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The yeast Saccharomyces cerevisiae system: A powerful tool to study the mechanism of protein synthesis initiation in eukaryotes
Biochimie (1994) 76, 853-861
© Soci6t6 franqaise de biochimieet biologic mol~culaire/ Elsevier, Paris
853
The yeast Saccharomyces cerevisiae system: A powerful tool to study the mechanism of protein synthesis initiation in eukaryotes M Altmann, H Trachsel lnstitut fiir Biochemie und Molekularbiologie, UniversitiitBern, Biihlstrasse 28, CH-3012 Bern, Switzerland
Summary - - This review summarizesrecent progress in the study of initiationof protein synthesis in the yeast Saccharomyces cerevisiae. Biochemicaland genetic approaches provide new insight into the recognitionof the 5'-end of mRNA by initiation factors and 40S ribosomes, unwindingof mRNA secondary structures in the untranslatedregion and proper recognitionof the AUG start codon. Experiments with initiation factor-dependentcell-free systems have facilitated studies of factor functionsand factor requirements for translation of different mRNAs. The analysis of mutations which suppress the inhibitoryeffect on translation of RNA secondary structure in the 5'-untranslatedregion of yeast mRNAs has led to the identificationof gene products which may be involvedin both transcription and translation. initiation of protein synthesis / eukaryotic initiation factors / factor-dependent extracts / yeast
Introduction In recent years studies on the mechanism of initiation of protein synthesis have received a strong impetus from experiments with the unicellular eukaryotic organism Saccharomyces cerevisiae (for recent reviews, see [1, 2]). Most of the initiation factors already known from mammalian and plant cells, and a few additional factors, have also been isolated from yeast, At least 19 genes encoding polypeptides involved in initiation of translation have been cloned (see table i). Gene replacement experiments with truncated versions of cloned genes (in yeast such mutants are easy to obtain due to the high efficiency of homologous recombination) have shown that most of these factors are essential for the survival of the cell (see table I). This is in contrast to the finding that less than 10% of all yeast genes cloned so far have been shown to be essential and demonstrates the importance of the process of initiation of protein synthesis for cellular metabolism and proliferation. Perhaps the most important conclusion from the work with Saccharomyces cerevisiae is that despite the evolutionary distance between this lower eukaryote and vertebrate organisms, the mechanism of protein synthesis is remarkably conserved. This is shown by the fact that mammalian initiation factors like elF4E (also called elF-4Fot) [3], elF5A [4] and elF-2a kinases [5] can replace their yeast homologues in vivo.
This allows us to draw conclusions from experiments with yeast that are of general validity for the process of eukaryotic protein synthesis. In the following sections we will summarize data describing the following processes: 1) recognition of the cap-structure of mRNA by the elF-4F complex; 2) unwinding of the 5'-untranslated region of mRNA by factors elF-4A (also called elF-4FI3), Tif3 (the yeast homologue of mammalian elF-4B) and the gene products of SSLI and SSL2; and 3) recognition of the AUG start codon of protein synthesis. The examples chosen should illustrate the potential of combining classical genetic, molecular biological and biochemical approaches when working with Saccharomyces cerevisiae. In vitro studies using extracts from mutant strains
The ultimate proof that a polypeptide is involved in initiation of protein synthesis can be obtained with the help of yeast mutants in which the function of the factor in question is repressed under certain conditions. Extracts from mutant strains should prove inactive in protein synthesis unless the wild-type protein (obtained, for example, by over-expressing the cloned gene in E coli or from yeast wild-type extracts) is added to restore its translational capacity. This approach seems easier if the gene is non-essential for
854 Table i. Translation initiation factors and their genes from Saccharomyces cerevisiae. Factor
Gene
Estimated molecular mass (kDa)
Involved in
Function
elF-4E (elF-4Fot)
TIF45 (CDC33)
24
Cap recognition
Essential
elF-4A
TIFi/TIF2
46
RNA-unwinding
Essential (double disruption)
elF-4Fy
TIF4631/ TIF4632
1071104
Cap recognition
Essential (double disruption)
elF-4B
TIF3
48.5
RNA-unwinding
Dispensable
elF-2o~
SUI2
35
Met-tRNAf binding and AUG start condon recognition
Essential
!F-213
sui3
32
Met-tRNAf binding and AUG start condon recognition
Essential
elF-2y
GCDII
58
GTP and met-tRNAf binding
Essential
SUIi
12
AUG start codon recognition
Essential
elF-2B subunit
GCD6
8I
elF-2.GTP-regeneration
Essential
elF-2B subunit
GCD2
7I
el F-2.GTP-regeneration
Essential
elF-2B subunit
GCD!
68
elF-2.GTP-regeneration
Essential
elF-2B subunit
GCD7
43
el F-2.GTP-regeneration
Essential
elF-2B subunit
GCN3
34
eIF-2.GTP-regeneration
Dispensable
elF-3 subunit
PRTI
88
?
Essential
elF-5
TIF5
45
Subunit joining
Essential
elF-SA (former elF-4D)
TIFSIA/ TIFSI8
17
First peptide bond formation ?
Essential (double disruption)
(elF-4F~)
the survival of the cell, but if the gene is essential - as is true for most genes encoding initiation factors conditionally lethal mutants have to be obtained that allow the shut-off of gene function when shifting cells to non-permissive conditions (eg raising the temperature fi'om 25°C to 37°C or changing the growth medium from galactose to glucose). Experiments with translational extracts deprived of a particular initiation factor activity also allow the determination of factor requirement for efficient translation of particular mRNAs, the isolation of intermediates in the initiation pathway, analysis of partial reactions and factor structure-function studies. Four examples of yeast strains from which factor-dependent extracts can be obtained are presented below.
Extracts lacking initiation factor Tif3
Surprisingly, deletion of the gene encoding Tif3, the homologue of mammalian elF-4B, is non-lethal for the yeast. However, TIF3-deleted strains show a coldsensitive slow growth phenotype. Even though no homologous genes have been detected by DNA-hybridization techniques [6, 7], the possibility of other factors supplementing the function of Tif3 in the genedisrupted strains cannot be excluded. Extracts from a TIF3-deleted strain show similar translational activity to wild-type extracts when incubated at temperatures above 27°C. At lower temperatures - and nicely corroborating the in vivo phenotype translation in the mutant extract is 2-3-fold reduced in
855 comparison to the wild-type extract. The translational defect can be 'cured' by adding wild-type yeast extract (but not mutant extract), purified Tif3 from wild-type yeast (M Altmann, B Wittmer, H Trachsel, unpublished results) or even mammalian elF-4B expressed in E coli, showing that the factors are interchangeable despite the relatively limited homology [6]. No difference in the pattern of newly synthesized polypeptides can be observed in the Tif3-dependent extract programmed with total yeast m R N A when compared to wild type extract. This indicates that the majority of yeast mRNAs require this protein for efficient translation. It remains to be investigated if mRNAs devoid of secondary structure in their 5'-UTR (untranslated region) are translated with higher efficiency in the absence of Tif3 as indicated by in vivo experiments with reporter mRNAs of increasing secondary structure [6].
E.rtracts dejicient in initiation factor Prt l activity One of the 'oldest' temperature-sensitive mutants of yeast exhibiting rapid inhibition of protein synthesis at 37°C is strain ts-187 [8]. It was shown to have a defect in the formation of the elF2.Met-tRNAf.GTP. 40S preinitiation complex [91 due to a mutationin the PRTI gene. The gene has been cloned and sequenced [ 10, 11]. It encodes a protein product of Mr 88 000. Most of PRT! protein has been found associated with a complex of Mr 500000 [12]. This complex is assumed to be the yeast initiation factor elF-3. In vitro extracts derived from the prtl-I mutant strain are rapidly inhibited in their translational capacity when preincubated at 37°C. Addition of purified yeast elF-3 restores the translational activity of the heat-inactivated extract (T Naranda, H 'rrachsel, JWB Hershey, unpublished results).
