ISSN 00268933, Molecular Biology, 2010, Vol. 44, No. 2, pp. 268–276. © Pleiades Publishing, Inc., 2010. Original Russian Text © O.A. Murina, S.E. Moskalenko, G.A. Zhouravleva, 2010, published in Molekulyarnaya Biologiya, 2010, Vol. 44, No. 2, pp. 301–310.
CELL MOLECULAR BIOLOGY UDC 575.2:582.282.23
Overexpression of Genes Encoding tRNATyr and tRNAGln Increases the Viability of Saccharomyces cerevisiae Strains with Nonsense Mutations in the SUP45 Gene O. A. Murina, S. E. Moskalenko, and G. A. Zhouravleva Department of Genetics and Breeding, St. Petersburg State University, St. Petersburg, 199034 Russia; email:
[email protected] Received July 17, 2009 Accepted for publication August 4, 2009
Abstract—At present, the machinery supporting the viability of organisms possessing nonsense mutations in essential genes is not entirely understood. Nonsense mutants of Saccharomyces cerevisiae yeast containing a premature translation termination codon in the essential SUP45 gene are known. These strains are viable in the absence of mutant suppressor tRNAs; hence, the existence of alternative mechanisms providing nonsense suppression and mutant viability is conjectured. Analysis of clones obtained by transformation of a strain bearing a nonsensemutant allele of SUP45 with a multicopy yeast genomic library revealed three genes encoding wildtype tRNATyr and four genes encoding wildtype tRNAGln, which increased nonsense mutant viability. Moreover, overexpression of these genes leads to an increase in the amount of the fulllength eRF1 protein in cells and compensates for heat sensitivity in the nonsense mutants. Probable ways of tRNATyr and tRNAGln influence on the increase in the viability of strains with nonsense mutations in SUP45 are discussed. DOI: 10.1134/S0026893310020123 Key words: translation termination, yeast, nonsense suppression, SUP45, sup45 nonsense mutants, eRF1, tRNA
Protein factors eRF1 and eRF3 play the key role in translation termination in eukaryotes (for review, see [1]). As any of the three termination codons, UAA, UAG, or UGA, is transformed into the A site, it is rec ognized by the eRF1 protein, and the peptidyl–tRNA bond is cleaved [2]. Factor eRF3 is GTPase. It stimu lates eRF1 to allow the release of the new polypeptide chain from the ribosome [3, 4]. Translation termina tion requires the cooperation of the eRF1 and eRF3 proteins [3, 5]. In Saccharomyces сerevisiae yeast, translation termination factor eRF1 is encoded by the SUP45 gene [6, 7], and eRF3 is encoded by SUP35 [8–10]. Although the SUP35 and SUP45 genes are essential for the cell, they can contain nonsense muta tions [11–15]. These mutations are not lethal, but they cause translation termination aberrations. The first nonsense mutation in the yeast SUP45 gene was obtained in the presence of the dominant SUQ5 suppressor, which encoded a serine tRNA with a mutant anticodon [11]. The nonsensesuppressing ability of the mutant serine tRNA allowed isolation of four allosuppressor sup45 alleles with premature ter mination codons (PTCs) UAA in different regions of the SUP45 S. сerevisiae coding sequence. Nonsense mutations in the S. cerevisiae SUP45 gene (sup45n) are not lethal even without the SUQ5 dominant suppressor [13]. The survival of the mutant strains is explained by the presence of the functional
fullsize eRF1 protein. Its amount is less than in wild type cells; therefore, it is suggested that even such a small amount is sufficient to support cell viability. The main translation product of SUP45 in sup45n mutants is a truncated eRF1 variant, formed by termi nation at the PTC. The absence of mutant suppressor tRNAs able to “sensify” the termination codon in sup45n strains indicates that there are alternative pathways supporting the viability of the mutants. In our previous studies of pathways responsible for the survival of nonsense mutants, we observed a low frequency of the initial loss of the wildtype SUP45 allele in cells possessing this allele together with a mutant sup45n allele [13]. However, higher frequency of secondary loss was observed in cells that had ini tially lost the wildtype SUP45 allele but were then transformed with the same allele [13]. The increase in the frequency of wildtype SUP45 allele loss is likely to be determined by either a specific regulation pathway, which is triggered by the reduced level of eRF1 in the cell, or by the selection of additional mutations during the first loss, which would increase the viability of cells with nonsense mutations in SUP45. Later studies showed that nonsense mutations had elevated levels of endogenous tRNAs with potential nonsensesup pressing activity. This observation argued for the effect of regulatory pathways favoring the survival of such mutants [16].
