translational level in response to amino acid availability. gcn3 mutations block derepression of GCN4 expression in starvation conditions. gcdl andgcd 12 ...
Copyright 0 1987 by the Genetics Society of America
Interactions Between Positive and Negative Regulators of GCN4 Controlling Gene Expression and Entry Into the Yeast Cell Cycle Satoshi Harashima,’ Ernest M. Hannig and Alan G. Hinnebusch2 Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 Manuscript received April 30, 1987 Accepted July 24, 1987
ABSTRACT The GCN4 gene encodes a transcriptional activator in yeast whose expression is regulated at the translational level in response to amino acid availability. gcn3 mutations block derepression of GCN4 expression in starvationconditions.gcdl andgcd 12 mutationsrestore derepressionof GCN4 expression in gcn3 deletion mutants, suggesting that GCN3 positively regulates GCN4 indirectly by antagonism of these GCD functions. gcdl and g c d l 2 mutations also lead to temperature-sensitivearrest in the G, phase of the cell cycle in gcn3 deletion mutants. The GCN3 allele completely suppresses both derepression of GCN4 expression and the temperature-sensitive growth conferred by gcd 12 mutations and partially suppresses these phenotypes in gcdl mutants. This suggests that the GCN3 product can promote or provide GCD function in nonstarvation conditions even though it opposes GCD function when cells are starved for amino acids. The gcn3-102 allele is completely defective for positive regulation of GCN4 expression; however, it mimics GCN3 in suppressing gcdl and g c d l 2 mutations and thus retains the ability to restore GCD function in nonstarvation conditions. These data suggest that GCN3, GCDl and G C D l 2 have closely related functions required for regulation of GCN4 expression and entry into the cell cycle. We suggest that GCN3 antagonizes the regulatory functions of GCDl and G C D l 2 in starvation conditions either by competing with these factors for the same sites of action or by modifying their structures by physical interaction.
S
ACCHAROMYCES cerevisiae responds to starvation for any single amino acid by derepressing a large number of enzymes in several amino acid biosynthetic pathways. This cross-pathway regulatory response, known as general amino acid control, operates at the level of transcription of the structural genes that encode the coregulated enzymes. Trans-acting factors encoded by GCNl-GCN9 are required for derepression of enzyme expression in starvation conditions, whereas the products of G C D l - G C D l 3 are needed for enzyme repression in nonstarvation conditions. Most gcd mutations are pleiotropic and lead to either temperature-sensitive or unconditional slow growth. Some gcdl alleles are also defective for the killer response, failing to maintain M dsRNA at the permissive temperature (reviewed in HINNEBUSCH 1986; NIEDERBERGER, AEBI and HUETTER1986; GREENBERG et al. 1986; HARASHIMA and HINNEBUSCH 1986). Genetic analysis has led to a model in which G C N l is the proximal positive effector in the general control system and is subject to negative regulation by multiple GCD genes in nonstarvation conditions. T h e GCD factors are in turn negatively regulated by G C N l , GCN2 and GCN3 in response to amino acid starvation
’
Laboratory of Microbial Genetics, Department of Fermentation Technology, Osaka University, Yamada-oka 2-1, Suita-shi, Osaka, 565, Japan. To whom correspondence should be addressed. Genetics 117: 409-419 (November, 1987)
(WOLFNER et al. 1975; HINNEBUSCH and FINK 1983; NIEDERBERGER, AEBI and HUETTER1986; GREENBERG et al. 1986; HARASHIMA and HINNEBUSCH 1986).3 In support of this model, it was shown that GCN4 protein binds specifically to cis-acting DNA sequences required for the derepression of structural genes subject to the general control (HOPE and STRUHL1985; ARNDTand FINK 1986). In addition, several GCN and GCD genes have been shown to control the expression of a GCN4-lacZ fusion in the manner predicted by the genetic model (HINNEBUSCH 1985; HARASHIMA and HINNEBUSCH1986). Four AUG codons are present in the 5’ leader of GCN4 mRNA and act in cis to repress translation of GCN4 protein in nonstarvation conditions. GCDl and GCDIO-GCD13 are required for the inhibitory effects of the upstream AUG codons on GCN4 expression, whereas GCN2 and GCN3 are needed to suppress the effects of these sequences in starvation conditions (THIREOS, PENNand GREER1984; HINNEBUSCH 1984; HINNEBUSCH 1985; MUELLERand HINNEBUSCH 1986; MUELLER,HARASHIMA and HINNEBUSCH 1987). In contrast to this translational mechanism, it has been Gene names and allele numbers for gcn and certain gcd mutations have been changed from those used in the original reports describing the mutations. The previous designations for mutations discussed in this report are as follows: gcn2-101 = aasl-1; gcn3-101 = aasZ-1; gcn3-102 = aas2-2;gcn4-101 = aas3-1;gcdl-101 = tra3-1.