Extracts dej~cient in initiation factor elF.4E activity If not available, a temperature-sensitive mutation in an essential gene can be constructed. The strategy adc,pted to obtain a collection of yeast mutants expressing temperature-sensitive elF-4E is shown in figure 1 [13]. The yeast TIF45 gene was originally cloned from a k-gti I yeast DNA library and shown to be essential for the survival of the cell [14]. Copies of the cloned gene on a plasmid were chemically mutagenized in vitro and transformed into a yeast strain that could only grow on galactose as elF-4E was only expressed from the TIF45 ORF (open reading frame) fused to the galactose-regulated GALl promoter (located on a second plasmid; see fig 1). Transformed yeast clones unable to grow at 37°C on glucose but growing on galactose containing media (wild type elF-4E being expressed from GAL1-TIF45) were further analysed and confirmed as carders of temperature sensitive elF-4E mutations [ 13].
Extracts from one of the elF-4E mutants obtained were already quite inactive in protein synthesis when cells were grown at 250C on glucose-containing media. Preincubation of the extract at 37°C for 10 min rendered it even less active in translation. The extracts could be stimulated 5-7-fold by addition of purified yeast elF-4E expressed in E coli. Translation of total yeast mRNA in the inactivated extract was dependent on added factor. In contrast, mRNAs devoid of secondary structure in their 5'-UTR (untranslated region) like alfalfa mosaic virus AMV-4 RNA [13] or in vitro transcribed CAT-mRNA with the D-sequence of tobacco mosaic virus (TMV) [15] were translated equally well in the absence and presence of exogenous elF-4E. These observations support a model in which the first step of protein synthesis is the interaction of elF4E with the mRNA cap structure followed by the binding of further factors like the 'g-subunit of elF-4F, elF-4A and elF-4B, mRNAs with unstructured leader sequences may not need elF-4E (or at least much lower levels of the factor) to bind these initiation factors for initiation of translation.
Translational extracts dejicient in initiation factor elF-4A The genes TIFI and TIF2 encode elF-4A [16]. The deletion of one gene renders viable yeast cells while the double-deletion is lethal [17]. A conditionally lethal strain was obtained that grows on galactose, but not on glucose containing media, by placing the only elF-4A gene in the cell on a plasmid under the control of the GALl promoter. When growing cells were shifted from galactose- to glucose-containing media, growth was greatly retarded after four to six generations. Extracts prepared from these cells were inactive in translation of either total yeast mRNA or in vitro transcribed and capped CAT-mRNA. Upon addition of purified yeast elF-4A, translation of these mRNAs could be stimulated 15-20-fold. At that time, this was the ultimate proof that TIFI and TIF2 products encode the homologue of mammalian elF-4A [181. Interestingly, synthetic CAT-mRNA with the TMV ~)-sequence (unstructured 5'-leader) also showed a strong dependence on exogenous factor in the elF-4Adependent system, though not as strong as control CAT-mRNA [15]. The elF-4A requirement is not dependent on the length of the leader sequence, as a synthetic mRNA with a very short 5'-leader of only eight nucleotides also required the factor for its translation [ 19]. So far, and contrary to the case with elF-4E, no mRNAs have been identified whose translation is independent of elF-4A.
856
Unwinding of RNA secondary structure in the 5'-UTR of yeast mRNAs About 70% of yeast mRNAs have a 5'-UTR of 20 to 80 nucleotides with an average length of 52 nucleotides and with adenosine residues clearly predominating (for position -25 to position -1 relative to the AUG initiation codon the average nueleotide composition is 46% A, 21% C, 21~ U, 12% G; [20]). That translation in yeast is very sensitive to RNA secondary structure is evident from several independent analyses fn vitro mutagenesis of TIF45 g~ne .encoding elF-4E
Transformation into a GAL1/TIF45 mutant strain
) 3,
Replicaplate and select those that do not grow at 37'C on glucose-, but do grow on gslactose.containing media
Fig 1. Strategy employed to obtain temperature sensitive yeast mutants modified in the TIF45 gene encoding elF-4E. 1) A plasmid preparation containing the TIF45 gene was randomly mutagenized in vitro by treatment with hydroxylamine and (2) transfolmed into a yeast strain in which the chromosomal copy of the TIF45 gene has been disrupted by the LEU2 gene. This strain carries a wild-type TIF45 gene under the control of the GALl promoter allowing on galactose containing media the expression of the essential eiF4E-activity. 3) Transformed cells were plated on a glucosecontaining medium to stop the expression of the wild-type gene and to reveal the activity of the mutated TIF45 gene. Temperature-sensitive mutants among the transformants were identified by comparison of their growth at low and high temperature (adapted from [13]). TIF45::LEU2 denotes the disruption of the elF-4E gene locus by the LEU2-gene. The asterisk indicates random mutations introduced into TIF45.
showing that the insertion of palindromic structures into the 5'-UTR with a free energy of more than - 2 0 kcal/mol inhibits mRNA translation by more than 98% [6, 21-23]. A comparable strong inhibition in mammalian cells requires much stronger secondary structures o f - 5 0 to --60 kcal/mol [24], suggesting that factors involved in the unwinding of the 5'-UTR of mRNA are more limited in activity and/or amount in yeast than in mammalian cells. A remarkable exception is the 5'-UTR of the PMA1mRNA (H+-ATPase) whose 233 nucleotide leader sequence forms a very strong secondary structure with a free energy o f - 5 0 kcai/mol [251. While PMAImRNA is very efficiently translated in yeast [23] its translation in a reticulocyte lysate is very poor. Interestingly, the sequence from position -14 t o - 2 1 relative to the start AUG codon (UAAUUAUC) is complementary to the nucleotide sequence at the 3'-end of yeast 18S rRNA providing the possibility that a ShineDalgarno-like mechanism facilitates translation of this mRNA.
Suppressors of stem loop structures Since RNA secondary structure in the 5'-UTR strongly inhibits translation of most mRNAs, Donahue and coworkers designed all elegant approach to clone factors that might be involved in RNA unwinding. So far, the suppressor genes SSL! and SSL2 have been characterized [26, 27]. The strategy employed to obtain these mutants and the phenotypes of these mutants is presented in figure 2. They introduced a 36 nucleotide sequence with perfect dyad symmetry 10 nt downstream of the 5'-end of the ttlS4-mRNA and 50 nt upstream of the first AUG codon. 'This construct confers a His--phenotype on cells expressing it because the strong secondary structure inhibits the translation of the yeast HIS4-mRNA by more than 99% [22]. Spontaneous revertants of this mutant strain were selected according to their capacity to grow on a synthetic minimal medium lacking histidine. Genetical analysis of the His+ revertants identified four distinct suppressor genes whose products were capable (in its mutated form) of melting or otherwise bypassing the stem-loop structure and to translate the HIS4-mRNA. They therefore named the suppressor genes SSLI-SSL4 (SSL = suppressor of stem-loop) [26]. The haploid suppressor strains carrying ssll-I and ssl2-1 are phenotypically different in that ssil-I has an additional ts (temperature sensitive)-phenotype (fig 2). Interestingly, when crossing ssll-I and ssl2-1 haploids of the opposite mating type, the haploid spores with both ssll-I ssl2-1 (obtained by dissecting tetrade spores from diploids that had undergone meiosis) were not only ts but also His- (fig 2), indicating a physical interaction of both gene products.