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This work is dedicated to the search for factors increasing the viability of S. сerevisiae strains with non sense mutations in SUP45. The search was conducted with a yeast genomic library in a multicopy vector. We managed to identify genes whose overexpression increased the survival rate of strains with SUP45 muta tions and to recognize pathways compensating for nonsense mutations. EXPERIMENTAL Strains. Experiments were performed with S. cere visiae strains: 31BD1656 (МАTα ade114 trp1289 ura352 leu23,112 sup45105 [pRS316/SUP45]); 1BD1606 (МАТα ade114 his71 lys9A21 trp1289 ura352 leu23,112) and its derivatives bearing non sense alleles sup45104, sup45105, and sup45107; and 1AD1628 (MATa ade114 his3 lys2 trp1289 ura352 leu23, 112 SUP45::HIS3 [pRS315/SUP45]) [13]. The ade114, his71, lys9A21, and trp1289 alleles bear nonsense mutations UGA, UAA, UAA, and UAG, respectively. Escherichia сoli strains: DH5α and C600 [17]. Plasmids. We used centromeric pRS316/SUP45 plasmid, bearing a fragment of yeast DNA with the fullsize SUP45 allele under the control of its own pro moter [13], the pRS425 multicopy vector [18], and a commercial S. сerevisiae genomic library in the YEp13 multicopy vector (American Type Culture Collection, ATCC no. 37323; http:// www.lgcstandardsatcc.org). The library consisted of plasmids bearing 5–20 kb fragments of yeast genomic DNA obtained by diges tion of the genome with Sau3AI restriction endonu clease and cloning into YEp13 linearized with BamHI restriction endonuclease. The pRS425tRNATyr plas mid, bearing the wildtype allele of tY(GUA)J1 con trolled by its own promoter, was constructed on the basis of pRS425 multicopy vector. The pRS125 RNATyr multicopy plasmid bearing the wildtype allele of tU(UUG)/L controlled by its own promoter. The mutant alleles sup45102Gln, sup45105Tyr, and sup45105Gln were constructed as described in [19]. Strain growth. Strains were grown on standard cul ture media for yeast: complete YAPD medium, mini mal MD, synthetic SC, and selective media lacking certain SC components. Elimination of the pRS316 plasmid with the URA3 marker from a strain was per formed by growing in presence of 1 g/l 5fluoroorotic acid (5FOA, Sigma). Bacteria were grown on com plete LB medium. Bacterial clones transformed with YEp13 bearing a genomic DNA fragment were detected on minimal M9 medium [21]. Ampicillin resistant transformants were selected on LB and M9 media supplemented with 100 μg/ml ampicillin. Yeast strains were grown at 26°С and bacteria was grown at 37°С. Manipulations with Saccharomyces yeast were done by conventional methods [22–24]. Highly effi cient yeast transformation and isolation of plasmid DNA from yeast were performed as in [25, 26]. MOLECULAR BIOLOGY
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Stability of the pRS316/SUP45 plasmid. To assess the rate of pRS316/SUP45 loss, 31BD1656 cells bearing this plasmid and the pRS425 vector, or pRS425tRNATyr, or pRS425tRNAGln were picked from SCUraLeu medium and inoculated to dishes with YAPD, 200 cells per dish. The dishes were incu bated for 4 days, and the colonies were replicaplated to SCUra and SCLeu. Clones with the Ura–Leu+ genotype were counted. Five hundred to one thousand colonies of each segregant were analyzed for evalua tion of plasmid loss rate. Sequencing of the terminal regions of inserts in YEp13 was performed at the Taxon Center for Meth ods of Animal Molecular Systematics, Zoological Institute, Russian Academy of Sciences, in an ABI PRISM sequencer with an ABI PRISM BigDye Ter minator v3.1 Ready Reaction Cycle Sequencing Kit. Primers: PSB and ycp50r2 [27]. Protein content. Preparation of cell lysates, SDS PAGE of proteins, and Western blotting with poly clonal antibodies against eRF1 and monoclonal anti bodies T6074 against tubulin (Sigma) were carried out as in [13]. The photofilm was scanned, and signal intensity was evaluated with TotalLab software. Statistical evaluation. Error of the mean was calcu lated according to the equation:
