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. 2016 Jul 12;44(19):9231–9244. doi: 10.1093/nar/gkw630

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Codon engineering of genes regulated by ablates their sensitivity to rare glutamine codon concentration. (A) Three target mRNAs known to be under-expressed in a sup70-65 yeast were selected. In silico, all CAG codons within each ORF were replaced by synonymous CAA codons. Using the two-state TASEP model, their translation efficiency was compared in a wild-type and sup70-65 simulation (dark and light grey bars respectively). (B) Synthetic variants of the same three ORFs were prepared (e.g. FAR7^CAA) in which all CAG codons were replaced by CAA. Their expression levels were compared in wild-type (dark bars) and sup70-65 mutant yeast (light bars) using quantitative Western blotting normalised for mRNA expression level as in Figure 3 (n = 3, ± SE). For reference, the expression level of the progenitor, CAG-containing allele, (Figure 3B data) is shown by the dashed lines. (C) Expression levels of -sensitive target genes YIL152W and FAR7 were compared in wild-type and sup70-65 mutant yeast using quantitative Western blotting normalised by measured mRNA levels, in the presence and absence of ectopically-expressed SUP70 wild-type (n = 3, ± SE). The tRNA-encoding SUP70 gene was transformed on a multicopy plasmid to ensure high-level expression.

Inline graphic — Codon engineering of genes regulated by ablates their sensitivity to rare glutamine codon concentration. (A) Three target mRNAs known to be under-expressed in a sup70-65 yeast were selected. In silico, all CAG codons within each ORF were replaced by synonymous CAA codons. Using the two-state TASEP model, their translation efficiency was compared in a wild-type and sup70-65 simulation (dark and light grey bars respectively). (B) Synthetic variants of the same three ORFs were prepared (e.g. FAR7^CAA) in which all CAG codons were replaced by CAA. Their expression levels were compared in wild-type (dark bars) and sup70-65 mutant yeast (light bars) using quantitative Western blotting normalised for mRNA expression level as in Figure 3 (n = 3, ± SE). For reference, the expression level of the progenitor, CAG-containing allele, (Figure 3B data) is shown by the dashed lines. (C) Expression levels of -sensitive target genes YIL152W and FAR7 were compared in wild-type and sup70-65 mutant yeast using quantitative Western blotting normalised by measured mRNA levels, in the presence and absence of ectopically-expressed SUP70 wild-type (n = 3, ± SE). The tRNA-encoding SUP70 gene was transformed on a multicopy plasmid to ensure high-level expression.