Decoding accuracy in eRF1 mutants and its correlation with pleiotropic quantitative traits in yeast

Abstract

Translation termination in eukaryotes typically requires the decoding of one of three stop codons UAA, UAG or UGA by the eukaryotic release factor eRF1. The molecular mechanisms that allow eRF1 to decode either A or G in the second nucleotide, but to exclude UGG as a stop codon, are currently not well understood. Several models of stop codon recognition have been developed on the basis of evidence from mutagenesis studies, as well as studies on the evolutionary sequence conservation of eRF1. We show here that point mutants of Saccharomyces cerevisiae eRF1 display significant variability in their stop codon read-through phenotypes depending on the background genotype of the strain used, and that evolutionary conservation of amino acids in eRF1 is only a poor indicator of the functional importance of individual residues in translation termination. We further show that many phenotypes associated with eRF1 mutants are quantitatively unlinked with translation termination defects, suggesting that the evolutionary history of eRF1 was shaped by a complex set of molecular functions in addition to translation termination. We reassess current models of stop-codon recognition by eRF1 in the light of these new data.

Figure 1.

eRF1 point mutants affect viability and termination efficiency in yeast. Yeast strain YTH82 was used to generate data for this figure. (A) Amino acids mutated in this study are shown in spacefill in a ribbon model of yeast eRF1. The yeast model was generated by homology modelling based on the published structure of human eRF1 (48). Phenotypes of the respective mutants are indicated in red (inviable or genetically unstable), orange (viable but detectable termination defect on at least one of the three stop codons) or green (viable and no significant termination defect on any of the analysed stop codons). (B) The relative conservation of amino acids in the eRF1 sequence is indicated by colour. Amino acids mutated in this study are shown in spacefill as in panel A. (C) Some eRF1 mutants alter the intracellular abundance of the protein. A representative western blot is shown for the three mutants that showed significantly different changes in abundance compared to wild-type. Numbers under the blots give the abundance relative to wild-type, as average of three independent experiments. ‘Loading’ indicates a high molecular weight band arising from a cross reaction of the anti-eRF1 antibody, which was used as loading control.

Figure 2.

Strain specificity of stop codon read-through. (A) Bar graphs on the left show basal stop codon read-through levels for three SUP45 shuffling strains. Bar graphs on the right show changes in stop codon read-through compared to wild-type for two different mutants. Error bars indicate the variability in data from four independent transformants. Strains used are indicated. See text for discussion. (B) Western blots indicate that there are no differential stability effects that could give rise to the strain specific effect of the M48I mutant observed in panel A.

Figure 3.

Relationship between stop codon read-through and growth in yeast eRF1 mutants. Yeast strain YTH82 was used to generate data for this figure. (A) plots of growth rates versus termination efficiency for individual strains for three stop codons with ‘C’ as fourth base context. Growth data are from the same experiment in all three graphs. Error bars (x-axis) indicate the standard error for the growth rate of three independent transformants, y-axis error bars indicate the standard error for termination efficiency of four independent transformants. Pearson product moment correlation coefficients for the correlation between termination defects and growth are given. Data points for eRF1 mutants analysed in panel B are indicated. (B) The change in stop codon read-through was measured for all possible tetranucleotide stop signals in two eRF1 mutants and [PSI⁺] cells. Error bars indicate the standard deviation of data from six independent transformants. These data indicate that loss of growth in the N58A mutant (panel A) is unlikely to stem from general termination defects, since the [PSI⁺] strain shows more severe termination defects for most stop signals.

Figure 4.

Pleiotropic phenotypes in sup45 mutants. Yeast strain YTH82 was used to generate data for this figure. (A) Observed growth phenotypes of a selection of eRF1 mutants. (B) Cell shape phenotypes observed in I32F and D110G eRF1 mutants. (C) A heat map summarizing all observed phenotypes for all mutants and control strains. Growth phenotypes were quantified by measuring colony diameters after 48 h of growth under the respective conditions, and normalized to values between one (fittest observed phenotype) and zero (worst observed phenotype).

Figure 5.

eRF3 is a dosage suppressor of eRF1-related phentoypes. Yeast strain YTH82 was used to generate data for this figure. (A) eRF3 overexpression partially suppresses the colony colour change observed in eRF1 mutants. Patched colonies of cells containing either an empty 2µ plasmid, or a 2µ plasmid also containing the eRF3 gene, are shown. Numbers next to the panels indicate the red-shift in the presence of high-copy eRF3, as evaluated from computerized scans of the plates. Positive numbers indicate a shift towards white colour, negative numbers a shift towards red. (B) Effects of high copy expression of eRF3 in the eRF1 N58A mutant. High copy number expression of eRF3 partially suppresses the colony colour and paromomycin phenotypes. It also very weakly suppresses the hydroxyurea and high osmolarity phenotpyes, while exacerbating the temperature sensitivity observed in this mutant. (C) Western blots demonstrate that eRF3 is overexpressed 3-fold in the presence of high copy plasmids encoding its gene, and that neither the stability of wild-type eRF1 nor of N58A eRF1 are affected by this overexpression. (D) Summary of suppression by high copy number eRF3. Paromomycin, hydroxyurea and high osmolarity phenotypes can be co-suppressed together with colony colour in many sup45 (eRF1) mutants.

Figure 6.

Correlation between eRF1-related phenotypes. Yeast strain YTH82 was used to generate data for this figure. The linear distances between individual data points approximate the degree of correlation between the severities of the two phentoypes, i.e. data points located more closely together show stronger correlation in the rank order of the phenotypes. See text for discussion.

Figure 7.

Stop codon specificity of read-through in eRF1 N-domain mutants. The boxed panel shows as reference a summary of residues identified in previous studies. Cavities 1–3 refers to residues identified by Bertram et al. (4), pocket 1/2 to residues identified by Hatin et al. (6). The K and S residues of the TASNIKS motif, and the C124 residue identified by Fan-Minogue et al. (5), are also shown. The other three panels show the same view of the eRF1 N-domain, with residues mutated in this study shown in spacefill and colour coded according to the level of stop codon read-through observed. Residues of mutants conferring strong UGA-specific read-through are indicated.