∑ ( x – x ) , 2
S=
i
n(n – 1)
where n is the number of samples, xi is the parameter value in the i sample, and x is the mean value of the parameter [28]. The homogeneity of the material was assessed by the multifield Chisquare method: χ2 = Σ(ftheor – fexper)2/ftheor ;
ftheor = ni × nj /Ntotal,
where ni is the total in the ith row and nj is the total in the jth column. The Chisquare value was estimated by comparison with the predicted value for the level of significance α = 0.05 and the number of the degrees of freedom ν = (n – 1)(k – 1), where n is the number of columns and k is the number of rows [28]. RESULTS Transformation and Analysis of Transformants To detect factors increasing the survival rate of nonsense mutants for SUP45, we used strain 31B D1656, which bears the nonsense allele in the chro mosome and harbors the sup45105 centromeric plas mid. This strain is characterized by a low rate of initial loss of pRS316/SUP45 and a considerable increase in the rate of secondary plasmid loss after retransformation (Fig. 1). The change in pRS316/SUP45 loss rate in the presence of the sup45105 mutant allele was considered to be a measure of the influence of an unknown factor involved on nonsense mutant viability.
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Identified genes
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Three distinct genes coding for tRNATyr
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Fig. 2. Schematic presentation of transformant screening.
0
Fig. 1. The increase in the rate of secondary pRS316/SUP45 plasmid loss by S. cerevisiae 31BD1656 cells after its first loss. (a) is growth of strain 31BD1656 on SCUra medium and in medium with 5FOA after the first (I) and second (II) events of pRS316/SUP45 loss. For either culture, typical data for 1 of 12 transformants ana lyzed are presented. (b) is quantitative assessment of the rate of pRS316/SUP45 loss by 31BD1656 after the first (I) and second (II) replacements. Data are presented with standard errors of the mean [28].
Strain 31BD1656 [pRS316/SUP45] was trans formed with the yeast genomic library in the YEp13 vector (see EXPERIMENTAL). Five thousand trans formants were selected on SCUraLeu medium and replicaplated to the medium with 5FOA without leucine. The rate of pRS316/SUP45 loss was estimated from the growth pattern. Strain 31BD1656 [pRS316/SUP45] transformed with the empty YEp13 vector was used as a control. We selected 1000 of the 5000 transformants, where the rate of pRS316/SUP45 loss differed significantly from the control value after two tests on the medium with 5FOA without leucine. Elevated loss rates were recorded in 115 of them. We isolated plasmid DNA from the transformants to check the stability of the phenotype. The mixtures of isolated plasmids contained both the pRS316/SUP45 [URA3] plasmid and YEp13 [LEU2] with genomic inserts. To obtain YEp13 alone, E. сoli strain C600 was transformed with the plasmid mixture. Clones harboring YEp13 with inserts were selected by complementation of the leuB6 mutation in E. сoli. Plasmids were isolated from two independent clones of the yeast genomic library and used for 31BD1656 retransformation. The rate of pRS316/SUP45 loss was assessed from the pattern of transformant growth on 5FOA without leucine. Elevated rates of the sec ond loss of pRS316/SUP45 were observed only in 8 of
