Viable nonsense mutants for the essential gene SUP45 of Saccharomyces cerevisiae
© Moskalenko et al; licensee BioMed Central Ltd. 2003
Received: 29 November 2002
Accepted: 10 February 2003
Published: 10 February 2003
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.
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°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.
Nonsense mutations in the essential gene SUP45 can be isolated in the absence of tRNA nonsense suppressors.
Termination of protein synthesis occurs when the ribosome elongation machinery encounters an in-frame termination (stop) codon, either UAG, UGA or UAA, in the mRNA. This stop codon located in the A-site of the ribosome is recognized by a release factor (RF1/RF2 in prokaryotes and eRF1 in eukaryotes), which triggers release of the nascent peptide from the ribosome. Termination efficiency is enhanced by the GTPase release factor, RF3 in prokaryotes and eRF3 in eukaryotes [reviewed in [1–4]].
Release factor eRF1 recognizes all three stop codons , in contrast to prokaryotes where RF1 catalyses translation termination at UAG and UAA codons, and RF2 at UGA and UAA codons . All these proteins are encoded by essential genes: prfA (for RF1), prfB (for RF2) in bacteria [7–9] and SUP45 (for eRF1) in S. cerevisiae [10, 11]. Although the sequences of prokaryotic and eukaryotic release factors differ significantly [see  for review], a "tRNA-mimicry" model generalizing the mechanisms of prokaryotic and eukaryotic translation termination, was proposed . Peptide determinants of RF1 and RF2 involved in the specificity of stop codon recognition have been identified: in both cases a tripeptide (PAT for RF1 and SPF for RF2) was located in the homologous region of both proteins .
Recently the crystal structures of human eRF1 and bacterial RF2 have been determined and found to be different [15, 17]. However, when associated to the ribosome RF2, conformation is modified and mimics a tRNA molecule [16, 18]. Thus both eRF1 and RF2 may have comparable structures both of which are compatible with tRNA mimicry model as proposed previously . The eRF1 is composed of three domains. Domain 3 corresponds to the C-terminal part of eRF1 that is necessary for the interaction with eRF3 although there are some discrepancies in the precise localization of the region of eRF1 that interacts with eRF3 [19–22]. Domain 2 is responsible for the peptidyl transferase hydrolytic activity and includes a GGQ motif that has been highly conserved through evolution . Mutations of GGQ (Gly residues) are dominant-negative in vitro  and lethal in vivo in S. cerevisiae cells . The function of domain 1 remains unknown although it was proposed that together with domain 2, it could form a functionally active "core" domain . The role of N-terminal domain of eRF1 in stop codon recognition has been proposed  and supported by mutational approach  and crosslinking experiments .
In eukaryotes eRF1 interacts with eRF3 in vivo [27, 28]. This interaction is mediated by the C-terminus of eRF1 [20, 21]. The deletion of the 19 C-terminal amino acids abolishes the interaction with eRF3, causes an enhancement of nonsense suppression, but does not destroy viability. In contrast, eRF1 with a 32 C-terminal amino acid deletion is unable to support viability . Also it has been shown that nonsense mutations (UAA) in the SUP45 gene lead to lethality in the absence of SUQ5 suppressor . However, a viable nonsense mutant has been isolated for the prfB gene of S. typhimurium encoding RF2 release factor. It has been proposed that this mutation reduces the cellular amount of RF2 leading to inefficient termination of translation and autosuppression .
In the present work we have isolated five spontaneous nonsense mutations in the SUP45 gene that confer viability in different genetic backgrounds. These mutations result in a decreased level of full-length eRF1, thermosensitivity and lethality in meiotic segregation of heterozygous diploids.
Isolation of nonsense mutations in the SUP45 gene
After selection for histidine (his7-1 (UAA)) – lysine (lys9-A21 (UAA)) prototrophy, sixteen SUP45 mutants and eighty-four SUP35 mutants among 400 His+ Lys+ revertants were obtained. All sup45 mutant alleles together with the wild-type SUP45 allele from parent strain 1B-D1606 were cloned using PCR. The DNA sequence for each of the cloned alleles was determined.
