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. 2015 Apr 20;43(9):4591–4601. doi: 10.1093/nar/gkv376

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© The Author(s) 2015. 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. — Growth complementation assays for the stop codon specificities of the Hs-eRF1 L126 mutants in the presence of Sc- or Hs-eRF3. (A) Growth complementation assay for single vector transformants of wild type (WT) and L126 mutants of Hs-eRF1 in the presence of heterologous Sc-eRF3 using Sc-eRF1 tet-off strain (S13-D09). Transformants of p416ADH (URA3 marker) (38) harboring each Hs-eRF1 variant grown under permissive conditions were streaked on the SC-Uracil plates containing the stable tetracycline analog (Dox) doxycycline (10 μg/ml) and their colony formation was monitored. (B) Growth complementation assay for dual vector transformants of L126 variants of Hs-eRF1 on p416ADH (URA3 marker) and Hs-eRF1 mutants T32A and V71L (26) on p415GPD (LEU2 marker) in the presence of homogeneous Hs-eRF3 on p414GPD (TRP1 marker) using the Sc-eRF1-Sc-eRF3 double tet-off strain (Y138) (38). Transformants grown under permissive conditions were streaked on the SC-Uracil-Leucine-Tryptophan plates containing the stable tetracycline analog doxycycline (Dox; 10 μg/ml) and their colony formation was monitored. Dox, doxycycline. Codon specificities are indicated: (all) omnipotent, i.e. UAG, UGA, UAA-specific (UAA/UAG) and (UGA) biased to be favorable to ‘UAA and UAG dual-specific’ and ‘UGA-specific’, respectively. Colony growth is indicated as (–) no growth at all, (±) very severe inhibition of growth with several revertant colonies and (+) normal growth.