857 The phenotype of this haploid double mutant allowed the simple cloning of both wild type SSL1 and SSL2 genes: when transforming double mutants with a wild type yeast DNA library and selecting for cells with a His+-phenotype, two types of transformants were obtained: His+/tr (temperature re~,,istant) due to the SSLl-gene (which is dominant over ssll-1) and His+/ts due to transformation with the SSL2-gene. Both genes are essential for the survival of the cell and surprisingly, both gene products are involved in DNA-repair: both ssll- and ssl2-mutant strains show an UV irradiation hyper-sensitive phenotype. When incubated at the non-permissive temperature, ssll tsmutant extracts rapidly show inactivation of translation, indicating the involvement of the protein in initiation of protein synthesis [27]. However, to our knowledge, recovery of translation in a heat-inactivated ssil-mutant extract by addition of the wild type SSLl-protein has not been reported, and therefore final proof for the physiological involvement of SSLI (or other SSL-factors) in initiation of protein synthesis is still lacking. While the SSLl-gene product is a protein with multiple zinc fingers and does not show any significant homology to any other known protein sequence, SSL2 is the homologue of the human gene ERCC3 (now called XPB). This protein was proposed to be a DNAhelicase whose defective forms are unable to repair UV-induced DNA damage and cause diseases like xeroderma pigmentosum and Cockayne's syndrome. The SSLI protein has recently been found to be part of the yeast RNA polymerase II initiation factor b (homologue of human TFIIH) while SSL2 protein interacts with the complex but is not part of it [28]. The intriguing question of whether SSL gene-products are proteins with more than one function or whether, when mutated or overexpressed, they tbrtuitously
Fig 2. Strategy employed to obtain suppressors of the inhibitory effect on translation of stem-loop (SSL) structures, their phenotypes and the cloning of the SSLI and SSL2 genes. 1) The expression of the HIS4 gene was inhibited at the translational level by introducing a strong RNA secondary structure into the 5' untranslated region of the mRNA. This results in a His--phenotype. 2) Spontaneous revertants (His+-phenotype) were selected on medium lacking histidine. Two revertants, ssll-I and ssl2-1, were further characterized. The mutant ssll.! showed an additional temperature-sensitive phenotype. 3) Mating of the two mutants, sporulation of diploids and dissection of haploid tetrades allowed the obtention of ssll-I ssl2-1 double mutants. 4) Both phenotypic properties of the double mutant (His- and temperature-sensitivity) allowed for the cloning of the SSLI and SSL2 genes by transformation with cloned wild-type DNA and selection for His+ and either temperature-resistance or temperature-sensitivity (adapted from [26, 27]).
Introduction of secondary structure at the 5'-UTR of HIS4-mRNA
His', tr
2.
Selection of spontaneous His÷-revertants
/
\
His+, ts
His+, tr
\/ 3.
Mating of His+-revertants, sporulation and selection of haploid cells carrying both the ssl1-f and ssl2-/ mutation
.-)
His', ts Transformation with yeast DNA library and selection of His+-transformants
His+, tr
His+, ts
858 1.
Creation of a his4"-mutant due to a modified AUG start codon
His', Z.
white
Selection of spontaneous His+-revertant;
/ I-
\ ,
/ His+ , white
His+ , blue
HIs+, t$
His+, tr
Fig 3, Strategy employed to obtain suppressors of initiator (SUI) codon mutations and their phenotypes. I) The AUG translation initiation codons of the HIS4 gene and the ~-galactosidase reporter gene were mutated to UUG. This results in a His and white pilenotype (no production of [3-galactosidase). 2) Three spontaneous revertants for both phenotypes (His+ and blue colour) were selected. They can initiate translation 'at the UUG codon due to mutations in SUll, SUI2 or SUI3 gene. The suii and sui2 mutants have an additional temperature-sensitive-phenotype (adapted from [32-34]). Asterisks indicate mutated versions of the SUl-proteins. potentiate the unwinding of mRNA remains to be answered.
Recognition of the AUG start codon for protein synthesis In yeast the consensus sequence fbr the start codon with its flanking sequences is 5'-A/Y AA/UAAUGUC U-3' [201 as opposscd to the sequence 5'-CCACC .AU GG-3' in mammals [2911. The sequences flanking the
start AUG codon in yeast HIS4-mRNA do not seem to play a very important role. Only an exchange at position - 3 (relative to the start AUG codon) from A to U reduced the translational efficiency of the HIS4-1eader by 50% (mutations A ~ C or A ~ G didn't have any effect) [22]. Mutational analysis of the leader sequence of HIS4-mRNA has not revealed any particular features capable of significantly enhancing or inhibiting the translational efficiency of the mRNA besides the inhibitory effect of RNA secondary structures. The only mutations identified that affected recognition of the AUG start codon are alterations of the start AUG codon itself: any single or double base-exchange in the HIS4-mRNA start codon inhibited translation by 98% or more [30].
The importance of codon-anticodon base-pairing When the AUG start codon of HIS4-mRNA is mutated to AGG translation drops to 0.4% of the control level, rendering cells His4- [30]. However, when the anticodon sequence of initiator-tRNA was mutated (5'CAU-3' ---> 5'-CCU-3') to allow base-pairing with AGG and the mutated tRNA was overexpressed (expression from a wild-type copy of the initiatortRNA gene was also maintained) a His+-phenotype was obtained. Under conditions of low expression the mutated initiator-tRNA was also able to complement the His--phenotype in a yeast strain with an additional mutation in the gene MESI encoding the methionyltRNA synthetase. The mutant mesl gene product has the capacity of enhanced charging of the mutated tRNA while maintaining its ability to charge the wildtype initiator and elongator Met-tRNA species [31]. These findings corroborate the importance of proper codon-anticodon interaction at the start AUG codon tbr translation.
Suppressors of initiator codon mutations Met-tRNAf is bound to elF-2.GTP on the 40S ribosome. Therefore elF-2 may participate in AUG codon recognition. Indeed, the genes encoding the o~- (SUl2) and the [3-subunit tSUi3) of elF-2 have been cloned as suppressors of initiator codon mutations in the HIS4mRNA which (when mutated) confer the ability to start translation efficiently at an UUG-codon. The selection of SUI (suppressor of initiator codon)-mutants and their phenotypes is presented in figure 3. Spontaneous suppressors of a his4--yeast strain that were able to use UUG as start codon were selected. In order to discriminate against revertants that recreated the AUG codon, the mutated 5'-UTR of HIS4-mRNA was also fused in-frame to the ,8-galactosidase open reading frame. Recreation of the AUG codon resulted in a white colony phenotype when
859 cells were incubated in the presence of the chromogenie substrate X-Gal (5-bromo-4-chloro-3-indolyl-13D-galactoside). In contrast, mutations in a SUI gene should not only render a His+-phenotype, but mutant strains should also form blue colonies on X-Gal plates. This easy selectable phenotype permitted the isolation of SUIs in three different complementation groups, suil- and sui2-mutants were both recessive and conferred a ts-phenotype to the cell, a dominant SUl3-mutant was temperature-resistant (fig 3). In order to clone the SUI3 gene, cloned genomic DNA fragments from the SUl3-mutant were transformed into the original SUl31his4- strain and His+-transformants selected. The cloning of the SUII and SUI2 genes was even easier: transformation of the temperature-sensitive suppressor strains with a wildtype yeast DNA library and selection of temperatureresistant transformants was used to clone the genes. All three SUI genes are essential for the survival of the cell. SUI3 is 42% identical with the [3-subunit of mammalian elF-2 and like its homologue contains two potential nucleic acid-binding domains, three polylysine domains at the amino-terminal end and a Zn(lI) 'finger' motif, Cys-X2-Cys-Xzg-Cys-X2-Cys at the carboxyi end [32]. SUI2 is 58% identical with the asubunit of human elF-2. Both proteins are conserved in the amino-terminal region and contain a serine residue which is the target of a kinase that down-regulates translation [33]. SUII has been shown biochemically aot to be a subunit of elF-2. Its ability to use a non-AUG codon (UUG) for translation initiation suggests that it encodes an additional factor that cooperates with elF-2 and Met-tRNAf in AUG start codon recognition [34].