The sequencing of the parent strain 1B-D1606 revealed 6 nucleotide substitutions compared with the published sequence : A69G, G519T, T687C, T702A, G927A, T1008C. All of these, except T702, are the same as those described earlier  for mutant alleles that obviously had a different origin regarding the wild-type strain. All nucleotide substitutions are localized in the third codon position and do not change the amino acid residues. The reason for these differences could be the origin of the strain from Petergoff Breeding Stocks (XII race S. cerevisiae) that has an independent origin  compared to the strains used in other laboratories .
Nonsense mutations in the SUP45 gene lead to readthrough of the mutated stop codon
To determine the level of nonsense suppression in the different SUP45 mutants a vector based assay system for quantification of nonsense codon readthroughin vivo was employed . β-galactosidase activities measurements in the mutants are consistent with the results obtained for auxotrophy (Figure 2A): the parent strain does not possess any suppressor activity. In the omnipotent mutants tested, nonsense suppression levels of all stop codons were high (between 8- and 28% for UAA; between 10- and 25% for UAG, and between 22- and 42% for UGA) (Figure 2B).
Since the C-terminal region of eRF1 is essential for cell viability [10, 11], some full-length eRF1 should be present even in strains bearing sup45 nonsense mutations (which will be referred to as sup45-n, where n equals n onsense), probably by readthrough of the stop codon. An affinity-purified anti-eRF1 antibody was used in the Western blot analysis to examine eRF1 levels in SUP45 mutants. As shown in Figure 2C, there was a considerable amount of full-length eRF1 in sup45-n strains. Comparison with a wild-type strain revealed a significant decrease in the amount of eRF1 protein. Ribosome fractions isolated from sup45-104, sup45-105, sup45-107 contained a truncated protein with molecular mass consistent with the predicted one (31.5, 43.0 and 35.2 kDa, respectively) (Figure 2C).
Western blot analysis did not reveal the presence of truncated protein either in ribosome fractions, or in lysates prepared from sup45-101 strain (Figure 2C and data not shown). The inability to detect such truncated protein with a predicted molecular mass of 29.6 kDa could be explained by its proteolytic sensitivity possibly because of the absence of the ribosome-binding site. The small truncated protein (5.9 kDa) in sup45-102 strain could not be detected. Relative to the level of eRF1 in wild-type strain 1B-D1606 (set to 1), the amount of full-length eRF1 in 101-D1606, 102-D1606, 104-D1606, 105-D1606 and 107-D1606 strains were 0.32, 0.08, 0.13, 0.14 and 0.17, respectively. The highest level of eRF1 in sup45-101 strain corresponds to the lowest level of suppression as measured by β-galactosidase assay (Figure 2B). These results suggest that a very small amount of eRF1 is required for cell viability.
Do sup45-n mutations lead to lethality in different genetic backgrounds?
The viability of strains bearing nonsense mutations in the essential SUP45 gene may be explained in several ways. It is possible that the parent strain itself contains weak tRNA suppressor. However, the level of readthrough in this strain, measured in a quantitative termination codon readthrough assay, is negligible (Figure 2B).
To further confirm that viability of sup45-n mutations could not be explained by the presence of weak tRNA suppressor mutation in the parent strain plasmid, shuffle analysis was performed on the derivative of S288C  whose entire genomic sequence does not contain mutations in tRNA genes . A haploid strain 1A-Y23282 bearing SUP45 disruption and the [URA3 SUP45] plasmid pRS316/SUP45 was transformed with [LEU2 sup45-n] plasmids and the resulting transformants were plated on 5-FOA medium. All transformants were able to grow in the presence of 5-FOA (Figure 4D).