410
S. Harashima, E. M. Hannig and A. G. Hinnebusch
suggested that GCNG and GCN7 positively regulate GCNI at the transcriptional level (GREENBERG et al. 1986). Some of the known GCN-encoded factors may be involved in recognition of the starvation signal (uncharged tRNA) that triggers the derepression of GCN4 expression (MESSENGUY and DELFORGE 1976). Others may be activated by this signal to antagonize the negative effectors encoded by GCD genes. We are using a genetic approach to identify GCN-encoded factors that might interact directly with GCD gene products. In a previous study, we observed allelespecific effects among gcdl and gcn3 mutations. T h e gcd 1-101 mutation leads to temperature-sensitive (Tsm-) growth at 36”. T h e gcn3-102 mutation was isolated as a partial suppressor of the Tsm- phenotype of the gcdl-101 mutation. By contrast, a gcn3-IO1 gcd 1-101 double mutant is inviable. T h e observation of markedly different phenotypes for these two double mutants was taken as an indication that the GCN3 and GCDl proteins might physically interact (HINNEBUSCH and FINK 1983). In this communication, we report additional instances of allele-specific effects involving gcdl and gcn3 mutations, and similar, more dramatic interactions occurring between gcn3 and gcdl2 mutations. Our findings suggest that GCN3, GCDl and GCD12 have closely related functions in the regulatory pathway that couples GCNI expression to amino acid availability. In addition, it seems likely that the regulatory and the essential functions carried out by GCDl and GCD12 are closely associated. We speculate that GCN3 competes with or modifies the products of GCDl and GCD12 in starvation conditions in a way that alters the translational complex to permit more efficient translation of GCN4 mRNA. MATERIALS AND METHODS Yeast strain construction: T h e genotypes of all yeast strains employed are given in Table 1. Strain H96 (MATa
GCD gcn2-101 gcn3-101 hisl-29 ura3-52 [HISI::lucZ,ura352, integrated at uru3-52) (HARASHIMA and HINNEBUSCH 1986) is sensitive to the amino acid analogue 3-aminotriazole (AT“) as the result of its gcn2 and gcn3 mutations. T h e gcd strains in Table 2 were isolated from H96 as 3-aminotriazole-resistant (ATr) revertants. T h e gcd mutations thus obtained also lead to temperature-sensitivity for growth (Tsm-) (HARASHIMA and HINNEBUSCH 1986). Strains shown in Table 3 are either gcd revertants isolated from H96 o r AT’ Tsm- meiotic segregants obtained from a back-cross between a gcd gcn2-101 gcn3-101 revertant of H96 and strain H I 17, which has the same genotype as H96 except at MAT. Construction of the gcdl-501 strains in Table 4 began with H424 (MATagcdl-501 gcn2-101 gcn3-101 hisl-29 inol ura352 [HIS4::LacZ,URA3 integrated at ura3-52]),an AT’ Tsmmeiotic segregant from a back-cross of revertant H56 (HARASHIMA and HINNEBUSCH 1986) with H 1 17. H460 was derived from H424 by “inositolless death” to eliminate the URA3-containing plasmid carrying the HIS4::LacZ fusion integrated at uru3-52 (HARASHIMA and HINNEBUSCH 1986). H424 was crossed with strain H3 (MATa ura3-52 Leu2-3
Leu2-112) and a Ura- Tsm+’- meiotic segregant (H469) was selected with the presumptive genotype MATa uru3-52 inol gcdl-501. T h e GCN genotype of H469 was confirmed by subsequent crosses to H602 (MATagcn2-101 lys2) and H603 (MATa gcn3-101 lys2). 4 AT’ : 0 AT”, 3 AT’ : 1 AT” and 2 AT’ : 2 AT“ segregation was observed in both crosses, as expected for heterozygosity at GCDl and GCN2 o r GCN3 (GCD GCN strains are AT’). Strain H629 is a Tsm+’- meiotic segregant from a 4 AT‘ : 0 AT” tetrad produced by the hybrid H469 X H602; H628 was obtained as a Tsm- AT‘ meiotic segregant from a 4 AT’ : 0 A T s tetrad produced by H469 X H603. H562 is a Tsm+’- AT’ segregant isolated from a cross between H469 and H37 (MATa gcn4-101 met14 ura3-52). T h e gcd12-503 strains in Table 4 were derived from H427, a MATa gcd1.2-503 gcn2-101 gcn3-101 hisl-29 inol ura3-52 [HZS4::lacZ,URA3, integrated at ura3-521 meiotic segregant from a cross between revertant H64 (HARASHIMA and HINNEBUSCH 1986) and H I 17. H463 was obtained by inositolless death from H427. H626, H625 and H454 are meiotic segregants obtained from a hybrid between H427 and H4 (MATa ura3-52 leu2-3 Leu2-112). T h e genotypes of these strains were confirmed by crosses to GCD gcn2-101, GCD gcn3-101 and GCD GCN tester strains: T h e presence of gcd12-503 in the GCN3 strains H626 and H454 was verified by the reappearance of Tsm- meiotic segregants from crosses to GCN2 gcn3-101 testers. T h e presence of gcn2-101 in H626 was shown by the 0 AT‘ : 4 A T s segregation in all asci produced by a hybrid formed with a gcn2101 tester. T h e presence of gcn3-101 in H625 was verified by the 2 AT’ Tsm- : 2 ATs Tsm+ segregation observed in all asci produced by a hybrid formed with agcn3-101 tester. Construction of strains H652 and H653 in Table 5 began with strain H647 (MATa gcd12-503 leu2-3 leu2-112), obtained from the same cross described above for obtaining H454. H647 was crossed with H650 (MATa gcn3::LEUZ ura3-52 leu2-3 leu2-112) and H651 (MATa gcn3::LEUZ gcd12-503 ura3-52 leu2-3 leu2-112) was isolated as a Leu+ AT‘ Tsm- meiotic segregant from the resulting hybrid. H651 was crossed to H I 7 (MATa gcn3-102 ura3-52 leu2-3 leu2-112) and H652 was isolated as a Leuf AT’ Tsmmeiotic segregant; H653 was isolated as a Leu- AT“ Tsm+ segregant from the same cross. H824 was also a segregant from cross H647 X H650 described above. Construction of H654 and H660 in Table 5 began with a cross between H628 and H649 (MATa gcn3::URA3 ura352 leu2-3 leu2-112) from which H657 (MATa gcn3::URA3 gcdl-501 u r d - 5 2 ) was isolated as a Ura+ AT‘ Tsm- segregant. H657 was crossed with H229 (MATa gcn3-102 ura352 inol) from which H654 was obtained as a Ura- Tsm+’segregant. H825 (MATa gcdl-501 gcn3-101 ura3-52 leu2-3 leu2-112 Lys2) was isolated as a Ura- AT’ Tsm- segregant from the cross between H628 and H649. H825 was crossed with H650 to obtain H660 as a Leu+ AT‘ Tsm- segregant. EY 152 and EY 153 are meiotic segregants from a cross between H654 and H562. EY152 is Tsm+’- and failed to complement a gcn3 tester for its AT” phenotype; EY153 is Tsm- and complements a gcn3 tester for A T sensitivity. Plasmids: Plasmid Ep69 is a stable, low-copy plasmid containing the GCN3 gene on a 3.9-kb fragment obtained and FINK1983) inserted from plasmid pAH 16 (HINNEBUSCH in YCp50. Plasmid Epl50 contains a frameshift mutation in the GCN3 coding region and was derived from Ep69 by filling in a ClaI restriction site with Klenow DNA polymerase and religating the plasmid. As expected, this procedure destroyed the ClaI site and created a new NruI site at the same location. T h e CluI site is located 60% of the coding region from the amino terminal end of the GCN3 coding