115 transformants harboring plasmids isolated from the yeast genomic library (Fig. 2). Identification of Genes Whose Overexpression Increases the Loss Rate of the Plasmid Bearing the SUP45 Gene in the Presence of the Mutant sup45105 Allele To detect genes that increase the rate of pRS316/SUP45 loss, we sequenced regions adjacent to the 5' and 3'ends of the inserts in plasmids obtained from the library. The nucleotide sequences of the inserts were identified by using the database on the complete genome of S. cerevisiae yeast (http:// www.yeastgenome.org). Four of the eight inserts were found to contain genes for tRNATyr, and the other four, for tRNAGln. As each insert of the wildtype genome contained a gene for tRNATyr or tRNAGln, we suggested that overexpression of these genes was responsible for the elevated pRS316/SUP45 loss rate in 31BD1656 transformants. We tested our suggestion by transforming 31B D1656 [pRS316/SUP45] with multicopy plasmids pRS425tRNATyr and pRS425 tRNAGln and per forming qualitative and quantitative tests of the rates of pRS316/SUP45 loss from the transformants grown on 5FOA without leucine (Figs. 3a and 3b). Strain 31B D1656 [pRS316/SUP45] transformed with pRS425 served as a control. It was found that plasmids bearing tRNA genes increased the pRS316/SUP45 loss rate significantly. Thus, the increase in the rate of pRS316/SUP45 loss by 31BD1656 strain in case in each of the eight analyzed inserts was caused by the presence of genes encoding tRNATyr or tRNAGln. Sequencing of the inserts allowed identification of the tY(GUA)J1 and tQ(UUG)L genes, which encoded wild type tRNATyr and tRNAGln, respectively. MOLECULAR BIOLOGY
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Effect of the Overexpression of Genes for tRNATyr or tRNAGln on the Manifestation of sup45n Mutations The effect of the overexpression of genes for tRNATyr or tRNAGln on the efficiency of nonsense sup pression in strain 31BD1656 was assessed from the phenotypes of transformants tested on selective media. Prior to the test, the pRS316/SUP45 plasmid was eliminated from 31BD1656 transformants har boring pRS425tRNATyr or pRS425tRNAGln. Strain 31BD1656 transformed with pRS425 and cured of pRS316/SUP45 was used as a control. Strain 31BD1656 has nonsense mutations of two types (UGA (ade 114) and UAG (trp 1289)) and the nonsense mutation sup45105 (UAA). No increase was detected in the level of nonsense suppression under the conditions of overexpression of genes coding for tRNATyr or tRNAGln. Strain 31BD1656 bears no UAA nonsense muta tions, whose suppression could be assessed from growth on the corresponding selective medium. The effect of overexpression of genes coding for tRNATyr or tRNAGln on the efficiency of nonsense suppression of UAA was assessed in strain 1051BD1606, whose chro mosome possessed the sup45105 mutant allele. We also used strains 1041BD1606 and 1071BD1606, having the mutant alleles sup45104 and sup45107, respec tively. The genomes of these strains had nonsense mutations of all the three types: UGA (ade 114), UAG (trp 1289), and UAA (his71, lys9A21). Strains 104, 105, and 1071BD1606 were trans formed with pRS425tRNATyr or pRS425tRNAGln plasmids or with the pRS425 vector. Suppression of MOLECULAR BIOLOGY
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5FOA
103 106 (b)
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14 12 10 8 6 4 2 0
105
104
103
10.8 ± 1.72%
8.3 ± 1.96%
0.5 ± 0.05%
tRNAGln
Control tRNATyr
tRNAGln cells/ml
Control
An increase in the degree of suppression of the UAA PTC in the sup45105 allele can be one of the signs of overexpression of genes encoding tRNATyr or tRNAGln. As a result, the cell would accumulate more fullsize eRF1. The eRF1 protein was quantified by Western blotting with purified polyclonal antibodies in strain 31BD1656 with overexpressed genes for tRNATyr or tRNAGln. The amount of protein in 31B D1656 was measured after loss of pRS316/SUP45. For reference, we determined protein content in strain 1B D1606, possessing the wildtype SUP45 allele, and strain 1051BD1606, with the mutant sup45105 allele. We detected elevated amounts of fullsize eRF1 in 31BD1656 against the backgrounds of overexpressed tRNA genes: tY(GUA)J1, encoding tRNATyr, or tQ(UUG)L, encoding tRNAGln (Fig. 3c). The amount of truncated 43kDa eRF1 was also elevated. This molecular weight corresponds to the polypeptide pro duced after recognition of the UAA PTC in the coding region of SUP45 by the termination factor.