The efficiency of maintenance of pRS315/SUP45 plasmid on SUP45::HIS3 [sup45-n] background
Transformants of 1A-D1628 bearing plasmids a:
% of Ura+ Leu- colonies after rounds of selection:
pRS315 [LEU2] with:
pRS316 [URA3] with:
9.5 ± 1.1
11.0 ± 0.7
6.3 ± 0.8
10.9 ± 1.1
2.2 ± 0.7
4.3 ± 0.9
2.5 ± 0.9
12.1 ± 2.2
0.3 ± 0.2
15.6 ± 1.7
2.0 ± 0.3
5.0 ± 0.6
Ura+ Leu- colonies were selected and tested for adenine and tryptophan auxotrophy. In all cases sup45-n mutations led to suppression of ade1-14 and trp1-289 (Figure 4B). Cells bearing pRS316/sup45-n plasmid were re-transformed with pRS315-SUP45 plasmid and the experiment repeated (Table 1, second column). The results show that after growth in the presence of mutant alleles of SUP45 the ability to lose the plasmid with the wild-type allele of SUP45 increased. To determine whether such an increase could be explained by reversions of sup45-n alleles to wild-type allele during shuffle experiments in yeast, the corresponding plasmids were prepared from yeast transformants and sequenced. In all tested cases the presence of sup45-n mutation was confirmed (data not shown).
Do nonsense mutations in the SUP45 gene lead to lethality in meiosis?
Previously it has been shown that sup45 nonsense mutants, selected in the presence of SUQ5, are not viable in the absence of the SUQ5 tRNA suppressor . In our work such mutants are viable for three distinct yeast strains all of which do not contain SUQ5. Thus the main difference between our data and previously published data  may be explained by different approaches to selection the mutants used. In addition, in our work, plasmid shuffle analysis was used to test the viability of sup45-n mutants as analysis of meiotic products has been used to characterize the viability of sup45-0 (0 because of o chre) mutants .
Nonsense mutations in SUP45 lead to decreased ascospore viability
D1628 transformed with plasmids:
Plasmid stability %
Total number of tetrads
Tetrads with indicated number of viable spores
% of His+Ura+ segregants a
Microscopic examination of the lethal spores from the control strain showed that all were unable to carry out mitotic divisions and stayed at the one cell stage. This result could be explained by loss of SUP45 bearing plasmids. In contrast, in the case of diploids bearing mutant sup45-n alleles most lethal spores form microcolonies of 4 to 20 cells (Table 3), many of which were abnormally shaped buds or abnormally elongated. Apparently, the SUP45::HIS3 [sup45-n] spores can germinate but the cells fail to divide after a few cell cycles.
Nonsense mutations in SUP45 lead to the block of cell divisions after spore formation
D1628 transformed with plasmids:
Total number of non-viable spores
Percentage of non-viable spores forming microcolony with following numbers of cells:
3 – 4
Nonsense mutations in SUP45 support ascospore viability in presence of SUQ5
Strain D1638 transformed with plasmids:
Total number of tetrads
Number of tetrads with viable spores
% of His+Ura+ segregants
Nonsense mutations in SUP45 are recognized as stop codons in the presence of wild-type SUP45 allele
Western blot analysis revealed the presence of fusion protein as well as wild-type eRF1 protein for all transformants, except sup45-102 (Figure 5B). No readthrough proteins were observed for any of the plasmids bearing sup45-n mutations except sup45-101, for which a small amount of readthrough fusion protein was detected. Thus, in the presence of a normal amount of wild-type eRF1 the readthrough of sup45-n alleles is completely repressed.
In this study we have shown that yeast containing nonsense mutations in the SUP45 gene can be selected for as viable strains. The product of this gene, translation termination factor eRF1, is essential and even small deletions from its C-terminus (about 30 amino acids) are lethal [20, 21]. Consistent with this, we have shown that the full-length eRF1 protein is present in all mutants bearing sup45-n alleles, although in smaller amounts.