Interactions Among GCN4 Regulators
41 1
TABLE 1 S.cerarisiae strains Strain
H4 H17 H54 H56 H57 H58 H59 H6 1 H62 H63 H64 H70 H75 H79 H96 H211 H454
Genotvw
MATa ura3-52 leu2-3 leu2-112 M A T a gcn3-102 ura3-52 leu2-3 leu2-112 MATagcdlO-501 gcn2-101 gcn3-101 hisl2 9 ura3-52 [HIS4::lacZ,ura3-52, integrated at ura3-521 M A T a gcdl-501 gcn2-101 gcn3-101 hisl-29 ura3-52 [HIS4::lacZ,ura3-52, integrated at ura3-521 MATO GCDll-501 gcn2-101 gcn3-101 hisl29 ura3-52 [HIS4::lacZ,ura3-52, integrated at ura3-521 MATagcd12-501 gcn2-101 gcn3-101 hisl29 ura3-52 [HIS4::lacZ,ura3-52, integrated at ura3-521 M A T a GCDll-502 gcn2-101 gcn3-101 hisl29 ura3-52 [HIS4::lacZ,ura3-52, integrated at ura3-521 M A T a G C D l l - 5 0 3 gcn2-101 gcn3-101 hisl29 ura3-52 [HIS4::lacZ,ura3-52, integrated at ura3-521 M A T a gcd10-503 gcn2-101 gcn3-101 hisl29 ura3-52 [HISI::lacZ,ura3-52, integrated at ura3-521 MATagcd12-502 gcn2-101 gcn3-101 hisl29 ura3-52 [HIS4::lacZ,ura3-52, integrated at ura3-521 M A T a gcd12-503 gcn2-101 gcn3-101 hisl2 9 ura3-52 [HIS4::lacZ,ura3-52, integrated at ura3-521 MATagcdl-505 gcn2-101 gcn3-101 hisl-29 ura3-52 [HISI::lacZ,ura3-52, integrated at ura3-521 M A T a gcdl3-501 gcn2-101 gcn3-101 hisl29 ura3-52 [HISI::lacZ,ura3-52, integrated at ura3-521 M A T a g c d l l - 5 0 5 gcn2-101 gcn3-101 hisl29 ura3-52 [HISI::lacZ,ura3-52, integrated at ura3-521 M A T a GCD gcn2-101 gcn3-101 hisl-29 ura3-52 [HlS4::EacZ,ura3-52, integrated at ura3-52 ] MATa gcdl-101 gcn3-102 ura3-52 M A T a gcd12-503 ura3-52
sequence (E. M. HANNICand A. G. HINNEBUSCH, unpublished data). gcn3 deletion plasmids were constructed by replacing a 782-bp HindIII-ClaI fragment which contains 60% of the GCN3 coding sequences and 0.22 kb of 5’ flanking DNA with the 2.18-kb LEU2 XhoI-Sal1 fragment (Ep146) or with the 1.1-kb URA3 Hind111 fragment (Ep149) unpublished data). (E. M. HANNIGand A. G. HINNEBUSCH, Plasmid C102-9 is a low-copy plasmid containing the GCN2 gene (DRISCOLL PENN,THIREOS and GREER1984) on a 7.1PE”. kb fragment, kindly provided by MONICA DRISCOLL Genetic methods: Yeast genetic techniques and culture media (SHERMAN, FINK and LAWRENCE 1974) as well as procedures for determination of 3-aminotriazole resistance and temperature sensitivity (HARASHIMA and HINNEBUSCH 1986) were described previously. 3-aminotriazole was used at 30 mM. Yeast transformation was done according to the
Strain
H460 H463 H466 H562 H595 H601 H605 H625 H626 H628 H629 H651 H652 H653 H654 H658 H659 H660 H748 H821 H824 F69 F98 EY 125 EY152 EY 153
GenotvDe
M A T a gcdl-501 gcn2-101 gcn3-101 hisl-29 inol ura3-52 M A T a gcd12-503 gcn2-101 gcn3-101 hisl29 inol ura3-52 M A T a gcn2-101 gcn3-101 hisl-29 inol ura3-52 M A T a gcdl-501 ura3-52 inol MATa gcdl-505 gcn2-101 gcn3-101 hisl-29 ura3-52 inol [HIS4::lacZ,ura3-52 integrated at ura3-52 ] MATagcn2-101 lysl M A T a gcd11-505 gcn2-101 gcn3-101 hisl29 ura3-52 inol [HIS4::lacZ,ura3-52 integrated at ura3-52 ] M A T a gcd12-503 gcn3-101 ura3-52 M A T a gcd12-503 gcn2-101 ura3-52 leu2-3 leu2-112 M A T a gcdl-501 gcn3-101 ura3-52 lys2 M A T a gcdl-501 ura3-52 lys2 M A T a gcdl2-503 gcn3::LEUZ ura3-52 leu23 leu.?-112 M A T a gcd12-503 gcn3::LEUZ ura3-52 leu2-3 leu2-112 MATa gcdl2-503 gcn3-102 ura3-52 leu2-3 leu2-112 M A T a gcdl-501 gcn3-102 ura3-52 gcdl-504 gcn2-101 gcn3-101 his129 ura352 [HISI::lacZ,ura3-52 integrated at ura3-521 gcdl-506 gcn2-101 gcn3-101 hisl-29 ura352 [HIS4::lacZ,ura3-52 integrated at ura3-521 M A T a gcdl-501 gcn3::LEUZ ura3-52 lys2 leu2-3 leu2-112 M A T a gcn3-IO1 ura3-52 M A T a gcn3-101 lys2 M A T a gcd12-503 gcn3::LEUZ ura3-52 leu2-3 leu2-112 M A T a gcn3-102 inol ura3-52 [HISI::lacZ, URA3 integrated at ura3-521 M A T a gcdl-101 ura3-52 M A T a gcn3::LEUZ ura3-52 leu2-3 leu2-112 M A T a gcdl-501 gcn3-102 inol ura3-52 M A T a gcdl-501 ura3-52
method of Ito et aE. (1983). Gene replacements a t GCN3 were conducted by the method of ROTHSTEIN (1 983) using the 4-kb NruI-BglI fragment of Ep146 o r the 4-kb EcoRIPvuI fragment of Ep149 for yeast transformation. DNA blot-hybridization analysis of total transformant DNA was conducted to confirm all gene replacements (E. M. HANNIC and A. G. HINNEBUSCH, unpublished data). T h e gcn3::URA3 andgcn3::LEU2 deletion alleles conferred an ATs phenotype in GCD strains and this phenotype segregated 2 A T s Ura+ (or Leu+) : 2 AT‘ Ura- (or Leu-) in meiotic analysis of hybrids formed between the deletion strains and GCN ura352 leu2-3 leu2-112 strains. Assays of lacZ fusions: Culture conditions and 8-galactosidase assays used for measuring steady-state levels of GCN4::lacZ fusion enzyme expression were described previously (LUCCHINI et al. 1984).
S. Harashima, E. M. Hannig and A. G. Hinnebusch
412
Cell-cycle studies: For each strain analyzed, one-half o f an asynchronous culture growing exponentially in liquid YPD medium at 23' was shifted to 36' while the other half was maintained at 23". Six hours after the shift, an aliquot was taken from each culture, sonicated for 20 sec and the percentage o f unbudded cells was measured by light microscope examination of 300 cells.