(a)
–Ura–Leu
Frequency of pRS316/SUP45 loss, %
The Level of the eRF1 Protein in the sup45105 Nonsense Mutant Increases with Overexpression of Genes Encoding tRNATyr or tRNAGln
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tRNATyr
tRNAGln
(c)
eRF1, kDa 49.0 43.0
Fig. 3. Overexpression of either of the tY(GUA)J1 or tQ(UUG)L genes, coding for tRNATyr or tRNAGln, respec tively, increases the rate of pRS316/SUP45 plasmid loss and the level of the fullsize eRF1 protein in strain 31B D1656. (a) is growth of strain 31BD1656 transformed with plasmids pRS425 (control), pRS425tRNATyr, or pRS425tRNAGln on SCUraLeu and on 5FOA without leucine. For each plasmid, typical data for 1 of 12 transfor mants analyzed are presented. (b) is quantitative assess ment of the rate of pRS316/SUP45 loss in the same trans formants. Data are presented with standard errors of the mean [28]. (c) is western blotting with polyclonal antibod ies against eRF1. Arrows indicate the fullsize eRF1 pro tein (49.0 kDa) and the truncated variant produced by the mutant (43.0 kDa).
nonsense mutations was tested on a panel of selective media. No effect of overexpression of genes coding for tRNATyr or tRNAGln on the efficiency of nonsense sup pression was found in the derivatives of strain 1B D1606 bearing the mutant sup45n allele (data not shown). We also analyzed the growth of transformants with pRS425tRNATyr or pRS425tRNAGln on complete YAPD medium at 37°С, because it is known that non sense mutants for SUP45 are heatsensitive [29]. Strains transformed with pRS425 served as a control. In addition, growth of heattolerant 1BD1606 trans formants was studied. It was found that transformants of strains 104 and 1051BD1606 grew at the elevated temperature with the presence of pRS425tRNATyr or pRS425tRNAGln (Fig. 4). Transformants of 1071B D1606 did not grow at nonpermissive temperatures.
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YAPD 37°C 1 2
Control SUP45
tRNATyr tRNAGln Control
sup45104 (UAA)
sup45105 (UAA)
sup45107 (UGA)
tRNATyr tRNAGln Control tRNATyr tRNAGln Control tRNATyr tRNAGln 106 105 106 105 106 105 106 105
Fig. 4. Overexpression of either of the tY(GUA)J1 or tQ(UUG)L genes, coding for tRNATyr or tRNAGln, respec tively, compensates for the sensitivity of sup45n (UAA) strains to elevated temperatures. The growth of strains 1B D1606 (SUP45), 1041BD1606 (sup45104), 1051B D1606 (sup45105), and 1071BD1606 (sup45107), transformed with plasmids pRS425 (control), pRS425 tRNATyr, or pRS425tRNAGln, was estimated on com plete YAPD medium at 25 and 37°C. Typical data for 2 of 12 transformants analyzed (1, 2) are presented. The PTCs for each nonsense mutation are parenthesized.
