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Tunable translational control using site-specific unnatural amino acid incorporation in Escherichia coli.

Kato Y - PeerJ (2015)

Bottom Line: Here, we report that this translational switch can control the translational efficiency at any intermediate magnitude by adjustment of the 3-iodo-L-tyrosine concentration in the medium, as a tunable translational controller.The translational efficiency of a target gene reached maximum levels with 10(-5) M 3-iodo-L-tyrosine, and intermediate levels were observed with suboptimal concentrations (approximately spanning a 2-log10 concentration range, 10(-7)-10(-5) M).Such intermediate-level expression was also confirmed in individual bacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences , Tsukuba, Ibaraki , Japan.

ABSTRACT
Translation of target gene transcripts in Escherichia coli harboring UAG amber stop codons can be switched on by the amber-codon-specific incorporation of an exogenously supplied unnatural amino acid, 3-iodo-L-tyrosine. Here, we report that this translational switch can control the translational efficiency at any intermediate magnitude by adjustment of the 3-iodo-L-tyrosine concentration in the medium, as a tunable translational controller. The translational efficiency of a target gene reached maximum levels with 10(-5) M 3-iodo-L-tyrosine, and intermediate levels were observed with suboptimal concentrations (approximately spanning a 2-log10 concentration range, 10(-7)-10(-5) M). Such intermediate-level expression was also confirmed in individual bacteria.

No MeSH data available.


Related in: MedlinePlus

Schematic of the translational switch using the amber codon-specific IY.An amber stop codon is inserted next to the ATG translational start codon in the target gene (egfp). MJR1 is an amber suppressor tRNA. IYRS is an aminoacyl-tRNA synthetase that orthogonally recognizes IY and MJR1. Extracellular IY is taken up by the bacteria. The addition of IY in the media results in amber stop codon read-through and translation of the target gene. Translation is interrupted in the absence of IY. RF1, peptide chain release factor 1.
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fig-1: Schematic of the translational switch using the amber codon-specific IY.An amber stop codon is inserted next to the ATG translational start codon in the target gene (egfp). MJR1 is an amber suppressor tRNA. IYRS is an aminoacyl-tRNA synthetase that orthogonally recognizes IY and MJR1. Extracellular IY is taken up by the bacteria. The addition of IY in the media results in amber stop codon read-through and translation of the target gene. Translation is interrupted in the absence of IY. RF1, peptide chain release factor 1.

Mentions: We previously demonstrated a translational switch using site-specific unnatural amino acid (UAA) 3-iodo-L-tyrosine (IY) incorporation in natural amber suppressor-free strains of Escherichia coli (Minaba & Kato, 2014). Although IY is not perfectly “unnatural” and found in very specific biological tissues such as the thyroid cells (Tietze et al., 1989) and the sponge skeletons (Ueberlein et al., 2014), we cannot detect IY ubiquitously in natural environments. The translational switch is based on conditional read-through of the UAG amber stop codons that are inserted in target genes (Fig. 1). A variant of aminoacyl-tRNA synthetase (aaRS) IYRS that was derived from the archaebacterium Methanocaldococcus jannaschii specifically recognizes both IY and an amber suppressor tRNA (tRNACUA) MJR1 (Sakamoto et al., 2009). Extracellular IY is taken up and incorporated into proteins at sites of the amber codons in the IYRS/MJR1-expressing cells. The target gene transcripts with the amber stop codons inserted next to the AUG translational start codon are translated only in the presence of IY. The absence of IY prevents translation of the target genes.


Tunable translational control using site-specific unnatural amino acid incorporation in Escherichia coli.

Kato Y - PeerJ (2015)

Schematic of the translational switch using the amber codon-specific IY.An amber stop codon is inserted next to the ATG translational start codon in the target gene (egfp). MJR1 is an amber suppressor tRNA. IYRS is an aminoacyl-tRNA synthetase that orthogonally recognizes IY and MJR1. Extracellular IY is taken up by the bacteria. The addition of IY in the media results in amber stop codon read-through and translation of the target gene. Translation is interrupted in the absence of IY. RF1, peptide chain release factor 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4419535&req=5

fig-1: Schematic of the translational switch using the amber codon-specific IY.An amber stop codon is inserted next to the ATG translational start codon in the target gene (egfp). MJR1 is an amber suppressor tRNA. IYRS is an aminoacyl-tRNA synthetase that orthogonally recognizes IY and MJR1. Extracellular IY is taken up by the bacteria. The addition of IY in the media results in amber stop codon read-through and translation of the target gene. Translation is interrupted in the absence of IY. RF1, peptide chain release factor 1.
Mentions: We previously demonstrated a translational switch using site-specific unnatural amino acid (UAA) 3-iodo-L-tyrosine (IY) incorporation in natural amber suppressor-free strains of Escherichia coli (Minaba & Kato, 2014). Although IY is not perfectly “unnatural” and found in very specific biological tissues such as the thyroid cells (Tietze et al., 1989) and the sponge skeletons (Ueberlein et al., 2014), we cannot detect IY ubiquitously in natural environments. The translational switch is based on conditional read-through of the UAG amber stop codons that are inserted in target genes (Fig. 1). A variant of aminoacyl-tRNA synthetase (aaRS) IYRS that was derived from the archaebacterium Methanocaldococcus jannaschii specifically recognizes both IY and an amber suppressor tRNA (tRNACUA) MJR1 (Sakamoto et al., 2009). Extracellular IY is taken up and incorporated into proteins at sites of the amber codons in the IYRS/MJR1-expressing cells. The target gene transcripts with the amber stop codons inserted next to the AUG translational start codon are translated only in the presence of IY. The absence of IY prevents translation of the target genes.

Bottom Line: Here, we report that this translational switch can control the translational efficiency at any intermediate magnitude by adjustment of the 3-iodo-L-tyrosine concentration in the medium, as a tunable translational controller.The translational efficiency of a target gene reached maximum levels with 10(-5) M 3-iodo-L-tyrosine, and intermediate levels were observed with suboptimal concentrations (approximately spanning a 2-log10 concentration range, 10(-7)-10(-5) M).Such intermediate-level expression was also confirmed in individual bacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences , Tsukuba, Ibaraki , Japan.

ABSTRACT
Translation of target gene transcripts in Escherichia coli harboring UAG amber stop codons can be switched on by the amber-codon-specific incorporation of an exogenously supplied unnatural amino acid, 3-iodo-L-tyrosine. Here, we report that this translational switch can control the translational efficiency at any intermediate magnitude by adjustment of the 3-iodo-L-tyrosine concentration in the medium, as a tunable translational controller. The translational efficiency of a target gene reached maximum levels with 10(-5) M 3-iodo-L-tyrosine, and intermediate levels were observed with suboptimal concentrations (approximately spanning a 2-log10 concentration range, 10(-7)-10(-5) M). Such intermediate-level expression was also confirmed in individual bacteria.

No MeSH data available.


Related in: MedlinePlus