TABLE 2
Tetrad segregation from hybrids between gcd gcn2-101 gcn3-I01 revertants and GCD gcn2-101 GCN3 and GCD GCN2 gcn3-101 tester strains Ascus type'
RESULTS gcd mutation
g c d l and g c d l 2 mutations are suppressed by GCN3: gcn2 and gcn? mutants are unable to derepress amino acid biosynthetic enzymes in starvation conditions. As a result, they are sensitive to 3-aminotriazole, an inhibitor of the HIS3 gene product. We previously described the isolation of gcd mutants by reversion of the 3-aminotrazole-sensitive (AT') phenotype of a gcn2-I01 gcn?-101 double mutation. The gcd mutations we obtained also confer temperature-sensitive or unconditional slow growth (HARASHIMA and HINNEBUSCH 1986). In the course of our genetic analysis, we crossed the gcd gcn2 gcn3 revertants by GCD GCN strains and analyzed the meiotic products of the resulting hybrids. T h e simple expectation was to find in each ascus two Tsm-, 3-aminotriazole-resistant (AT') spores containing the gcd mutation involved in the cross. In most asci, the remaining two spores were expected to have a Tsm+ ATs phenotype since they lack a gcd mutation but contain either gcn2-I01 or gcn3-101. This proved to be the case in asci produced by hybrids containing g c d l 0 , g c d l l and gcdl? mutations; however, the crosses involving gcdl and g c d l 2 mutations gave rise to a significant number of asci in which either three or all four spores had a Tsm+ AT" phenotype (data not shown). This suggested that expression of the phenotypes of gcdl and g c d l 2 mutations in our revertants might be dependent upon the presence of gcn2-I01 or gcn?-l01. We examined this possibility in two ways. T h e first approach was to cross the AT' Tsm- gcd gcn2-I01 gcn3-I01 revertants by GCD gcn2-I01 GCN? and GCD GCN2 gcn?-l01 strains, thus making either gcn2-I01 or gcn?-I01 homozygous in each cross. In the case of gcdl and g c d l 2 revertants, we observed 2 AT' Tsm-: 2 ATs Tsm+ segregation in nearly every ascus produced by hybrids in which gcn?-I01 is homozygous. By contrast, hybrids which are homozygous for gcn2-I01 and heterozygous at GCN3 also gave rise to a significant number of 1 AT' Tsm-: 3 ATs Tsm+ and 0 AT' Tsm-: 4 ATs Tsm+ asci (Table 2). All hybrids constructed from revertants containing mutations in GCDIO, G C D l l or GCDl? gave rise to only 2 AT' Tsm-: 2 AT" Tsm+ asci. These results suggest that the gcdl and g c d l 2 mutations present in the revertants are suppressed by the GCN? allele. In the case of the g c d l 2 mutations, suppression appeared to be complete; for gcdl mutations, suppression of both mutant phenotypes appeared to be incomplete (Table
2)-
Strain"
involved
Testerb
No.of asci tested
2AT'
1AT'
OAT'
Tsm-:
2AT' Tsm+
Tsm-: 3AT" Tsm+
Tsm-: 4AT' Tsm+
6
3
7
0
0 0
H56 H56
gcdl-501 gcdl-501
gcn2 gcn?
9 7
H70 H70
gcdl-505 gcdl-505
gcn2 gcn3
8 9
2
4
2
8
1
0
H54 H54
gcd10-501 gcd10-501
gcn2 gcn3
7
7
0
0
8
8
0
0
H62 H62
gcdI0-503 gcd10-503
gcn2 gcn?
9
9
0
4
4
0
0 0
H57 H57
GCDII-501 GCDII-501
gcn2 gcn?
9
7
9 7
0 0
0 0
H79 H79
gcd11-505 gcdll-505
gcn2 gcn?
11 11
11 11
0 0
0 0
H58 H58
gcdl2-501 gcd12-501
gcn2 gcn3
5
0
3
2
4
4
0
0
H64 H64
gcd12-503 gcd12-503
gcn2 gcn3
12 12
2 12
7
3
0
0
H75 H75
gcd13-501 gcdl3-501
gcn2 gcn3
6 6
6
0
6
0
0 0
All gcd strains have the genotype gcd gcn2-101 gcn3-101 hisl29 ura?-52 [HIS4::lacZ,URA? integrated at ura3-521. The gcn2 and gcn3 tester strains are H601 (MATa GCD gcn2I01 GCN3 l y s l ) and H821 (MATa GCD GCN2gcn3-101 lys2). For the gcdl revertants, the actual ascus types observed from left to right were 2 AT' Tsm-: 2 ATs Tsm+, 1 AT' Tsm-: 1 AT'/" Tsm+/-: 2 ATs Tsm+ and 2 AT'" Tsm+/-: 2 AT" Tsm+, where AT" and Tsm+/- designate weak AT resistance and weak temperature sensitivity, respectively.
Suppression of g c d l and g c d l 2 mutations by the cloned GCN3 gene: In the second approach, we transformed gcd gcn2-101 gcn?-I01 strains with single-copy plasmids carrying the cloned GCN2 or GCN? genes. The results in Table 3 indicate that the GCN3 gene completely suppressed the AT' and Tsm- phenotypes of three g c d l 2 mutants and partially suppressed both phenotypes in five gcd I mutants. Transformation of the same strains with the GCN2 gene (Table 3) or with a gcn? frameshift mutation constructed in vitro (data not shown) had no effect on the phenotype of the gcdl and g c d l 2 mutants. Transformation of two out of five g c d l l gcn2-I01 gcn3-I01 mutants with the GCN? gene gave the opposite result of exacerbating the slow growth phenotype of these mutants. T h e phenotypes associated with all other mutant alleles of GCDIO, G C D l l and GCDl? were unaffected by introduction of the GCN2 or GCN3 gene. GCN4::lacZ expression is regulated by interactions between GCN3, G C D l and GCD12: T o investigate the effect of these genetic interactions on the expression of GCN4, we measured GCN4::lacZexpres-
Interactions Among GCN4 Regulators
413
TABLE 3
TABLE 4
Suppression of gcdl andgcdl2 mutations by the cloned GCN3 gene in gcd gm2 gcn3 strains
Constitutive derepression of GCN4lacZ expression in gedl2503 and gedl-501 mutants is suppressed by GCN3
~~
~
GCN4::lacZ enzyme activity (units)b
Phenotype of transformant harboring plasmid-borne geneb ~
gcd mutation
Strains"
involved
H56 H70 H595 H658 H659 H62 H605 H57 H59 H6 1 H79 H58 H63 H64 H75 H96
gcdl-501 gcd 1-502 gcdl-505 gcd 1-504 g ~ 1-506 d gcd 10-502 gcd 11-505 GCDl l-501 GCDll-502 GCDI 1-503 GCDll-504 gcd 12-50 1 gcd 12-502 gcd 12-503 gcd13-501 GCD
Vector alone
GCN?
GCNP
AT'
Tsm
AT'
Tsm
AT'
Tsm
+ + + + + + + +
-
+ + +
-
+ + + + + + + + +
-
+/+/+/+/+/-
+/+/+/+/+/-
+ +
+ + + + + -
-
+/-c
+/+/+/+/-
-
+
+ +
+
+I-
-
+/+/+/+/+/-
+
+ + + + + + -
+ -
-
+/+/+/-
+ + + +
All strains contain gcn2-101, gcn3-101, hisl-29, ura3-52 and a HIS4::lacZ fusion integrated at ura3-52. H595 also contains an inol mutation. GCN2 and GCN3 were contained on stable, low-copy, autonomously replicating plasmids C102-9 and Ep69, respectively. YCp50 was used as the "Vector alone." ' For g c d l l mutants, a Tsm phenotype of +/- indicates unconditional slow growth.
