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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 6. — Reductions in the global translation initiation rate ameliorate the mRNA-specific translational inefficiencies caused by reduced concentration. (A) Cell cytometry was used to assay the prevalence of pseudohyphal chains that typify the sup70-65 mutant. Forward scatter measurements indicated the extent of formation of large chains of cells. Pseudohyphal growth was assessed in wild-type and sup70-65 yeast in the presence of either a wild-type ectopic GCN2 allele (dark-shaded frequency plot), or a constitutively-active gcn2^c allele (light-shaded). Population sizes of large chains and single budded cells were quantified using the cytometry data and plotted in the bar chart. (B) To confirm these observations, the degree of pseudohyphal chains formation in wild-type or sup70-65 cells, transformed with a plasmid expressing either CGN2 or gcn2^c was assessed by direct microscope observation. Pseudohyphal chains were counted, and a chain formation index used to capture the extent of pseudohyphal growth during growth on complete (YPD) or minimal medium (SC) (6). (C) HA-tagged FAR7 was expressed in wild-type and sup70-65 yeast transformed with ectopically expressed CGN2 or gcn2^c genes to either maintain, or reduce, global translation initiation rates respectively. Three different gcn2^c alleles were used with increasing degrees of constitutive eIF2 phosphorylation activity (E1522K < E1573G < M719V, E1522K). FAR7 expression was quantified using Western blotting, normalised for mRNA expression level as in Figure 3 (n = 3, ± standard error of the mean). The Far7p expression level in the mutant sup70-65 was expressed as a percentage of the expression level in a wild-type cell.

Inline graphic — Reductions in the global translation initiation rate ameliorate the mRNA-specific translational inefficiencies caused by reduced concentration. (A) Cell cytometry was used to assay the prevalence of pseudohyphal chains that typify the sup70-65 mutant. Forward scatter measurements indicated the extent of formation of large chains of cells. Pseudohyphal growth was assessed in wild-type and sup70-65 yeast in the presence of either a wild-type ectopic GCN2 allele (dark-shaded frequency plot), or a constitutively-active gcn2^c allele (light-shaded). Population sizes of large chains and single budded cells were quantified using the cytometry data and plotted in the bar chart. (B) To confirm these observations, the degree of pseudohyphal chains formation in wild-type or sup70-65 cells, transformed with a plasmid expressing either CGN2 or gcn2^c was assessed by direct microscope observation. Pseudohyphal chains were counted, and a chain formation index used to capture the extent of pseudohyphal growth during growth on complete (YPD) or minimal medium (SC) (6). (C) HA-tagged FAR7 was expressed in wild-type and sup70-65 yeast transformed with ectopically expressed CGN2 or gcn2^c genes to either maintain, or reduce, global translation initiation rates respectively. Three different gcn2^c alleles were used with increasing degrees of constitutive eIF2 phosphorylation activity (E1522K < E1573G < M719V, E1522K). FAR7 expression was quantified using Western blotting, normalised for mRNA expression level as in Figure 3 (n = 3, ± standard error of the mean). The Far7p expression level in the mutant sup70-65 was expressed as a percentage of the expression level in a wild-type cell.