There are several processes known that can overcome the effect of nonsense codons, such as suppression by either mutated or natural cellular RNA [reviewed in ]. The readthrough of in-frame stop-codon could be also programmed by weak termination context and/or RNA-secondary structure elements [see  for recent review].
Many of the commonly used E. coli laboratory strains as well as wild-type populations contain UAG suppressor tRNAs . For S. cerevisiae such data are unknown. The existence of a mutated nonsense-suppressed tRNA gene in the genetic background of the parent strain employed in our study is unlikely, because of the codon-specificity of such suppressors: in our work both ochre (UAA) and opal (UGA) sup45-n mutations have been isolated. Further the parent strain as well as strains used for testing viability of sup45-n mutations, do not possess any suppressor activity. Moreover, no mutations in the tRNA encoding genes were found in strain S288C , the derivative of that used in our plasmid shuffle experiments.
Viable opal nonsense mutation (supK584) in the essential gene prfB encoding bacterial release factor RF2 had been described in S. typhimurium . It has been shown that this mutation reduces the cellular amount of RF2. Such a reduction in RF2 level leads to inefficient termination of translation and thus to autosuppression. Nonsense mutations have been also isolated in the essential SUP35 gene encoding translation termination factor eRF3, both (sup35-2 (UAG) and sup35-21 (UAA)) [41, 42] being viable. Thus, different translation termination factors, such as RF2, eRF1 and eRF3, have a common property – the viability of nonsense mutants for their encoding genes. Nonsense mutations in the SUP45 gene (sup45-0) in SUQ5-ochre suppressing background have been previously isolated; nevertheless, they could not support viability in the absence of SUQ5 mutation leading to lethality in the crosses with wild-type strain .
The efficiency of UAA-readthrough of sup45-n mutations depends on the base following stop codon
Level of eRF1 (% from wild type)
Natural occurrence in highly expressed genes (%) 
Interestingly, all sup45-0 mutations, that led to the appearance of an in-frame UAA stop codon , were situated in the strongest +4 context where purine was presented in fourth position. However the discrepancy between our data and that of Stansfield et al. cannot be explained by different context influence because one viable sup45-n mutation which is completely identical to sup45-18  has been isolated (Borchsenius, Zhouravleva, unpublished). Also, the readthrough of premature stop codons in the SUP45 gene cannot be explained only by weak termination context because in the presence of wild-type eRF1 protein there was no readthrough of fusion Gal4-Sup45-n mRNAs. Interestingly, the possibility of isolating viable nonsense mutations in the SUP45 gene due to the existence of wild-type tRNAs in the yeast cytoplasm has been predicted . Natural nonsense suppression is a widespread mechanism of translation regulation [reviewed in ]. To date, the ability of different yeast tRNA to translate all three stops codons has been shown [46–48]. Experiments are in progress to identify the precise mechanism leading to readthrough of sup45-n mutations.
Although sup45-n mutations do not lead to lethality in different genetic backgrounds, their viability increased after growth in the absence of wild-type allele. The maintenance of plasmid containing a wild-type copy of the SUP45 gene changed: the frequency of its loss was very low compared with plasmid bearing sup45-n mutation, however, this frequency significantly increased after reshuffle of SUP45 bearing plasmid in sup45-n mutants. In crosses between the sup45-n strains and the wild-type strain the viability of segregants bearing sup45-n alleles was higher than in meiotic progeny of diploid cells heterozygous for SUP45 disruption. Possibly, additional mutations were selected during the first stage of selection, or, alternatively, a decreased level of eRF1 could influence expression of other components of the translation termination complex. Further studies are necessary to identify the precise mechanism of this phenomenon.