Exceptions to the rule In more than 95% of the mRNAs of Saccharomyces cerevisiae the first AUG from the 5'-end is used as start codon for translation. How does the recognition of an AUG start codon by scanning 40S ribosomes carrying elF-2.GTP.Met-tRNAf function in those mRNAs where it is not the first AUG which serves as the start codon for translation? Reinitiation
Ribosomes can resume translation after passing an upstream open reading frame by scanning the mRNA until they find a downstream ORE This translational mechanism, called reinitiation, has been described for mRNAs with small upstream ORFs in their 5'-UTR which regulate the expression of the downstream open reading frame, eg C P A I - m R N A encoding the gluta-
minase subunit of carbamoyl-phosphate synthetase and GCN4-mRNA encoding a transcription factor that activates the expression nf many genes involved in amino acid and nucleotide biosynthesis. The translation of both mRNAs is stimulated under conditions of nutritional deprivation (in the case of CPAI by low arginine concentration) because ribosomes can efficiently resume the translation at a downstream start codon. In the case of C P A I - m R N A an upstream ORF encodes a 25 amino acid peptide that is assumed to repress translation of the downstream ORF in the presence of arginine [35]. In the case of GCN4-mRNA amino acid deprivation leads to phosphorylation and inactivation of elF-2, allowing ribosomal subunits to bypass the upstream ORFs and resume translation at the fifth open reading frame which encodes the GCN4 protein. Under conditions where overall mRNA translation is downregulated, translation of GCN4-mRNA is not only maintained but enhanced to provide the expression of vital enzymatic activities. This exceptional regulatory mechanism has facilitated the cloning of several genes involved in recycling of elF-2.GTP and the regulation of elF-2 activity (for recent reviews see [36, 41, 42]). Internal initiation
On certain RNAs with unusual leader structures (several upstream AUGs and strong secondary, structures) like those of picornaviruses or some mammalian mRNAs, ribosomes can bypass the ~'-end of the mRNA and bind in a cap-independent way directly to internal ribosome entry sites (IRES). Cellular factors required for this process have been isolated from mammalian cells and characterized (for recent reviews see [37, 38]). So far, evidence for internal initiation of translation in yeast is scarce. In an elF-4E-dependent lysate (see above) translation of the second ORF of a dicistronic mRNA was observed when the poliovirus 5'-UTR was used as internal ribosome binding site. Translation of the second ORF was more efficient when the bicistronic mRNA was uncapped and was inhibited by the addition of elF-4E. This suggests that under conditions where ribosomal subunits are not directed to the cap structure of the mRNA, cap-independent internal initiation may occur [39]. Surprisingly, a small yeast RNA has been isolated that inhibits in trans the translation of mRNAs carrying poliovirus sequences in yeast or mammalian extracts. The mode of action and the biological significance of this inhibitory RNA is not known [40]. Recent data suggest that the translation of certain viral as well as certain yeast RNAs may be initiated by a cap-independent internal ribosome binding mechanism (N Iizuka, L Najita, A Franzusoff, P Sarnow, personal communication).
860 Conclusion The possibility of combining biochemical with powerful genetical experiments in the yeast Saccharomyces cerevisiae has made this organism a model system to study initiation of protein synthesis in eukaryotes. In addition to the translation factors already known from the mammalian system, several new factors like SUII and like SSLI and SSL2 were found. Their function in initiation of protein synthesis has still to be established. Mutants that suppress the inhibitory effect of non-AUG codons on translation have demonstrated the central role of elF-2 and Met-tRNAr in the recognition of the AUG start codon.
Acknowledgments We thank Patrick Linder for helpful comments on this manuscript and Peter Sarnow for communication of unpublished results. References I Linder P. Prat A (1990) Baker's yeast, the new work horse in protein synthesis studies: analyzing eukaryotic translation initiation. BioEssays 12.519526 2 Linder P (1992) Molecular biology of translation in yeast. Anemic van Leeuwenhoek 62.47,-62 3 Altmann M. Muller PP, Pelletier J. Sonenber8 N. Trachsel tl (1989) A mammalian translation initiation factor can substitute for its yeast homologue in riro. J Biol Chem 264. 12145-12147 4 Schwelhcrger HG. Kant; iIA. Hershey JWB (1993)Translation initiation Ihctor elF-SA expressed from either of two yeast genes or from human eDNA-functional identity under aerobic and anaerobic conditions. J Biol Chem 268. 14018-14025 5 Dever TE. Chen JJ. Barber GN. Cigan AM. Feng L. Donuhue TF. London IM, Kalze MG. Hinnebusch AG (1993) Mammalian eukaryotic initiation factor 2¢g kinases functionally substitute for GCN2 protein kinase in the GCN4 translational control mechanism of yeast. Pmc Natl Aead Sci USA 90, 4616-.4620 6 Altmann M. Muller PP, Wittmer B, Ruchti F, Lanker S, Trachsel H (1993) A Sacchawmyce,v cerevisiae homologue of mammalian translation initiation factor 4B contributes to RNA helicase activity. EMBO J 12, 3997-4003 7 Coppolecchia R. Buscr R Stotz A, Linder P (1993) A new yeast translation initiation factor suppresses a mutation in the elF-4A RNA helicase. EMBO J 12, 4005-4OI I 8 Hartwell Ltl, McLaughlin CS (19fi8) Temperature,sensitive mutants of yeast exhibiting a rapid inhibition of protein synthesis. J 8acterio! 96, 16641671 9 Feinberg B. McLanghlin CS. Moldave K (1982) Analysis of temperature-sensitive mutant Is 187 of Saccharomyees cel~vi,~iae altered in a component required for the initiation of protein synthesis. J 8iol Chem 257, 10846-10851 I0 Keierleber C. Wittekind M. Qin S, McLaughlin CS (1986) Isolation and characterization of PRTI. a gent required for the initiation of protein biosynthesis in Sa( chmomyces ('ereri.~iae. Mol Cell Bird 6. 4419-4424 I I HanicJoyce PJ. Singer RA. Johnston GC (1987) Molecular characterization of the yeast PRTI gene in which mutations affect translation initiation and r~gulati~n of cell proliferation. J 8 M Chem 262. 2845-285 I 12 Cigan AM. Bushman JL. Boal TR. Hinnchusch AG (1993) A protein complex of translational regulators of GCN4 mRNA is the guanine nucleotideexchange factor for translation initiation factor 2 in yeast. Ptv~(.Na# Acad S¢i USA 90. 5350-5354