'
sion in the gcd mutants shown in Table 4. T h e results demonstrate an excellent correlation between the degree of A T resistance, temperature-sensitivity and derepression of GCN4::lacZ expression in the various gcd strains. As expected, the gcn3-I01 mutation blocked efficient derepression of GCN4::lacZ expression in the ATs GCD gcn3-101 strain H748 compared to AT' GCD GCN strain H4. By contrast, gcn3-I01 led to higher levels of GCN4::lacZ expression in the presence of the gcd 1-501 or gcd 12-503 mutations compared to the expression observed in the corresponding gcd GCN3 strains: gcd12-503 gcn3-101 strain H625 exhibited constitutive derepression of GCN4::lacZ expression at a level 40- to 50-fold higher than the repressed level observed in fully wild-type strain H4. By contrast, gcd12-503 GCN3 strain H454 exhibited GCN4::lacZ expression very similar to that seen in GCD GCN strain H4. Therefore, the G C N 3 allele completely suppressed the effect of gcd 12-503 on GCN4::lacZ expression. In the case of gcdl-501, the GCN3 allele did not completely eliminate constitutive derepression of GCN4::lacZ expression, but led to 3to 5-fold lower expression than observed in the gcdl5 0 1 gcn3-101 strain. By contrast to the effects of gcn3101, the gcn2-101 allele led to lower GCN4::lacZ expression relative to the GCN2 allele in the presence or absence of mutations in GCDl or GCDI2. Disruption of GCN3 leads to efficient expression
Strain"
Genotvue
H463 H626 H625 H454
gcdl2 gcn2 gcn3 gcd 1 2 gcn2 GCN3 g c d l 2 GCN2 gcn3 gcdl2 GCN2 GCN3
H460 H629 H628 H562
gcd 1 gcn2 gcn3 gcd 1 gcn2 GCN3 gcdl GCN2 gcn3 gcd 1 GCN2 GCN3
H466 H748 H4
GCD gcn2 gcn3 GCD GCN2 gcn3 GCD GCN2 GCN3
AT'
+ + + + +
+ + +
Tsm
R
DR
-
240 21 1000 35
340 33 830 150
-
+/-
1300 390 2300 450
1500 290 3200 730
+ + +
27 44 20
15 69 225
+ + +/-
-
a Each strain was transformed with plasmid p180, a stable, lowcopy, autonomously replicating plasmid containing a GCN4::lacZ fusion. * These values are the averages of 2-3 assays on independent transformants that varied from the average by 30% or less. The repressing (R) conditions were 6 hr of growth to mid-exponential phase in minimal medium supplemented with leucine, isoleucine, valine and inositol as described (HINNEBUSCH 1985); the derepressing (DR) conditions were 6 hr of culturing in the same medium containing 5-methyltryptophan at 0.5 mM, following 2 hr of growth in the absence of 5-methyltryptophan.
of gcdl and gcdl2 mutations: We replaced the gcn3101 mutation in several gcd strains with a gcn3 allele containing a deletion of 60% of the coding region plus an insertion of the yeast URA? or LEU2 gene. In GCD haploid strains, the gcn3::URA3 allele confers an ATs Tsm+ phenotype similar to that conferred by gcn3-101. This suggests that G C N 3 is not essential in GCD cells grown in non-starvation conditions and is only required for derepression in starvation conditions (E. M. HANNIGand A. G. HINNEBUSCH, unpublished data). When we replaced gcn3-101 with gcn3::URA3in twogcd I 2 gcn2-I01 gcn3-I01 revertants (H63 and H64) and in the gcdl-501 gcn2-I01 gcn3101 revertant H56, we found that the transformants exhibited an AT' Tsm- phenotype similar to that of the untransformed parent strains containing gcn3101. This suggests that the gcn3-101 mutation is similar to a null allele of G C N 3 and that elimination of the G C N 3 gene product is the necessary condition for full expression of gcdl and g c d l 2 mutant phenotypes. We observed only one instance of allele specificity in these gene replacement experiments: gcd 12-502 gcn2-I01 gcn3::URA3 transformants exhibit a more severe growth defect at the semipermissive temperature than their isogenic parent strain containing the gcn3-101 allele (Figure 1). This more extreme growth defect was always found to be associated with gcn3::URA3 in 10/10 asci produced by a hybrid between GCD GCN2 gcn3::URA3 strain H649 and gcdl2502 gcn2-101 gcn3-I01 ura3-52 revertant H63. In this
414
S. Harashima, E. M. Hannig and A. G. Hinnebusch
F I G U R E 1 .--Kepklcement of gcn3-1OI with gcn3::tih\3 in ii grd12-502 nilltilnt kads t o iln incre;ised growti drfiw. Strain t{ti:< (Rrd12-502 gm2-101gm3-101 ura3-52). shown on the left, and a Ura' transformant of H63 containinggcn3::UiRA3 in place ofgcn3-101. shown on the right. were plated for single colonies o n complete medium (YPD)and incubated at 30" for 3 days.