The Amount of eRF1 in Strain 1051BD1606 with Overexpression of Genes for tRNATyr or tRNAGln at the Nonpermissive Temperature 37°С Previous studies demonstrated that the incubation of heatsensitive yeast strains possessing nonsense mutations in the SUP45 gene at 37°С resulted in a decrease in the content of the truncated variant of the eRF1 protein, whereas the amount of fullsize eRF1 did not change [29]. We studied the effect of overex pression of genes for tRNATyr or tRNAGln on the amount of eRF1 in cells of nonsensemutant strain 1051BD1606 under nonpermissive conditions. The amounts of eRF1 were determined in 1051BD1606 derivatives with the pRS425, pRS425tRNATyr, or pRS425tRNAGln plasmids. The derivatives were grown at 37°С for 4 and 8 h (Fig. 5). The initial cell cultures were used as a control immediately before the incubation at 37°С. We also determined the protein level in strain 1BD1606 bearing the wildtype SUP45 allele. Tubulin was used as a loading control. In the sup45105 [pRS425] mutant, the amount of the Nterminal fragment decreased in the course of strain growth under nonpermissive conditions. This observation is in agreement with the data reported in [29]. The amount of the truncated eRF1 protein in
strains incubated at 37°С also decreased with overex pression of genes for tRNATyr or tRNAGln. We also compared the amounts of the fullsize eRF1 variant in strains 1051BD1606 [pRS425], 1051BD1606 [pRS425tRNATyr], and 1051B D1606 [pRS425tRNAGln] sampled immediately before the incubation at 37°С (0 h), that is, grown at 25°С. We found that the amount of eRF1 in strains overexpressing tRNATyr or tRNAGln was larger than in strain 1051BD1606 [pRS425]. The amount of full size eRF1 in transformants incubated under nonper missive conditions did not change. Functionality of the Mutant eRF1 Protein The S. cerevisiae nonsense mutants for the SUP45 gene produce a fullsize eRF1 variant, but we cannot be sure that it is the wildtype protein. The mutants have high levels of endogenous tRNAs, which can be suppressors of nonsense mutations [16]. It is conceiv able that these tRNAs ensure the recognition of a PTC in the SUP45 mRNA as a sense codon. In particular, when tRNATyr or tRNAGln are overexpressed, they can act as potential suppressors of nonsense mutations in the SUP45 gene. Such amino acid substitutions in the primary structure of eRF1 are suggested to modify its properties. For example, position 385 in the amino acid sequence of eRF1, which corresponds to glutamate in the wildtype sequence, is occupied by the UAA PTC in the sup45105 allele. Also, tRNATyr and tRNAGln can suppress UAA PTCs located at posi tions of the SUP45 mRNA other than 385; for exam ple, at position 53, as in the sup45102 allele. Substitution of glutamate for tyrosine at position 385 or substitution of tyrosine for glytamine at posi tion 53 can modify the properties of the eRF1 protein. This prompted us to investigate the effect of missense substitutions located at the same positions as in sup45 105 and sup45102 mutants on eRF1 properties. We made use of the mutant sup45102Gln and sup45 105Tyr alleles, in which the GAA codon for glutamate at position 385 of eRF1 was replaced by CAA and TAC for glutamine and tyrosine, respectively (Fig. 6a), and the mutant sup45105Gln allele, where TAT (Tyr53) was substituted for CAA (Gln) in eRF1. Properties of mutant eRF1 variants were studied in strain 1AD1628 [pRS315/SUP45], bearing an inac tive SUP45 variant. This strain has also suppressible alleles ade114 (UGA) and trp1289 (UAG). Strain 1A D1628 [pRS315/SUP45] was transformed with plas mids pRS316/sup45105(Gln), pRS316/sup45 105(Tyr), pRS316/sup45102(Gln), and pRS316/SUP45 and used for control. The transfor mants were grown on complete YAPD medium. Clones that had lost pRS315/SUP45 but kept pRS316/sup45105(Gln), pRS316/sup45105(Tyr), pRS316/sup45102(Gln), or pRS316/SUP45 were selected. The clones were tested on selective media (Fig. 6). The efficiency of ade114 and trp1289 sup MOLECULAR BIOLOGY
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8 eRF1, kDa 49.0 43.0 Tubulin
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1051BD1606 (sup45105)
Fig. 5. Assay of eRF1 protein in strains 1BD1606 and 1051BD1606 grown at 37°C for 4 and 8 h. Strain 1051BD1606 was transformed with plasmids pRS425 (control), pRS425tRNATyr, or pRS425tRNAGln and strain 1BD1606 was transformed with pRS425. (a) is western blotting with polyclonal antibodies against the fullsize eRF1 and monoclonal antibodies against tubulin. Arrows indicate the fullsize eRF1 protein (49.0 kDa), the truncated protein (43.0 kDa) produced by the mutant, and tubulin. (b) is levels of fullsize and truncated eRF1 variants in strains transformed with the plasmids. Tubulin was used as a load ing control. The amount of eRF1 in the sample taken immediately before the incubation at 37°C (0 h) was taken as 1.0. The amounts of eRF1 in subsequent samples are indicated with reference to this value.