13 Aitmann M. Sonenberg N. Trachsel H (1989) Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system. Mol Cell Biol 9. 4467--4472 14 Altmann M. Handschin C. Trachsel H (1987) mRNA cap-binding protein: cloning of the gene encoding protein synthesis initiation factor eIF-4E from Saccharomyces cerevisiae. Mol Cell Biol 7. 998-1003 15 Altmann M. Blum S. Wilson TM. Trachsel H (1990) The 5'-leader sequence of tobacco mosaic virus RNA mediates initiation-factor-4E-independent, but still initiation-factor-4A-dependent translation in yeast extracts. Gene 91. 127-129 16 Linder P. SIonimski PP (1988) Sequence of the genes TIFi and TIF2 from Sacharomyces cerevisiae coding for a translation initiation factor. Nucleic Acids Res 16. 10359 17 Linder P. SIonimski PP (1989) An essential yeast protein, encoded by duplicated genes TIF! and TIF2 and homologous to the mammalian translation initiation factor eiF-4A, can suppress a mitochondrial missense mutation. Proc Natl Acad Sci USA 86. 2286-2290 18 Blum S. Mueller M. Schmid SR. Linder P. Trachsel H (1989) Translation in Saccharomyces cerevisiae: initiation factor 4A-dependent cell-free system. Proc Natl Acad Sci USA 86. 6043--6046 19 Blum S. Schmid SR. Pause A. Buser P. Linder P. Sonenberg N. Trachsel H (1992) ATP hydrolysis by initiation factor 4A is required for translation initiation in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 89. 76647668 2D Cigan AM. Donahue TF (1987) Sequence and structural features associated with translational initiator regions in yeast - a review. Gene 59. I-18 21 Bairn SB. Sherman F (1988) mRNA structures influencing translation in the ycasi Saccharomyces cerevisiae. Mol Cell Biol 8. 1591-1601 22 Cigan AM. Pabich EK. Donahue TF (1988) Mutational analysis of the HIS4 translational initiator region in Saceharomyces cerevisi,e. Mol Cell Biol 8. 2964--2975 23 Sagliocco FA. Vega Laso MR. Zhu D. Tulle MF. McCarthy JEG. Brown AlP (1993) The influence of 5'-secondary structures upon ribosome binding to mRNA during translation in yeast. J B M Chem 268, 26522-26530 24 Pelletier J. Sonenberg N (1985) Insertion mutagenesis to increase secondary structure within the 5' non-coding region of a eukaryotic mRNA reduces translational efficiency. Cell 40. 515-526 25 Capieaux E, Vignais ML, Sentenac A, GolTeau A (1989) The yeast H÷ATPase gene is controlled by the promoter binding Ihctor TUE ,I 8iol Cheat 264. 7437-744fi 26 Gulyas KD, Donahue TF (1992) SSL2. a suppressor of a stem-loop matation in the !11S4 leader encodes the yeast homolo8 of human ERCC.3. Cell fig, 1031-1042 27 Yoon H, Miller SR Pabich EK, Donahue TF (1992) SSLI, a suppressor of a HIS4 5'-UTR stem-loop mutation, is essential t'or translation initiation and affects UV resistance in yeast. Genes Dev 6. 2463-2477 28 Fearer WJ, Svejstrup JQ, BardweU L, Bardwell AJ, Buratowski, Gulyas KD, Donahue TF, Friedberg EC, Komberg RD (1993) Dual roles of a multiprotein complex from S cewvisiae in transcription and DNA repair, Cell 75, 13791387 29 Kozak M (1986) Point mutations define a sequence flanking the AUG initi-ator codon that modulates translation by eukaryotic ribosomes, Cell 44, 283-292 30 Donahue TF, Cigan AM (1988) Genetic selection for mutations that reduce or abolish ribosomal recognition of the HIS4 translational initiator region, Mol Cell Biol 8, 2955-2963 31 Cigan AM, Feng L, Donahue TF (1988) tRNAi/met functions in directing the scanning rit)osome to the start site of translation. Science 242, 9396 32 Donahue TF, Cigan AM, Pabich EK, Castilbo Valavicius B (1988) Mutations at a Zn(il) finger motif in the yeast elF-2 gene alter ribosomal start-site selection during the scanning process. Cell 54, 621-4532 33 Cigan AM, Pabich EK, Feng L, Donahue TF (1989) Yeast translation initiation suppressor sni2 encodes the ~z subunit of eukaryotic initiation factor 2 and shares sequence identity with the human ct subunit. Proc Nail Acad Sci USA 86, 2784-2788 34 Yoon H, Donahue TF 0992) The sail suppressor locus in Saechm~myces eerevisiae encodes a translation factor thdt functions during tRNA,/mct recognition of the start codon. Mol Cell Biol 12, 248-260
861 35 Wemer M, Feller A, Messenguy F, P|6rard A (1987) The leader peptide of yeast gene CPA! is essential for the translational repression of its expression. Cell 49, 805-8 ! 3 36 Hinnebu~ch AG (1993) Gene-specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2. Mol Mic'robiol 10, 215-223 37 Altmann M, Trachsel H (1993) Regulation of translation initiation and modulation of cellular physiology. Trends Biochem Sci ! 8, 429-432 38 OH SK, Samow P (1993) Gene regulation:translational initiation by internal ribosome binding. Curr Opin Gen Devel 3,295-300
39 Altmann M, Blum S, Pelletier J, Sonenberg N, Wilson TMA, Trachsel H (1990) Translation initiation factor-dependent extracts from Saccharomyces cerevisiae. Biochim Biophys Acta 1050, 155-159 40 Coward P, Dasgupta A (1992) Yeast cells are incapable of translating RNAs containing the poliovirus 5' untranslated region: evidence for a translational inhibitor. J Viro166, 286-295 41 Pain VM (1994) Translational control during amino acid starvation. Biochimie 76, 7 i 8-728 42 Price N, Proud C (1994) The guanine nucleotide-exchange factor, eiF-2B. Biochimie 76, 748-760
© Soci6t6 franqaise de biochimieet biologic mol~culaire/ Elsevier, Paris
853
The yeast Saccharomyces cerevisiae system: A powerful tool to study the mechanism of protein synthesis initiation in eukaryotes M Altmann, H Trachsel lnstitut fiir Biochemie und Molekularbiologie, UniversitiitBern, Biihlstrasse 28, CH-3012 Bern, Switzerland
Summary - - This review summarizesrecent progress in the study of initiationof protein synthesis in the yeast Saccharomyces cerevisiae. Biochemicaland genetic approaches provide new insight into the recognitionof the 5'-end of mRNA by initiation factors and 40S ribosomes, unwindingof mRNA secondary structures in the untranslatedregion and proper recognitionof the AUG start codon. Experiments with initiation factor-dependentcell-free systems have facilitated studies of factor functionsand factor requirements for translation of different mRNAs. The analysis of mutations which suppress the inhibitoryeffect on translation of RNA secondary structure in the 5'-untranslatedregion of yeast mRNAs has led to the identificationof gene products which may be involvedin both transcription and translation. initiation of protein synthesis / eukaryotic initiation factors / factor-dependent extracts / yeast
Introduction In recent years studies on the mechanism of initiation of protein synthesis have received a strong impetus from experiments with the unicellular eukaryotic organism Saccharomyces cerevisiae (for recent reviews, see [1, 2]). Most of the initiation factors already known from mammalian and plant cells, and a few additional factors, have also been isolated from yeast, At least 19 genes encoding polypeptides involved in initiation of translation have been cloned (see table i). Gene replacement experiments with truncated versions of cloned genes (in yeast such mutants are easy to obtain due to the high efficiency of homologous recombination) have shown that most of these factors are essential for the survival of the cell (see table I). This is in contrast to the finding that less than 10% of all yeast genes cloned so far have been shown to be essential and demonstrates the importance of the process of initiation of protein synthesis for cellular metabolism and proliferation. Perhaps the most important conclusion from the work with Saccharomyces cerevisiae is that despite the evolutionary distance between this lower eukaryote and vertebrate organisms, the mechanism of protein synthesis is remarkably conserved. This is shown by the fact that mammalian initiation factors like elF4E (also called elF-4Fot) [3], elF5A [4] and elF-2a kinases [5] can replace their yeast homologues in vivo.