It is known that high levels of nonsense suppression in yeast are lethal . Most of sup45-n mutations lead to decreased spore viability. Spores bearing sup45-n mutations are characterized by limited growth after germination leading to the formation of micro-colonies of 4 to 20 cells. The arrest of cell division in sup45-n bearing spores cannot be explained by the presence of some residual eRF1 protein derived from the heterozygous diploid. It has been shown that disruption of the SUP45 gene leads to blockage of the cell cycle at the START of G1, manifested as the inability of spores to produce microcolonies . Interestingly, the presence of mutated suppressor tRNA also led to increase spore lethality  and to the arrest in cell division at approximately the 50-cell stage . The combination of sup45-n alleles with SUQ5 mutation restores the viability of sup45-n spores as well as sup45-0 mutations .
All sup45-n mutations (but not all missense mutations) led to thermosensitivity. The data concerning the efficiency of translation termination at different temperatures are controversial: for S. typhimurium it was higher at 42°C than at 37°C in both supK584 and wild-type strains . However for S. cerevisiae the efficiency of termination did not change in wild-type strain, but was lower at 33°C than at 30°C in the sup45 mutant . We did not find a significant difference in the amount of full-length eRF1 protein at the non-permissive temperature as compared with the permissive temperature both in the mutant sup45-104 strain and in the wild-type strain. Thus thermosensitivity of sup45-n mutants cannot be explained by the further reduction in the amount of eRF1 protein produced in the mutant at high temperature as proposed for RF2 protein in supK584 strain .
The sup45-n mutations lead to a high level of nonsense codon suppression (up to 42% for UGA stop codon when measured in the β-galactosidase assay) because of decreased level of eRF1. This result is consistent with previous findings [29, 53]. However, additional causes cannot be excluded as the full-length eRF1 protein produced in sup45-n mutants contains missense substitutions at the amino acid positions corresponding to the premature stop codons. Indeed, most of sup45-n mutations are situated in highly conserved regions of eRF1 (data not shown).
Previously it has been shown that truncated eRF1 proteins of 411, 360 and 359 amino acids were still able to bind to the ribosome . In our experiments truncated eRF1 proteins of 384, 316 and 282 amino acids, but not of 265 amino acids, bind to ribosome. This result suggests that the eRF1 ribosome-binding site is localized between amino acids 265 and 282. Consistent with these data, it has been previously proposed that for human eRF1 a ribosome-binding site includes amino acids 246 to 270 .
The eRF3-binding site on S. cerevisiae eRF1 is located in the C-terminal region, in particular the deletion from eRF1 of the 19 C-terminal amino acids abolish its interaction with eRF3 [20, 21]. The two-hybrid analysis used in our work confirms that loss of the extreme C-terminus of eRF1 abolishes its interaction with eRF3.
The studies described above raise a question about mechanisms leading to viability of nonsense mutations in essential genes. To date such mutations have been isolated in the several essential genes of S. cerevisiae [41, 42, 54, 55] and the existence of different mechanisms has been proposed. Viability of pol3 nonsense mutations has been explained by the presence of [PSI+] factor encoded by the SUP35 gene in the parent strain, which leads to readthrough of stop codons in the mutated POL3 gene . Analysis of snm1 nonsense mutations revealed that these mutations are suppressed by amplification of the plasmid carrying snm1 alleles. Plasmid amplification leads to an increase in SNM1 mRNA level, which, is believed could led to a partial readthrough resulting in the appearance of full length Snm1 protein. Interestingly, snm1 mutations do not result in translation infidelity suggesting that translation suppression was specific for the SNM1 gene . The viability of sup35-2 and sup35-21 mutations was explained by reduction of eRF3 level that causes inefficient termination of translation and leads to autosuppression [41, 42]. However, the precise mechanism leading to readthrough of these mutations, as well as sup45-n mutations, remains unclear. Future studies will include the identification of components that can participate in this process.
The data presented in this work show that nonsense mutations (sup45-n) in the SUP45 gene can be isolated and that such mutations support viability in different genetic backgrounds. All these mutations lead to a decreased level of full-length eRF1 protein and thus to the ability to suppress other nonsense mutations. It could appear that a very low level of eRF1 (up to 10% relative to wild type) is sufficient to support viability. All sup45-n mutations are situated in weak +4 termination context and the relative level of eRF1 full-length protein correlates with the strength of the four nucleotide translation termination signal. Most of sup45-n mutations lead to decreased spore viability and thermosensitivity. Our data confirm that the site of interaction with eRF3 is localized in the C-terminus of eRF1. Our results suggest that the ribosome-binding site of yeast eRF1 is localized between amino acids 265 and 282.