pression was assessed in strains 1AD1628 [pRS316/sup45105] and 1AD1628 [pRS316/sup45 102], which possessed omnipotent nonsensesup pressing mutations. No differences were found in the growth patterns of strains 1AD1628 [pRS316/SUP45], 1AD1628 [pRS316/sup45105(Tyr)], 1AD1628 [pRS316/sup45 105(Gln)], or 1AD1628 [pRS316/sup45102(Gln)]. For each strain, 24 independent clones were tested on selective media. The eRF1 protein possessing glutamine or tyrosine instead of glutamate at position 385 or glutamine instead of tyrosine at position 53 can support the survival of the strain with nonfunctional SUP45. Apparently, it is as functional as the wildtype eRF1 (data not shown). Thus, our results indicate that overexpression of genes for tRNATyr or tRNAGln per mits these tRNAs to suppress UAA nonsense muta tions in both the sup45105 and sup45102 alleles, pre serving the function of the eRF1 protein. DISCUSSION The realization of genetic information in the cell is regulated at several steps. One of them is protein syn thesis termination. In spite of the high accuracy of this process, some features make translation termination MOLECULAR BIOLOGY
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ambiguous to adapt the cell to environmental and internal changes (for review, see [30]). One of such features is nonsense suppression: recognition of a translation termination codon, generated by a muta tion in the coding region of a gene, as a sense codon. One of the causes of nonsense suppression is believed to be partial inactivation of termination factors eRF1 and eRF3. In particular, nonsense mutants for the SUP45 gene are characterized by the suppression of all the three termination codons, referred to as omnipo tent nonsense suppression [13]. It is known that mutant sup45n strains produce a fullsize eRF1 pro tein, but its level is much lower than in wildtype cells [13]. A decrease in the level of either eRF1 or eRF3 reduces termination efficiency in eukaryotes [11]. In this case, the preliminary termination codon resulting from a mutation is “sensified,” being readthrough by tRNA molecules, which compete with termination factors for attachment to the codon [31, 32]. We looked for factors increasing the survival rate of nonsense mutants for SUP45 that produce the par tially inactive translation termination factor eRF1. Studies of this sort allow not only identification of genes affecting termination but also understanding of pathways compensating for nonsense mutations in essential genes.
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SUP45 sup45105Gln sup45105Tyr sup45105 SUP45 sup45102Gln sup45102
cells/ml 106 105 104 103 106 105 104 103 106 105 104 103
Fig. 6. Missense mutations E385Q, E385Y, and Y53Q do not disturb the function of the eRF1 protein. (a) is sche matic presentation of missense mutations in the SUP45 gene. Original codons are indicated above. Codons with substitutions are underlined and indicated below. (b) is growth of strains 1AD1628 [pRS316/SUP45] and 1AD1628 [pRS316/sup45105(Tyr)], 1AD1628 [pRS316/sup45 105(Gln)], 1AD1628 [pRS316/sup45105] or 1AD1628 [pRS316/sup45102(Gln)], 1AD1628 [pRS316/sup45 102] on SCUra, SCAde, and SCTrp. Typical data for 1 of 24 transformants are presented for each strain.