This allows us to draw conclusions from experiments with yeast that are of general validity for the process of eukaryotic protein synthesis. In the following sections we will summarize data describing the following processes: 1) recognition of the cap-structure of mRNA by the elF-4F complex; 2) unwinding of the 5'-untranslated region of mRNA by factors elF-4A (also called elF-4FI3), Tif3 (the yeast homologue of mammalian elF-4B) and the gene products of SSLI and SSL2; and 3) recognition of the AUG start codon of protein synthesis. The examples chosen should illustrate the potential of combining classical genetic, molecular biological and biochemical approaches when working with Saccharomyces cerevisiae. In vitro studies using extracts from mutant strains
The ultimate proof that a polypeptide is involved in initiation of protein synthesis can be obtained with the help of yeast mutants in which the function of the factor in question is repressed under certain conditions. Extracts from mutant strains should prove inactive in protein synthesis unless the wild-type protein (obtained, for example, by over-expressing the cloned gene in E coli or from yeast wild-type extracts) is added to restore its translational capacity. This approach seems easier if the gene is non-essential for
854 Table i. Translation initiation factors and their genes from Saccharomyces cerevisiae. Factor
Gene
Estimated molecular mass (kDa)
Involved in
Function
elF-4E (elF-4Fot)
TIF45 (CDC33)
24
Cap recognition
Essential
elF-4A
TIFi/TIF2
46
RNA-unwinding
Essential (double disruption)
elF-4Fy
TIF4631/ TIF4632
1071104
Cap recognition
Essential (double disruption)
elF-4B
TIF3
48.5
RNA-unwinding
Dispensable
elF-2o~
SUI2
35
Met-tRNAf binding and AUG start condon recognition
Essential
!F-213
sui3
32
Met-tRNAf binding and AUG start condon recognition
Essential
elF-2y
GCDII
58
GTP and met-tRNAf binding
Essential
SUIi
12
AUG start codon recognition
Essential
elF-2B subunit
GCD6
8I
elF-2.GTP-regeneration
Essential
elF-2B subunit
GCD2
7I
el F-2.GTP-regeneration
Essential
elF-2B subunit
GCD!
68
elF-2.GTP-regeneration
Essential
elF-2B subunit
GCD7
43
el F-2.GTP-regeneration
Essential
elF-2B subunit
GCN3
34
eIF-2.GTP-regeneration
Dispensable
elF-3 subunit
PRTI
88
?
Essential
elF-5
TIF5
45
Subunit joining
Essential
elF-SA (former elF-4D)
TIFSIA/ TIFSI8
17
First peptide bond formation ?
Essential (double disruption)
(elF-4F~)
the survival of the cell, but if the gene is essential - as is true for most genes encoding initiation factors conditionally lethal mutants have to be obtained that allow the shut-off of gene function when shifting cells to non-permissive conditions (eg raising the temperature fi'om 25°C to 37°C or changing the growth medium from galactose to glucose). Experiments with translational extracts deprived of a particular initiation factor activity also allow the determination of factor requirement for efficient translation of particular mRNAs, the isolation of intermediates in the initiation pathway, analysis of partial reactions and factor structure-function studies. Four examples of yeast strains from which factor-dependent extracts can be obtained are presented below.
Extracts lacking initiation factor Tif3
Surprisingly, deletion of the gene encoding Tif3, the homologue of mammalian elF-4B, is non-lethal for the yeast. However, TIF3-deleted strains show a coldsensitive slow growth phenotype. Even though no homologous genes have been detected by DNA-hybridization techniques [6, 7], the possibility of other factors supplementing the function of Tif3 in the genedisrupted strains cannot be excluded. Extracts from a TIF3-deleted strain show similar translational activity to wild-type extracts when incubated at temperatures above 27°C. At lower temperatures - and nicely corroborating the in vivo phenotype translation in the mutant extract is 2-3-fold reduced in
855 comparison to the wild-type extract. The translational defect can be 'cured' by adding wild-type yeast extract (but not mutant extract), purified Tif3 from wild-type yeast (M Altmann, B Wittmer, H Trachsel, unpublished results) or even mammalian elF-4B expressed in E coli, showing that the factors are interchangeable despite the relatively limited homology [6]. No difference in the pattern of newly synthesized polypeptides can be observed in the Tif3-dependent extract programmed with total yeast m R N A when compared to wild type extract. This indicates that the majority of yeast mRNAs require this protein for efficient translation. It remains to be investigated if mRNAs devoid of secondary structure in their 5'-UTR (untranslated region) are translated with higher efficiency in the absence of Tif3 as indicated by in vivo experiments with reporter mRNAs of increasing secondary structure [6].
E.rtracts dejicient in initiation factor Prt l activity One of the 'oldest' temperature-sensitive mutants of yeast exhibiting rapid inhibition of protein synthesis at 37°C is strain ts-187 [8]. It was shown to have a defect in the formation of the elF2.Met-tRNAf.GTP. 40S preinitiation complex [91 due to a mutationin the PRTI gene. The gene has been cloned and sequenced [ 10, 11]. It encodes a protein product of Mr 88 000. Most of PRT! protein has been found associated with a complex of Mr 500000 [12]. This complex is assumed to be the yeast initiation factor elF-3. In vitro extracts derived from the prtl-I mutant strain are rapidly inhibited in their translational capacity when preincubated at 37°C. Addition of purified yeast elF-3 restores the translational activity of the heat-inactivated extract (T Naranda, H 'rrachsel, JWB Hershey, unpublished results).
Extracts dej~cient in initiation factor elF.4E activity If not available, a temperature-sensitive mutation in an essential gene can be constructed. The strategy adc,pted to obtain a collection of yeast mutants expressing temperature-sensitive elF-4E is shown in figure 1 [13]. The yeast TIF45 gene was originally cloned from a k-gti I yeast DNA library and shown to be essential for the survival of the cell [14]. Copies of the cloned gene on a plasmid were chemically mutagenized in vitro and transformed into a yeast strain that could only grow on galactose as elF-4E was only expressed from the TIF45 ORF (open reading frame) fused to the galactose-regulated GALl promoter (located on a second plasmid; see fig 1). Transformed yeast clones unable to grow at 37°C on glucose but growing on galactose containing media (wild type elF-4E being expressed from GAL1-TIF45) were further analysed and confirmed as carders of temperature sensitive elF-4E mutations [ 13].
Extracts from one of the elF-4E mutants obtained were already quite inactive in protein synthesis when cells were grown at 250C on glucose-containing media. Preincubation of the extract at 37°C for 10 min rendered it even less active in translation. The extracts could be stimulated 5-7-fold by addition of purified yeast elF-4E expressed in E coli. Translation of total yeast mRNA in the inactivated extract was dependent on added factor. In contrast, mRNAs devoid of secondary structure in their 5'-UTR (untranslated region) like alfalfa mosaic virus AMV-4 RNA [13] or in vitro transcribed CAT-mRNA with the D-sequence of tobacco mosaic virus (TMV) [15] were translated equally well in the absence and presence of exogenous elF-4E. These observations support a model in which the first step of protein synthesis is the interaction of elF4E with the mRNA cap structure followed by the binding of further factors like the 'g-subunit of elF-4F, elF-4A and elF-4B, mRNAs with unstructured leader sequences may not need elF-4E (or at least much lower levels of the factor) to bind these initiation factors for initiation of translation.
Translational extracts dejicient in initiation factor elF-4A The genes TIFI and TIF2 encode elF-4A [16]. The deletion of one gene renders viable yeast cells while the double-deletion is lethal [17]. A conditionally lethal strain was obtained that grows on galactose, but not on glucose containing media, by placing the only elF-4A gene in the cell on a plasmid under the control of the GALl promoter. When growing cells were shifted from galactose- to glucose-containing media, growth was greatly retarded after four to six generations. Extracts prepared from these cells were inactive in translation of either total yeast mRNA or in vitro transcribed and capped CAT-mRNA. Upon addition of purified yeast elF-4A, translation of these mRNAs could be stimulated 15-20-fold. At that time, this was the ultimate proof that TIFI and TIF2 products encode the homologue of mammalian elF-4A [181. Interestingly, synthetic CAT-mRNA with the TMV ~)-sequence (unstructured 5'-leader) also showed a strong dependence on exogenous factor in the elF-4Adependent system, though not as strong as control CAT-mRNA [15]. The elF-4A requirement is not dependent on the length of the leader sequence, as a synthetic mRNA with a very short 5'-leader of only eight nucleotides also required the factor for its translation [ 19]. So far, and contrary to the case with elF-4E, no mRNAs have been identified whose translation is independent of elF-4A.