Strains, media and reagents
The Saccharomyces cerevisiae strains used in this study are listed in Table 6 (see additional file 1). All strains were [psi-]. Strains BSC483/4c and D1628 have been described earlier [56, 57]. Yeast cultures were grown in standard rich (YPD) or synthetic (SC) media  at 25°C. Usually, omnipotent SUP45 mutations are selected by reversions of two nonsense mutations of different types (for example, UAA and UGA) [see  for review], this decreases the probability of obtaining mutations in tRNA genes. In this work we used reversions of two nonsense mutations of the same type (UAA). The yeast strain 1B-D1606 containing nonsense mutations of all types have been constructed. For selection of suppressor mutants, simultaneous spontaneous reversions to histidine and lysine prototrophy were employed. His+ Lys+ revertants were crossed with strains bearing sup35 or sup45 mutations to identify SUP45 mutants. Standard methods of yeast genetics were used . Transformants were grown in the media selective for plasmid maintenance (SC-Trp, SC-Leu, SC-Ura). Suppression of nonsense mutations was estimated by growth at 25°C on synthetic media lacking the corresponding amino acids. To determine the frequency of plasmid loss transformants were plated at 200 cells per Petri dish on YPD medium, grown at 25°C for several days and replica plated on SC-Ura or SC-Leu media. About 200 to 500 colonies from 3 independent transformants, were tested. For plasmid shuffle, selective medium containing 1 mg/ml 5-fluoroorotic acid (5-FOA, Sigma) was used. Yeast transformations were performed as described previously . 3-AT plates contained different concentrations of 3-amino-1,2,4-triazole (Sigma). The two-hybrid interactions were assessed by the ability to transactivate HIS3 reporter gene (to confer growth in the absence of histidine and in the presence of different concentrations of 3-AT).
Bacterial strains used in the work were JM109 (recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi Δ (lac-proAB) F' [traD36 proAB+lacIqlacZΔM15])  and XL1-Blue (recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1 [F', proAB, lacIq, Δ(lacZ)M15, Tn10(tet)]) .
For quantitative characterization of nonsense suppression in sup45 mutants β-galactosidase reporter system  was used. Mutants were transformed with pUKC815 plasmid containing PGK1-lacZ fusion (control) or with pUKC817, 818, 819 plasmids carrying TAA, TAG, TGA (termination codons), respectively, cloned in frame with lacZ. β-galactosidase activity was determined as described . Only in the case of nonsense suppression could active β-galactosidase be synthesized. Efficiencies of suppression were calculated as a ratio of β-galactosidase activity in cells harboring lacZ with premature termination codon to that in cells with a normal allele of lacZ. Values for liquid β-galactosidase assay represent the mean of at least three assays from each of three independent transformants.
Plasmid DNA was isolated as described . Yeast genomic DNA was prepared using a lyticase (Sigma) and genomic DNA purification kit (Promega).
The SUP45 ORF was amplified by PCR from the genomic DNA of yeast strains using oligonucleotides corresponding to the 5' of the ORF (82 – CATTTCGGCTTGTCTCC or 99 – TTTGTGATTCCATGAAGAGGATAACAGA CT) and an oligonucleotide, complementary to the 3' end of the ORF (83 – TCTGGCATCTAGTGATTAAATTC). PCR reactions were performed using Taq or Pfu polymerase (Promega) as recommended by the manufacturer. Sequences of PCR primers used for double strand sequencing were: 82, 83 (see above), 45 – CGACTGCACCTAAATCCAATG, 87 – CCTCATTATCCTCGGCATC, 92 – GTTCGATGTCAAAAGTGAC, 93 – GACGAAATTTCCCAGGACAC, 94 – CAAAAACTTCGGTGCTAC, 100 – GACATCGAACCTTACAAACCTATC, 101 – ctgctatcacttccacccaac, 104 – GAAAGGTCGCCGAAGTTGCTG, 105 – CAGCAACTTCGGCGACCTTTC. At least two independent PCR-products were sequenced and subcloned in pGEM-T vector (Promega). The presence of a mutated allele in pGEM-T plasmid was verified by sequencing.