cross we also observed roughly equal numbers of Uraand Ura+ segregants which have the AT' Tsm- phenotype indicative of the g c d l 2 mutation. This shows that g c d l 2 and gcn3 mutations a r e unlinked and rules out the possibility that g c d l 2 mutations a r e constitutively derepressing alleles of GCN3. T h e data shown in Table 5 indicate that gcn3::LEU2 behaves like gcn3-I01 and leads to constitutive derepression of GCN4::lacZ expression in a gcd12-503 strain. T h e degree of derepression is significantly lower in the gcd 1 2 gcn3::LEU2 strain compared to the g c d l 2 gcn3-I01 strain. However, GCN4::lacZ expression is clearly both constitutive and depressed in g c d l 2 gcn3::LEU2 cells by comparison with g c d l 2 GCN3 cells, where expression is very similar to that observed in wild-type cells. GCN4::lacZ expression was also constitutive in thegcdl g&3::LEU2 strain shown in Table 5 ; however, in this case, the degree of derepression is comparable to that seen in the gcdl GCN3 strain. gcn3-102 behaves like GCN3 and suppresses the gcdl and gcdl2 mutations: Like other gcn3 mutations we examined, gcn3-102 leads to an AT' phenotype
and blocks derepression of GCN4-lac2 expression in GCD cells (Table 5). However, gcn3-102 differs from gcn3-I01 and gcn3::URA3 in suppressing the phenotypes of gcdl and g c d l 2 mutations. GCD gcn3-102 strain F69 was crossed with the gcdl-501 gcn2-101 gcn3-I01 revertant H56 and the gcdl2gcn2-I01 gcn3IO1 revertants H58, H63 and H64. If gcn3-102 acts like gcn3-I01 and leads to expression of the gcdl and g c d l 2 mutant phenotypes (AT' Tsm-) all asci produced by these hybrids should exhibit 2 AT' Tsm-: 2 AT' Tsm+ segregation. However, in addition to such asci, we observed 1 AT' Tsm-: 3 ATs Tsm+ and 0 A T ' Tsm-: 4 ATs Tsm+ segregation, suggesting that suppression of thegcd mutations had occurred in these asci. Subsequently, we crossed gcd12-503 gcn3::LEU2 strain H 6 5 1 with G C D l 2 gcn3-102 strain H 17. As above, all three types of asci were produced by this hybrid and all Leu- segregants (which contain gcn31 0 2 ) were AT' Tsm+ whereas all AT' Tsm- segregants were Leu+. These results demonstrate directly that the mutant phenotypes associated with gcd12-503 a r e not expressed in gcd12-503 gcn3-102 recombi-
Interactions Among GCN4 Regulators TABLE 5 Allele-specific interactions between gcn3, gcdl and gcdl2 mutations affect expression of C C N 4 l a e Z GCN4::lac.Z enzyme activity (units)' Strain"
Genotvwb
H625 H652 H454 H653
gcd 12 gcn3-10 1 gcd 12 gcn3::LEUZ gcdl2 GCN3 gcdl2 gcn3-102
H628 H660 H562 H654
gcdl gcn3-101 gcdl gcn3::LEUZ gcdl GCN3 gcd lgcn3-102
H748 EY 125 H4 H17
GCD gcn3-101 GCD gcn3::LEUZ GCD GCN3 GCD gcn3-102
AT'
Tsm
+ + +
+
-
+ + +
+ + -
+
-
-
+/+/-
+
+ +
+
R
DR
1000 210 35 15
830 190 150 24
2300 550 450 230
3200 600 730 190
44 28 20 14
69 28 225 26
a Each strain was transformed with plasmid p180, a stable, lowcopy, autonomously replicating plasmid containing a GCN4::lacZ fusion. * All strains are GCN2. These values are the averages of 2-3 assays on independent transformants that varied from the average by 30% or less. Repressing (R) and derepressing (DR) growth conditions were the same as described in Table 4.
nants. As a final proof of this conclusion, we carried out a cross between gcdl2-503 gcn3::LEU2 strain H824 and gcd12-503 gcn3-102 strain H653 and observed 2 AT" Tsm- Leu': 2 ATs Tsm+ Leu- segregation in 10/10 asci produced by this hybrid. In agreement with these findings, Table 5 shows that GCN4::lacZ expression in a gcd12-503 gcn3-102 strain was 50-fold lower than that seen in a gcd12-503 gcn3-101 strain, and was indistinguishable from that seen in a GCD gcn3-102 strain. In agcn3-102 gcdl-501 double mutant, GCN4-lac2 expression was lower than that observed in any other gcdl-501 strain we examined, particularly in a gcdl-501 gcn3-101 mutant. We conclude that the gcn3-102 allele closely resembles G C N 3 in completely suppressing the phenotypes of the gcd 1 2 mutations and partially suppressing the gcd 1 mutations. Allele specificity in the interaction between gcn3102 andgcdl-101: gcn3-102 was isolated as a suppressor of the Tsm- phenotype of the gcdl-101 mutation in a G C N 3 strain (HINNEBUSCH and FINK 1983). As expected, a hybrid between gcdl-101 gcn3-102 double mutant H211 and gcdl-101 G C N 3 single mutant F98 gave rise predominantly to asci exhibiting 2 Tsm+/-: 2 Tsm- segregation. We showed above that gcn3-102 also partially suppresses the Tsm- phenotype of a gcdl-501 mutation. In a cross identical to H211 X F98 just described, except that gcdl-501 replaces gcdl-101 (EY 152 X EY 153),all four spores in every tetrad were Tsm+/-; however, two degrees of temperature-sensitivity were evident. Two ascospores in each tetrad
415
exhibited a less severe temperature-sensitivity than their two sister spores. We demonstrated by complementation testing for the ATs phenotype that all of the less temperature-sensitive ascospores from this cross contain the gcn3-102 allele. Therefore, gcdl-501 is also suppressed more efficiently by gcn3-102 than by GCN3. However, the degree of suppression o f g c d l 5 0 1 by gcn3-102 in cross EY 152 X EY 153 was significantly less than we observed for gcdl-101 in cross H211 X F98. As a result, at least one component of the suppressive interaction between gcn3-102 and gcdl-101 may be specific to these two alleles. gcdl and gcdl2 mutants arrest in the GI phase of the cell cycle at the restrictive temperature: Because the previously isolated gcdl-101 mutation leads to GI et al. 1975), we examined the arrest at 36" (WOLFNER terminal phenotype of our newly isolated temperature sensitive gcd revertants. Figure 2 shows that gcdl-501 strain H56 and gcd12-503 strain H64 accumulate as spherical unbudded cells after 6 h r of incubation at 36". By contrast, the Tsm- gcdlO (H62) and g c d l 3 (H75) mutants failed to accumulate with a characteristic morphology when treated in the same way (data not shown). DISCUSSION
At temperatures permissive for their growth, gcdl and gcd 1 2 mutants exhibit derepressed expression of genes encoding enzymes subject to general amino acid control. This phenotype is attributable to increased expression of GCN4, the direct positive regulator of these genes (HINNEBUSCH 1985; HARASHIMA and HINNEBUSCH 1986). T h e GCDl and GCD1.2 products repress GCN4 expression at the translational level by controlling the inhibitory effects of AUG codons located in the 5' leader of GCN4 mRNA (HINNEBUSCH 1985; MUELLERand HINNEBUSCH 1986; MUELLER, HARA~HIMA and HINNEBUSCH 1987). At the restrictive temperature, gcdl and g c d l 2 mutants arrest in the GI phase of the cell cycle. This temperature-sensitive phenotype remains evident after inactivation of G C N I , showing that at least one component of the Gcd- growth defect is unrelated to overexpression of GCN4 protein in the gcd mutants (HARASHIMA and HINNEBUSCH 1986). Therefore, it appears that GCDl and G C D l 2 either execute or regulate the expression of an essential function, in addition to their role in translational control of GCN4. T h e non-derepressible phenotype of GCD gcn3:: U R A 3 strains clearly demonstrates that inactivation of G C N 3 interferes with derepression of GCN4 in starvation conditions and verifies that G C N 3 is a positive effector of GCN4. Recessive mutations in either GCDl or G C D l 2 overcome the derepression defect in gcn3::URA3cells, supporting the idea that G C N 3 acts as a positive regulator by antagonizing GCDl and
416
S. Harashima, E. M. Hannig and A. G.Hinnebusch
FIGURE 2.-gcdl and gcd.12 mutants arrest as unbudded cells at 36". gcdl strain H56 (A and B), g c d l l strain H64 (C and D) and CCD strain H96 (E and F) were grown at 23" (A, C and E) or 36" (B. D and F) for 6 hr in complete medium (YPD).Shown here are representative fields from samples of each culture visualized by light microscopy. All three strains cultured at 23" and H96 cultured at 36" were in logarithmic growth at the time of sampling. Growth of H56 and H64 at 36" had arrested by 6 hr.