856
Unwinding of RNA secondary structure in the 5'-UTR of yeast mRNAs About 70% of yeast mRNAs have a 5'-UTR of 20 to 80 nucleotides with an average length of 52 nucleotides and with adenosine residues clearly predominating (for position -25 to position -1 relative to the AUG initiation codon the average nueleotide composition is 46% A, 21% C, 21~ U, 12% G; [20]). That translation in yeast is very sensitive to RNA secondary structure is evident from several independent analyses fn vitro mutagenesis of TIF45 g~ne .encoding elF-4E
Transformation into a GAL1/TIF45 mutant strain
) 3,
Replicaplate and select those that do not grow at 37'C on glucose-, but do grow on gslactose.containing media
Fig 1. Strategy employed to obtain temperature sensitive yeast mutants modified in the TIF45 gene encoding elF-4E. 1) A plasmid preparation containing the TIF45 gene was randomly mutagenized in vitro by treatment with hydroxylamine and (2) transfolmed into a yeast strain in which the chromosomal copy of the TIF45 gene has been disrupted by the LEU2 gene. This strain carries a wild-type TIF45 gene under the control of the GALl promoter allowing on galactose containing media the expression of the essential eiF4E-activity. 3) Transformed cells were plated on a glucosecontaining medium to stop the expression of the wild-type gene and to reveal the activity of the mutated TIF45 gene. Temperature-sensitive mutants among the transformants were identified by comparison of their growth at low and high temperature (adapted from [13]). TIF45::LEU2 denotes the disruption of the elF-4E gene locus by the LEU2-gene. The asterisk indicates random mutations introduced into TIF45.
showing that the insertion of palindromic structures into the 5'-UTR with a free energy of more than - 2 0 kcal/mol inhibits mRNA translation by more than 98% [6, 21-23]. A comparable strong inhibition in mammalian cells requires much stronger secondary structures o f - 5 0 to --60 kcal/mol [24], suggesting that factors involved in the unwinding of the 5'-UTR of mRNA are more limited in activity and/or amount in yeast than in mammalian cells. A remarkable exception is the 5'-UTR of the PMA1mRNA (H+-ATPase) whose 233 nucleotide leader sequence forms a very strong secondary structure with a free energy o f - 5 0 kcai/mol [251. While PMAImRNA is very efficiently translated in yeast [23] its translation in a reticulocyte lysate is very poor. Interestingly, the sequence from position -14 t o - 2 1 relative to the start AUG codon (UAAUUAUC) is complementary to the nucleotide sequence at the 3'-end of yeast 18S rRNA providing the possibility that a ShineDalgarno-like mechanism facilitates translation of this mRNA.
Suppressors of stem loop structures Since RNA secondary structure in the 5'-UTR strongly inhibits translation of most mRNAs, Donahue and coworkers designed all elegant approach to clone factors that might be involved in RNA unwinding. So far, the suppressor genes SSL! and SSL2 have been characterized [26, 27]. The strategy employed to obtain these mutants and the phenotypes of these mutants is presented in figure 2. They introduced a 36 nucleotide sequence with perfect dyad symmetry 10 nt downstream of the 5'-end of the ttlS4-mRNA and 50 nt upstream of the first AUG codon. 'This construct confers a His--phenotype on cells expressing it because the strong secondary structure inhibits the translation of the yeast HIS4-mRNA by more than 99% [22]. Spontaneous revertants of this mutant strain were selected according to their capacity to grow on a synthetic minimal medium lacking histidine. Genetical analysis of the His+ revertants identified four distinct suppressor genes whose products were capable (in its mutated form) of melting or otherwise bypassing the stem-loop structure and to translate the HIS4-mRNA. They therefore named the suppressor genes SSLI-SSL4 (SSL = suppressor of stem-loop) [26]. The haploid suppressor strains carrying ssll-I and ssl2-1 are phenotypically different in that ssil-I has an additional ts (temperature sensitive)-phenotype (fig 2). Interestingly, when crossing ssll-I and ssl2-1 haploids of the opposite mating type, the haploid spores with both ssll-I ssl2-1 (obtained by dissecting tetrade spores from diploids that had undergone meiosis) were not only ts but also His- (fig 2), indicating a physical interaction of both gene products.
857 The phenotype of this haploid double mutant allowed the simple cloning of both wild type SSL1 and SSL2 genes: when transforming double mutants with a wild type yeast DNA library and selecting for cells with a His+-phenotype, two types of transformants were obtained: His+/tr (temperature re~,,istant) due to the SSLl-gene (which is dominant over ssll-1) and His+/ts due to transformation with the SSL2-gene. Both genes are essential for the survival of the cell and surprisingly, both gene products are involved in DNA-repair: both ssll- and ssl2-mutant strains show an UV irradiation hyper-sensitive phenotype. When incubated at the non-permissive temperature, ssll tsmutant extracts rapidly show inactivation of translation, indicating the involvement of the protein in initiation of protein synthesis [27]. However, to our knowledge, recovery of translation in a heat-inactivated ssil-mutant extract by addition of the wild type SSLl-protein has not been reported, and therefore final proof for the physiological involvement of SSLI (or other SSL-factors) in initiation of protein synthesis is still lacking. While the SSLl-gene product is a protein with multiple zinc fingers and does not show any significant homology to any other known protein sequence, SSL2 is the homologue of the human gene ERCC3 (now called XPB). This protein was proposed to be a DNAhelicase whose defective forms are unable to repair UV-induced DNA damage and cause diseases like xeroderma pigmentosum and Cockayne's syndrome. The SSLI protein has recently been found to be part of the yeast RNA polymerase II initiation factor b (homologue of human TFIIH) while SSL2 protein interacts with the complex but is not part of it [28]. The intriguing question of whether SSL gene-products are proteins with more than one function or whether, when mutated or overexpressed, they tbrtuitously
Fig 2. Strategy employed to obtain suppressors of the inhibitory effect on translation of stem-loop (SSL) structures, their phenotypes and the cloning of the SSLI and SSL2 genes. 1) The expression of the HIS4 gene was inhibited at the translational level by introducing a strong RNA secondary structure into the 5' untranslated region of the mRNA. This results in a His--phenotype. 2) Spontaneous revertants (His+-phenotype) were selected on medium lacking histidine. Two revertants, ssll-I and ssl2-1, were further characterized. The mutant ssll.! showed an additional temperature-sensitive phenotype. 3) Mating of the two mutants, sporulation of diploids and dissection of haploid tetrades allowed the obtention of ssll-I ssl2-1 double mutants. 4) Both phenotypic properties of the double mutant (His- and temperature-sensitivity) allowed for the cloning of the SSLI and SSL2 genes by transformation with cloned wild-type DNA and selection for His+ and either temperature-resistance or temperature-sensitivity (adapted from [26, 27]).
Introduction of secondary structure at the 5'-UTR of HIS4-mRNA
His', tr
2.
Selection of spontaneous His÷-revertants
/
\
His+, ts
His+, tr
\/ 3.
Mating of His+-revertants, sporulation and selection of haploid cells carrying both the ssl1-f and ssl2-/ mutation
.-)
His', ts Transformation with yeast DNA library and selection of His+-transformants
His+, tr
His+, ts
858 1.
Creation of a his4"-mutant due to a modified AUG start codon
His', Z.
white
Selection of spontaneous His+-revertant;
/ I-
\ ,
/ His+ , white
His+ , blue
HIs+, t$
His+, tr
Fig 3, Strategy employed to obtain suppressors of initiator (SUI) codon mutations and their phenotypes. I) The AUG translation initiation codons of the HIS4 gene and the ~-galactosidase reporter gene were mutated to UUG. This results in a His and white pilenotype (no production of [3-galactosidase). 2) Three spontaneous revertants for both phenotypes (His+ and blue colour) were selected. They can initiate translation 'at the UUG codon due to mutations in SUll, SUI2 or SUI3 gene. The suii and sui2 mutants have an additional temperature-sensitive-phenotype (adapted from [32-34]). Asterisks indicate mutated versions of the SUl-proteins. potentiate the unwinding of mRNA remains to be answered.
