluation of Nonstandard Amino Acid Incorporation in <i>Escherichia coli</i>

Monk, Jordan W.; Leonard, Sean P.; Brown, Colin W.; Hammerling, Michael J.; Mortensen, Catherine; Gutierrez, Alejandro E.; Shin, Nathan Y.; Watkins, Ella; Mishler, Dennis M.; Barrick, Jeffrey E.

doi:10.1021/acssynbio.6b00192

Cited by 36 publications

(46 citation statements)

References 39 publications

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“…The structure of the resulting plasmid plpp5-oGFP-pA15-Spec was verified by restriction digest and sequencing. The rfp gene with P T5 promoter and T0 transcription terminator was PCR-amplified from the plasmid pRYG 39 , the orthogonal SD sequence was introduced by PCR and the resulting o-rfp construct was inserted into a unique SphI site of the plpp5-oGFP-pA15-Spc plasmid.…”

Section: Methodsmentioning

confidence: 99%

A fully orthogonal system for protein synthesis in bacterial cells

et al. 2020

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Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.

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Section: Methodsmentioning

confidence: 99%

A fully orthogonal system for protein synthesis in bacterial cells

et al. 2020

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“…Incorporation of nsAAs is then monitored by production of the full-length reporter protein as evaluated by standard gel electrophoresis or by fluorimetry. However, promiscuous aaRS-tRNA pairs can produce full-length target proteins even in the absence of the nsAA ( 9 – 13 ).…”

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confidence: 99%

Engineering posttranslational proofreading to discriminate nonstandard amino acids

Kunjapur

Stork

Kuru

et al. 2018

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

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SignificanceAccurate incorporation of nonstandard amino acids (nsAAs) is central for genetic code expansion to increase the chemical diversity of proteins. However, aminoacyl-tRNA synthetases are polyspecific and facilitate incorporation of multiple nsAAs. We investigated and repurposed a natural protein degradation pathway, the N-end rule pathway, to devise an innovative system for rapid assessment of the accuracy of nsAA incorporation. Using this tool to monitor incorporation of the nsAA biphenylalanine allowed the identification of tyrosyl-tRNA synthetase (TyrRS) variants with improved amino acid specificity. The evolved TyrRS variants enhanced our ability to contain unwanted proliferation of genetically modified organisms. This posttranslational proofreading system will aid the evolution of orthogonal translation systems for specific incorporation of diverse nsAAs.

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“…mRNA structure drives distal gene expression in a synthetic operon. To test the relation between mRNA secondary structure and translation re-initiation, a library of operons based on the pRXG plasmid 12 was assembled (Fig. 1a).…”

Section: Resultsmentioning

confidence: 99%

A possible universal role for mRNA secondary structure in bacterial translation revealed using a synthetic operon

et al. 2020

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In bacteria, translation re-initiation is crucial for synthesizing proteins encoded by genes that are organized into operons. The mechanisms regulating translation re-initiation remain, however, poorly understood. We now describe the ribosome termination structure (RTS), a conserved and stable mRNA secondary structure localized immediately downstream of stop codons, and provide experimental evidence for its role in governing re-initiation efficiency in a synthetic Escherichia coli operon. We further report that RTSs are abundant, being associated with 18%–65% of genes in 128 analyzed bacterial genomes representing all phyla, and are selectively depleted when translation re-initiation is advantageous yet selectively enriched so as to insulate translation when re-initiation is deleterious. Our results support a potentially universal role for the RTS in controlling translation termination-insulation and re-initiation across bacteria.

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Rapid and Inexpensive Evaluation of Nonstandard Amino Acid Incorporation in Escherichia coli

Cited by 36 publications

References 39 publications

A fully orthogonal system for protein synthesis in bacterial cells

A fully orthogonal system for protein synthesis in bacterial cells

Engineering posttranslational proofreading to discriminate nonstandard amino acids

A possible universal role for mRNA secondary structure in bacterial translation revealed using a synthetic operon

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