Viable nonsense mutants for the essential gene SUP45 of Saccharomyces cerevisiae

Abstract

Background: Termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs) - eRF1 and eRF3. The highly conserved translation termination factor eRF1 in Saccharomyces cerevisiae is encoded by the essential gene SUP45.

Results: We have isolated five sup45-n (n from nonsense) mutations that cause nonsense substitutions in the following amino acid positions of eRF1: Y53 --> UAA, E266 --> UAA, L283 --> UAA, L317 --> UGA, E385 --> UAA. We found that full-length eRF1 protein is present in all mutants, although in decreased amounts. All mutations are situated in a weak termination context. All these sup45-n mutations are viable in different genetic backgrounds, however their viability increases after growth in the absence of wild-type allele. Any of sup45-n mutations result in temperature sensitivity (37 degrees C). Most of the sup45-n mutations lead to decreased spore viability and spores bearing sup45-n mutations are characterized by limited budding after germination leading to formation of microcolonies of 4-20 cells.

Conclusions: Nonsense mutations in the essential gene SUP45 can be isolated in the absence of tRNA nonsense suppressors.

Figure 1

Schematic representation of sup45-n mutations isolated in present work. Position of the mutations is indicated above the SUP45 gene. The numbers under the SUP45 gene correspond to amino acid positions whose codons were changed to premature stop codons. Numbers on the right of arrows correspond to the size of truncated eRF1 protein.

Figure 2

Nonsense mutations in the SUP45 gene lead to omnipotent suppression. A. Plate assays showing the growth of yeast strains bearing nonsense mutations in the SUP45 gene (101-1B-D1606, 102-1B-D1606, 104-1B-D1606, 105-1B-D1606, 107-1B-D1606) in the synthetic medium without histidine (SC-His), lysine (SC-Lys), adenine (SC-Ade) or tryptophan (SC-Trp) at 25°C compared with the parent strain 1B-D1606. The types of nonsense mutations in parent strain are denoted under media indications. SUP45 mutant alleles are indicated. Ten serial dilutions of yeast suspension of the same density were used. Five independent clones were tested, representative results are shown. B. Determination of the level of nonsense suppression in sup45-n mutants. UAA, UAG and UGA suppression levels were quantified by measuring β-galactosidase levels in the strains 1B-D1606 (wild type) as control and in mutants transformed with plasmids pUKC817, pUKC818 or pUKC819 bearing a specific stop codon (TAA, TAG or TGA, respectively) in frame that precedes lacZ gene. The efficiency of nonsense codon readthrough (%) was quantified as a ratio of β-galactosidase activity in cells harboring lacZ with stop codon to that in cells without a stop codon in-frame with the lacZ gene in pUKC815 plasmids. The same strains as in Fig. 2A were used. Results are the means of three separate experiments. C. Western blot showing the synthesis of full-length eRF1 (49 kDa) and truncated eRF1 proteins with predicted molecular mass 43.0 kDa, 35.2 kDa and 31.5 kDa for mutants 105, 107 and 104, respectively. (*) Indicates a non-specific band. A search of yeast proteome with the N-terminal peptide sequence that was used for antibody production, revealed the presence of one protein (methionyl-tRNA synthetase, GenBank accession number CAA24627) containing this sequence that has an expected molecular weight near 85 kDa. Cultures of the same strains as in (A) were grown to mid-log phase in medium selective for plasmid and ribosome fractions were prepared. The same amount of each sample was loaded per lane. Immunoblot analysis was performed using polyclonal antibodies directed against the N-terminal part of eRF1.

Figure 3

Nonsense mutations in the SUP45 gene lead to thermosensitivity. Thermosensitivity of strains bearing sup45-n mutations. The same strains as in (2A) were tested on the rich medium (YPD) at 37°C.

Figure 4

Nonsense mutations in the SUP45 gene confer the viability in the different genetic backgrounds. A. Strain 1A-D1628 bearing SUP45 disruption was co-transformed by pRS316/SUP45 and pRS315/sup45-n plasmids. The cotransformants bearing pRS316/SUP45 and vector pRS315 only were used as a negative control. The transformants were assayed for growth by plating on 5-FOA medium to select against the URA3 plasmid pRS316/SUP45 carrying a wild-type copy of SUP45. Ten serial dilutions of yeast suspensions of the same density were used. Five independent transformants for each combination were tested. Representative results are shown. B. Plate assays showing the growth of transformants of 1A-D1628 bearing pRS316/SUP45 or pRS316/sup45-n plasmids on the synthetic medium without adenine (SC-Ade) or tryptophan (SC-Trp) at 25°C. The strains were tested in the same way as in Fig. 2A. C. Western blot showing the expression of full-length eRF1 (49 kDa) and truncated eRF1 proteins in the same strains as in (B). D. Complementation of the SUP45 gene disruption in the strain 13A-Y23282 by sup45-n mutations. The transformants with vector pRS315 were used as a negative control. The same plasmids as in (A) were used.

Figure 5

Expression of sup45-n mutations in the presence of wild-type amount of eRF1 protein does not lead to readthrough. A. Two-hybrid interactions. Strain HF7C was co-transformed with pGADGH plasmids carrying different sup45-n mutations and pGBT9/SUP35 plasmid. The interactions were tested by the extent of resistance to 3-AT. Ten serial dilutions of yeast suspensions of the same density were used. Five independent transformants for each combination were tested. Representative results are shown. B. Western blot showing the expression of full-length eRF1+Gal4 and truncated eRF1+Gal4 proteins with predicted molecular mass in the same transformants as at (A).