Interactions Among GCN4 Regulators G C D l 2 repressing functions in starvation conditions 1986). T h e surprising (HARASHIMA and HINNEBUSCH conclusion from the experiments presented here is that the GCN3 product also appears to promote the negative regulatory function and the essential function carried out by the products of these GCD genes in nonstarvation conditions. Thus, the gcn3-101 and gcn3::URA3 mutations enhance the derepression of GCN4::lacZ expression and the temperature sensitivity associated with gcd 1 mutations, and are absolutely required for expression of these phenotypes in g c d l 2 mutants. T h e GCN3 allele completely suppresses both phenotypes for all three gcd 1 2 alleles and partially suppresses each of five g c d l mutations we examined. These findings suggest that the absence of GCN3 gene product is the necessary condition for full expression of g c d l and g c d l 2 mutant phenotypes. Suppression of the gcd mutations by GCN3 could be explained in several ways. One possibility is that GCN3 can at least partially complement the loss of GCDl or G C D l 2 function. In this view, GCN3 and these two GCD genes carry out analogous functions, as in the case of the duplicated yeast histone H2B genes and the yeast RAS genes, to name only a few examples et al. 1981; KATAOKAet al. 1984; TATCH(RYKOWSKI ELL et al. 1984). Such functional complementation would imply that G C N 3 can provide GCD function in nonstarvation conditions even though it acts to oppose GCD function in starvation conditions. This suggests the interesting possibility that GCN3 acts as a positive effector of GCN4 by competing with GCDl and GCD12 in the regulatory mechanism in conditions of amino acid starvation. It then functions inefficiently in these circumstances as a GCD factor in repressing GCN4 expression (Figure 3A). gcn3-102 is completely defective for GCN3 positive regulatory function, yet it behaves like GCN3 in restoring a degree of GCD function to gcdl mutants and in completely suppressi n g g c d l 2 mutations. T h e complementation model can account for this finding by proposing that the gcn3102 mutation leaves intact the ability of the G C N 3 product to provide GCD function in repressing conditions. T h e gcn3-102 mutation impairs only the ability of the GCN3 protein to respond correctly to the inducer in derepressing conditions after it replaces the GCD gene product in the regulatory mechanism (Figure 3A). Another possible explanation for our results is that the GCN3 and gcn3-102 gene products suppress the phenotype of gcdl and g c d l 2 mutations by stabilizing thermolabile proteins encoded by these gcd alleles. According to this model, the G C N 3 protein forms a complex with the GCDl and G C D l 2 proteins. gcn3101, the allele present in the strains in which the gcdl and g c d l 2 mutations were isolated, and gcn3::URA3 would encode products incapable of complex forma-
417
tion with the GCD proteins. As a result, the thermolability of the gcd products would be expressed in gcn3101 and gcn3::URA3 strains, thus leading to a Gcdphenotype. By contrast, the products of the G C N 3 and gcn3-102 alleles would stabilize the mutant gcdl and g c d l 2 proteins, thus suppressing the Gcd- phenotype (Figure 3B). T h e idea of complex formation between GCN3 and GCD proteins is attractive because it offers a mechanism by which G C N 3 could antagonize GCD function in starvation conditions: Modification of the G C N 3 protein in response to starvation could alter the structure and activity of the GCD proteins with which it interacts. In this model, the gcn3-102 product would be capable of complex formation with GCD proteins and could therefore suppress the phenotypes of gcd mutations; however, it would be unable to antagonize the GCD factors in starvation conditions and incapable of promoting derepression of GCN4 expression. T h e gcn3-102 mutation suppresses the temperature sensitivity of the gcdl-101 mutation more efficiently and FINK 1983). than the GCN3 allele (HINNEBUSCH In addition, measurements of GCN4-lacZ expression suggest that gcn3-102 leads to greater repression of GCN4 in gcdl-101 strains than does the G C N 3 allele 1985). These findings suggest that the (HINNEBUSCH gcn3-102 product is more efficient than the G C N 3 product at restoring GCDl function in gcdl-101 cells. It was suggested previously that the gcn3-102 mutation leads to a specific change in the G C N 3 protein that compensates for a structural alteration in the product of gcdl-101 with which it interacts (HINNEBUSCH and FINK 1983). This explanation is in accord with the model we present in Figure 3B. However, we have shown here that gcn3-102 is also more efficient than G C N 3 in suppressing a different gcdl mutation isolated independently of gcn3-102. While the suppressor activity of gcn3-102 is considerably less for the new gcdl mutation than for gcdl-101, the absence of unequivocal allele specificity in these interactions prevents us from arguing more strongly in favor of a protein-protein interaction between G C N 3 and G C D l . T h e complementation model in Figure 3A can easily account for the suppressor activity of gcn3-102 by proposing that the product of this allele has greater GCD function than the wild-type G C N 3 product. A fact that is more difficult to explain by complex formation is that the gcn3::URA3 and gcn3-101 mutations have no effect on either the essential or regulatory functions of the GCDl and G C D l 2 gene products in GCD cells. To account for this observation, it could be proposed that the GCD gene products have associations with other proteins, in addition to the G C N 3 protein, that are sufficient to maintain the integrity of the complex in the absence of GCN3. Only the combination of a gcd mutation and inactivation of
S. Harashima, E. M. Hannig and A. G. Hinnebusch
418
B
A Starvation
Non-starvation GCD12 GCN3 GCD 12
1.GCN4mRNA
3'
rYnducer I I
gcdl2 gcn3: :URA3
6 !
gcd 12 GCN3
gcdl2 GCN3
L
-
Starvation
GCDl2 GCN3
/\GCN3
+=5I - .