Recognition of the AUG start codon for protein synthesis In yeast the consensus sequence fbr the start codon with its flanking sequences is 5'-A/Y AA/UAAUGUC U-3' [201 as opposscd to the sequence 5'-CCACC .AU GG-3' in mammals [2911. The sequences flanking the
start AUG codon in yeast HIS4-mRNA do not seem to play a very important role. Only an exchange at position - 3 (relative to the start AUG codon) from A to U reduced the translational efficiency of the HIS4-1eader by 50% (mutations A ~ C or A ~ G didn't have any effect) [22]. Mutational analysis of the leader sequence of HIS4-mRNA has not revealed any particular features capable of significantly enhancing or inhibiting the translational efficiency of the mRNA besides the inhibitory effect of RNA secondary structures. The only mutations identified that affected recognition of the AUG start codon are alterations of the start AUG codon itself: any single or double base-exchange in the HIS4-mRNA start codon inhibited translation by 98% or more [30].
The importance of codon-anticodon base-pairing When the AUG start codon of HIS4-mRNA is mutated to AGG translation drops to 0.4% of the control level, rendering cells His4- [30]. However, when the anticodon sequence of initiator-tRNA was mutated (5'CAU-3' ---> 5'-CCU-3') to allow base-pairing with AGG and the mutated tRNA was overexpressed (expression from a wild-type copy of the initiatortRNA gene was also maintained) a His+-phenotype was obtained. Under conditions of low expression the mutated initiator-tRNA was also able to complement the His--phenotype in a yeast strain with an additional mutation in the gene MESI encoding the methionyltRNA synthetase. The mutant mesl gene product has the capacity of enhanced charging of the mutated tRNA while maintaining its ability to charge the wildtype initiator and elongator Met-tRNA species [31]. These findings corroborate the importance of proper codon-anticodon interaction at the start AUG codon tbr translation.
Suppressors of initiator codon mutations Met-tRNAf is bound to elF-2.GTP on the 40S ribosome. Therefore elF-2 may participate in AUG codon recognition. Indeed, the genes encoding the o~- (SUl2) and the [3-subunit tSUi3) of elF-2 have been cloned as suppressors of initiator codon mutations in the HIS4mRNA which (when mutated) confer the ability to start translation efficiently at an UUG-codon. The selection of SUI (suppressor of initiator codon)-mutants and their phenotypes is presented in figure 3. Spontaneous suppressors of a his4--yeast strain that were able to use UUG as start codon were selected. In order to discriminate against revertants that recreated the AUG codon, the mutated 5'-UTR of HIS4-mRNA was also fused in-frame to the ,8-galactosidase open reading frame. Recreation of the AUG codon resulted in a white colony phenotype when
859 cells were incubated in the presence of the chromogenie substrate X-Gal (5-bromo-4-chloro-3-indolyl-13D-galactoside). In contrast, mutations in a SUI gene should not only render a His+-phenotype, but mutant strains should also form blue colonies on X-Gal plates. This easy selectable phenotype permitted the isolation of SUIs in three different complementation groups, suil- and sui2-mutants were both recessive and conferred a ts-phenotype to the cell, a dominant SUl3-mutant was temperature-resistant (fig 3). In order to clone the SUI3 gene, cloned genomic DNA fragments from the SUl3-mutant were transformed into the original SUl31his4- strain and His+-transformants selected. The cloning of the SUII and SUI2 genes was even easier: transformation of the temperature-sensitive suppressor strains with a wildtype yeast DNA library and selection of temperatureresistant transformants was used to clone the genes. All three SUI genes are essential for the survival of the cell. SUI3 is 42% identical with the [3-subunit of mammalian elF-2 and like its homologue contains two potential nucleic acid-binding domains, three polylysine domains at the amino-terminal end and a Zn(lI) 'finger' motif, Cys-X2-Cys-Xzg-Cys-X2-Cys at the carboxyi end [32]. SUI2 is 58% identical with the asubunit of human elF-2. Both proteins are conserved in the amino-terminal region and contain a serine residue which is the target of a kinase that down-regulates translation [33]. SUII has been shown biochemically aot to be a subunit of elF-2. Its ability to use a non-AUG codon (UUG) for translation initiation suggests that it encodes an additional factor that cooperates with elF-2 and Met-tRNAf in AUG start codon recognition [34].
Exceptions to the rule In more than 95% of the mRNAs of Saccharomyces cerevisiae the first AUG from the 5'-end is used as start codon for translation. How does the recognition of an AUG start codon by scanning 40S ribosomes carrying elF-2.GTP.Met-tRNAf function in those mRNAs where it is not the first AUG which serves as the start codon for translation? Reinitiation
Ribosomes can resume translation after passing an upstream open reading frame by scanning the mRNA until they find a downstream ORE This translational mechanism, called reinitiation, has been described for mRNAs with small upstream ORFs in their 5'-UTR which regulate the expression of the downstream open reading frame, eg C P A I - m R N A encoding the gluta-
minase subunit of carbamoyl-phosphate synthetase and GCN4-mRNA encoding a transcription factor that activates the expression nf many genes involved in amino acid and nucleotide biosynthesis. The translation of both mRNAs is stimulated under conditions of nutritional deprivation (in the case of CPAI by low arginine concentration) because ribosomes can efficiently resume the translation at a downstream start codon. In the case of C P A I - m R N A an upstream ORF encodes a 25 amino acid peptide that is assumed to repress translation of the downstream ORF in the presence of arginine [35]. In the case of GCN4-mRNA amino acid deprivation leads to phosphorylation and inactivation of elF-2, allowing ribosomal subunits to bypass the upstream ORFs and resume translation at the fifth open reading frame which encodes the GCN4 protein. Under conditions where overall mRNA translation is downregulated, translation of GCN4-mRNA is not only maintained but enhanced to provide the expression of vital enzymatic activities. This exceptional regulatory mechanism has facilitated the cloning of several genes involved in recycling of elF-2.GTP and the regulation of elF-2 activity (for recent reviews see [36, 41, 42]). Internal initiation
On certain RNAs with unusual leader structures (several upstream AUGs and strong secondary, structures) like those of picornaviruses or some mammalian mRNAs, ribosomes can bypass the ~'-end of the mRNA and bind in a cap-independent way directly to internal ribosome entry sites (IRES). Cellular factors required for this process have been isolated from mammalian cells and characterized (for recent reviews see [37, 38]). So far, evidence for internal initiation of translation in yeast is scarce. In an elF-4E-dependent lysate (see above) translation of the second ORF of a dicistronic mRNA was observed when the poliovirus 5'-UTR was used as internal ribosome binding site. Translation of the second ORF was more efficient when the bicistronic mRNA was uncapped and was inhibited by the addition of elF-4E. This suggests that under conditions where ribosomal subunits are not directed to the cap structure of the mRNA, cap-independent internal initiation may occur [39]. Surprisingly, a small yeast RNA has been isolated that inhibits in trans the translation of mRNAs carrying poliovirus sequences in yeast or mammalian extracts. The mode of action and the biological significance of this inhibitory RNA is not known [40]. Recent data suggest that the translation of certain viral as well as certain yeast RNAs may be initiated by a cap-independent internal ribosome binding mechanism (N Iizuka, L Najita, A Franzusoff, P Sarnow, personal communication).
860 Conclusion The possibility of combining biochemical with powerful genetical experiments in the yeast Saccharomyces cerevisiae has made this organism a model system to study initiation of protein synthesis in eukaryotes. In addition to the translation factors already known from the mammalian system, several new factors like SUII and like SSLI and SSL2 were found. Their function in initiation of protein synthesis has still to be established. Mutants that suppress the inhibitory effect of non-AUG codons on translation have demonstrated the central role of elF-2 and Met-tRNAr in the recognition of the AUG start codon.
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