Plasmid pRS316/ySUP45 was constructed by subcloning the XbaI-HindIII fragment from pYex-SUP45 (a gift of V. Kushnirov) in the same sites of pRS316 . Plasmid pRS315/SUP45 was described earlier . Plasmids pRS316/sup45-101, pRS316/sup45-102, pRS316/sup45-104, pRS316/sup45-107 were obtained by substitution of the BglII-EcoRI fragment of pRS316/SUP45 for BglII-EcoRI fragments from pGEM-T/sup45-101, pGEM-T/sup45-102, pGEM-T/sup45-104 and pGEM-T/sup45-107, respectively. Plasmid pRS316/sup45-105 was obtained by substituting the BglII-BamHI fragment of pRS316/SUP45 for BglII-BamHI fragment from pGEM-T/sup45-105. Plasmids pRS315/sup45-101, pRS315/sup45-102, pRS315/sup45-104, pRS315/sup45-105, and pRS315/sup45-107 were obtained by substituting BglII-BamHI fragment of pRS315/SUP45 for BglII-BamHI fragments from pRS316/sup45-101, pRS316/sup45-102, pRS316/sup45-104, pRS316/sup45-105 and pRS316/sup45-107, respectively.
Plasmids pGBT9/SUP35 and pGADGH/SUP45 were described previously . Plasmids pGADGH/sup45-101, pGADGH/sup45-102, pGADGH/sup45-104 and pGADGH/sup45-107 were obtained by substituting BglII-EcoRI fragment of pGADGH/SUP45 for BglII-EcoRI fragment from pGEM-T/sup45-101, pGEM-T/sup45-102, pGEM-T/sup45-104 and pGEM-T/sup45-107, respectively. Plasmid pGADGH/sup45-105 was obtained by substituting BglII-BamHI fragment of pGADGH/SUP45 for BglII-BamHI fragment from pGEM-T/sup45-105.
Yeast cultures were grown in YPD medium or in a medium selective for plasmid markers. Ribosomal fractions were prepared as described . From ten to twenty μg of the proteins were separated on 12 % SDS-polyacrylamide gel according to Laemmli , and transferred to a nitrocellulose membrane (Immobilon-P). Western blot analysis was performed with 1/100 diluted polyclonal rabbit antibodies that were obtained against mixture of peptides from the N-terminal (CLTDEYGTASNIKSRV) and the C-terminal (VDESEDEYYDEDEGS) regions of S. cerevisiae eRF1 and then purified against the N-terminal peptide of eRF1. Bound antibodies were detected using the Amersham ECF system (Amersham Pharmacia Biotech) and the Storm 840 PhosphorImager (Molecular Dynamics, USA). Signal intensities were quantified using ImageQuaNT 5.2 software (Molecular Dynamics, USA).
We are very grateful to M. Tuite and V. Kushnirov for yeast strains and plasmids; to C. Le Goff for her technical assistance and to M.D.Ter-Avanesyan for very helpful discussion. We thank H.B.Osborne for reading the manuscript. This work was supported by common grant from CNRS (PICS 1113), S.M., S.Ch., S.I-V., G.Z. from RFBR (00-04-22001NCNI_a), S.M., S.Ch., S.I-V. from CRDF No.ST-012-0, S.I-V., G.Z. from CRDF RB1-2336-ST-02. S.M from INTAS(YSF-00-19).
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