Non-starvation
I
3 '
ncd12 gcn3- 102
gcdl2 gcn3: URA3
8
v gcd 12 gcn3- 102
FIGURE3.-Two models to explain the interactions between GCDlZ and GCN3. (A) Functional complementation of a g c d l 2 mutation by G C N 3 and gcn3-102. Under nonstarvation conditions, the GCN3 product can substitute for the g c d l 2 product and repress translation of GCN4 mRNA. The positive regulatory function of G C N 3 in wild-type cells results from replacement of the G C D l 2 product by the functionally analogous GCN? product. This replacement depends upon the action of an inducer present only in starvation conditions. Interaction with the inducer also decreases the efficiency of the GCN? product in repressing translation, thus allowing increased expression of GCN4 protein. The gcn3-102 product can efficiently repress translation in a g c d l 2 mutant in non-starvation conditions and it retains this function in starvation conditions because it fails to interact with the inducer. (B) Direct interaction between the products of G C D l 2 and GCN? orgcn?102. Suppression of a g c d l 2 mutation by the GCN? product is explained by an interaction between the two proteins that corrects an instability in the g c d l 2 product. Only when GCN3 protein is missing is the g c d l 2 product instability manifested. In response to the influence of an inducer present in starvation conditions, the GCN3 product alters the configuration of the G C D l 2 or g c d l 2 products and renders them incapable of efficiently repressing translation of GCN4 mRNA. The gcn3-102 product retains the ability to interact with and stabilize the g c d l 2 product but cannot alter its configuration to allow derepression because it fails to interact with the inducer. In both models, interactions that lead to proper repression of GCN4 expression in nonstarvation conditions also suppress the temperature-sensitive growth defect associated with g c d l 2 mutations.
GCN3 would then result in destabilization of the complex and a Gcd- phenotype. T h e complementation model can account for the same observation by proposing that GCN3 can provide a degree of GCD function in the absence of GCDl or GCD12, but its contribution is dispensible in GCD cells grown in non-starvation conditions. Regardless of which model is correct, our data strongly suggest that GCN3, GCDl and GCD12 have closely related functions in the translational control of GCN4. This makes these gene products excellent candidates for factors that function at the interface between the positive and negative regulators controlling GCN4 expression. In addition, we have noted a good correlation between the effects of gcn3 mutations on the regulatory and the essential functions of G C D l and GCD12: With only one exception, the
severity of the Tsm- phenotype of a gcd mutant was directly correlated with the degree of constitutive derepression of GCN4::lacZ expression. T h e effects of GCN3 upon both phenotypes suggests that the essential and regulatory functions of the GCD proteins are interrelated. Given that GCN4 is regulated by these factors at the translational level (MUELLERet aE. 1987), we speculate that the essential functions carried out by the GCD factors affect protein synthesis. In this view, the role of GCN factors would be to alter or compete with one or more GCD factors whose functions in the translational complex are required to repress translation of GCN4 mRNA. Our genetic observations make GCN3 a likely candidate for a factor that participates directly in such interactions with the products of GCDl and GCD12. We thank JEFBOEKE,CHRISTOPHER PAD"
and PETERMUEL-
Interactions Among GCN4 Regulators for valuable comments on the manuscript, ALICE MA for technical assistance and DOTTIEALLORfor preparation of the manuscript. LER
LITERATURE CITED ARNDT,K., and G. R. FINK,1986 GCN4 protein, a positive transcription factor in yeast, binds general control promoters at all 5' TGACTC 3' sequences. Proc. Natl. Acad. Sci. USA 83: 8516-8520. DRISCOLL PE", M., G. THIREOS and H. GREER,1984 Temporal analysis of general control of amino acid biosynthesis in Saccharomyces cereuisiae: role of positive regulatory genes in initiation and maintenance of mRNA derepression. Mol. Cell. Biol. 4: 520-528. GREENBERG, M. L., P. L. MYERS,R. C. SKVIRSKY and H. GREER, 1986 New positive and negative regulators for general control of amino acid biosynthesis in Saccharomycescereuisiae. Mol. Cell. Biol. 6: 1820-1829. HARASHIMA, S., and A. G. HINNEBUSCH, 1986 Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cereuisiae. Mol. Cell. Biol. 6: 3990-3998. HINNEBUSCH, A. G., 1984 Evidence for translational regulation of the activator of general amino acid control in yeast. Proc. Natl. Acad. Sci. USA 81: 6442-6446. HINNEBUSCH, A. G., 1985 A hierarchy of trans-acting factors modulates translation of an activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. Mol. Cell. Biol. 5 2349-2360. HINNEBUSCH, A. G., 1986 The general control of amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. CRC Crit. Rev. Biochem. 21: 277-317. HINNEBUSCH, A. G., and G. R. FINK,1983 Positive regulation in the general amino acid control of Saccharomycescerevisiae. Proc. Natl. Acad. Sci. USA 8 0 5374-5378. HOPE,I. A., and K. STRUHL,1985 GCN4 protein, synthesized in vitro, binds HIS4 regulatory sequences: Implications for general control of amino acid biosynthetic genes in yeast. Cell 43: 177188. ITO, H., Y. FUKUDA,K. MURATA and A. KIMURA, 1983
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Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153: 163-168. KATAOKA, T., S. POWERS, C. MCGILL,0. FASANO,M. GOLFARB, J. STRATHERN, J. BROACHand M. WIGLER,1984 Genetic analysis of yeast R A S l and RASZ genes. Cell 37: 437-445. LUCCHINI,G., A. G. HINNEBUSCH, C. CHEN and G. R. FINK, 1984 Positive regulatory interactions of the HIS4 gene of Saccharomycescerevisiae. Mol. Cell. Biol. 4: 1326-1333. MESSENGUY, F., and J. DELFORGE, 1976 Role of transfer ribonucleic acids in the regulation of several biosyntheses in Saccharomyces cereuisiae. Eur. J. Biochem. 67: 335-339. MUELLER, P. P. and A. G. HINNEBUSCH, 1986 Multiple upstream AUG codons mediate translational control of GCN4. Cell 45: 20 1-207. MUELLER, P. P., S. HARASHIMA and A. G. HINNEBUSCH, 1987 A segment of GCN4 mRNA containing the upstream AUG codons confers translational control upon a heterologous yeast transcript. Proc. Natl. Acad. Sci. USA. 84: 2863-2867. NIEDERBERGER, P., M. AEBIand R. HUETTER,1986 Identification and characterization of four new GCD genes in Saccharomyces cerevisiae. Curr. Genet. 1 0 657-664. ROTHSTEIN, R. J., 1983 One-step gene disruption in yeast. Methods Enzymol. 101: 202-21 1. RYKOWSKI, M. C., J. W. WALLIS,J. CHOEand M. GRUNSTEIN, 1981 Histone 2B subtypes are dispensable during the yeast cell cycle. Cell 2 5 477-487. SHERMAN, F., G. R. FINKand C. LAWRENCE, 1974 Methods of Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. TATCHELL,K., D. CHALEFF,D. DEFEO-JONES and E. SCOLNICK, 1984 Requirement of either of a pair of ras-related genes of Saccharomyces cerevisiae for spore viability. Nature 3 0 9 523527. THIREOS,G., M. D. PENNand H. GREER,1984 5' Untranslated sequences are required for the translational control of a yeast regulatory gene. Proc. Natl. Acad. Sci. USA 81: 5096-5100. WOLFNER,M., D. YEP, F. MESSENGUYand G. R. FINK, 1975 Integration of amino acid biosynthesis into the cell cycle of Saccharomycescerevisiae. J. Mol. Biol. 9 6 273-290. Communicating editor: E. W. JONES