It is known that termination codon UAG can be readthrough as a sense codon by S. cerevisiae tRNATyr (GUA). This fact was discovered in studies of transla tion of the STE6 mRNA, which had a UAG codon in its open reading frame [33]. The suppression of UAA by tRNATyr (GUA) demands a hydrogen bond between G and A at position 3 of the codon. The G::A pair is not a canonical Watson–Crick one, but this interaction can be stabilized by the presence of pseudouridine at position 2 of the anticodon, which is typical of most tRNATyr (GUA) molecules [31]. Experiments in vitro demonstrate that the termination codon UAA of tobacco mosaic virus can be read through by tRNATyr (GUA) carrying modification mentioned above [34]. Overexpression of S. cerevisiae genes coding for tRNAGln (UUG) results in suppres sion of termination codon UAA in vivo by virtue of the formation of a hydrogen bond between G and U at position 1 of the codon [35]. Most tRNAGln (UUG) variants have an unmodified adenosine5'phosphate at the 3'end of the anticodon (position 37), which favors noncanonical G::U interaction [31].
Strains possessing the mutant sup45105 allele have elevated amounts of not only tRNATyr and tRNAGln but also some other tRNAs, whose ability to read through UAA has not been demonstrated [16]. The results can be explained by the coordinated regulation of all tRNA genes, resulting in higher levels of poten tial nonsensesuppressing and nonsuppressing tRNAs. Theoretically, UAA can be suppressed by various tRNAs [31]. We found genes encoding only tRNATyr (GUA) and tRNAGln (UUG). It is likely that these tRNAs are preferable nonsense suppressors owing to specific features of modification of their anticodon sequences and anticodon environment. Cells with nonsense mutations in SUP45 are tem peraturesensitive [29]. Study of 1BD1606 derivatives harboring the pRS425tRNATyr and pRS425tRNAGln plasmids showed that transformants of strains 1041B D1606 and 1051BD1606 grew at nonpermissive temperatures. Transformants of 1071BD1606 showed no growth. These differences are likely to be related to the fact that the mutant sup45n alleles pos sess UAA PTSs in the SUP45 ORFs of strains 1041B D1606 and 1051BD1606 and a UGA codon in 107 1BD1606 [13]. As mutations in the S. cerevisiae SUP45 gene not only cause omnipotent nonsense sup pression but also produce some pleiotropic effects, it is assumed that eRF1 plays some roles in addition to translation termination [30]. Possible explanation of the lethality of sup45105 nonsense mutants at ele vated temperatures is the fact that eRF1 enters protein aggregates essential for the adaptive response of the cell to elevated temperature; thus, the decrease in the amount of fullsize eRF1 causes cell death under the nonpermissive conditions. It is known that tRNATyr and tRNAGln are endogenous suppressor tRNAs able to readthrough only UAG and UAA but not UGA [31]. Assay of eRF1 in 1051BD1606, bearing a UAA PTC in the sup45105 allele, showed that this strain, when grown at 37°C, has a high level of fullsize eRF1 against the background of tY(GUA)J1 or tQ(UUG)L overexpression. It should be mentioned that the eRF1 protein produced with the presence of sup45 105(Tyr), sup45105(Gln), or sup45102(Gln) is prac tically as functional as the wildtype one, in spite of the fact that sup45105(Tyr) and sup45105(Gln encode eRF1 variants less stable than the wildtype protein [19]. Thus, overexpression of genes for tRNATyr and tRNAGln can compensate temperature sensitivity in nonsense mutants with the UAA PTS in the SUP45 mRNA and increase the survival rate of these strains owing to the larger amount of fullsize eRF1. ACKNOWLEDGMENTS We are grateful to O. Pashina for kindly provided plasmids. MOLECULAR BIOLOGY
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OVEREXPRESSION OF GENES ENCODING tRNAtyr AND tRNAGln
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