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Converting a eukaryotic transcriptional inhibitor into an activator
Cell, Vol. 56, 443-446,
November
4, 1988, Copyright
0 1968 by Ceil Press
onverting a Eukaryotic Transcriptional Inhibit “nto an Activator Jun Ma and Mark Btashne Department of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts 02138
ummary GALBO, an inhibitor of the yeast transcriptional activator GAL4, is converted into an activator by inserting an acidic activating sequence into it. This hybrid activator does not bind to DNA directly, but is brought to DNA by interacting with a derivative of GAL4 that interacts with both DNA and GAWO.
Recent experiments suggest that a typical eukaryotic transcriptional activator contains two functions: one directs DNA binding and the other (the activating sequence) interacts with some component of the transcriptional machinery (for reviews, see Ptashne, 1986; Maniatis et al., 1987; Guarente, 1988; Struhl, 1988; Ptashne, submitted). Here, we show that a eukaryotic negative regulator of transcription can be converted into an activator by inserting an activating sequence into it. Our experiment is formally analogous to that of Eiushman and Ptashne (1988) who converted a prokaryotic repressor into an activator by changing appropriate amino acids on one surface of the protein. In contrast to that case, the activator reported here does not bind DNA directly, but instead interacts with another protein that binds DNA. Our activator thus mimics a class of eukaryotic transcriptional activators believed to be brought to DNA by binding to a second DNA-bound protein. GAL4 is a transcriptional activator of the genes required for utilizing galactose in yeast (for reviews, see Oshima, 1982; Johnston, 1987). The amino-terminal 147 residues of this 881 amino acid protein (Laughon and Gesteland, 1984) contain the surface that binds to the DNA sequences in the UASo (the GAL upstream activating sequence; Keegan et al., 1986; also see Guarente et al., 1982; West et al., 1984; Giniger et al., 1985; Bram and Kornberg, 1985). There are two short regions in the more carboxy-terminal portion of GAL4 (region I: residues 148-196, and region It: residues 768-881), each of which can activate transcription when fused to the DNA binding portion of the molecule (Ma and Ptashne, 1987a; also see Brent and Ptashne, 1985). These transcriptional activating regions can be functionally replaced by a wide range of peptides encoded by E. coli genomic DNA fragments (Ma and Ptashne, 1987c) or by a peptide designed to form an amphiphilic a-helix (Giniger and Ptashne, 1987). One of these E. coli-derived sequences, 842, also activates transcription when attached to the DNA binding domain of the bacterial repressor LexA (Ma and Ptashne, 1987c). All these activating sequences, like those of GAL4 and an-
other yeast activator GCN4, are b~gh~y acidic (Ma and Ptashne, 1987a, 1987c; Hope and Struhl, 1986; Hope et al., 1988; also see Gill and Ptashne, 1987). GAL4 or its derivatives bearing one or more acidic activating sequences also activates transcription in mammalian plant and insect cells (Kakidani and Ptashne, 1988; Webster et al., 1988; Ma et al., 1988; Fisher et al., 1988). GAL80, a protein that does not bind DNA (Lue et al., 1987; J. M., unpublished data), inhibits the activity of GAL4 when yeast cells are grown in the absence of galactose (Nogi et al., 1984; for reviews, see Qshima, 1982; Johnston, 1987). GAL80 forms a complex with GAL4 (Lue et al., 1987; J. M., unpublished data) so that, although still bound to DNA (Giniger et al., 1985; Lohr et al., 1985; Selleck and Majors, 1987) GAL4 does not activate transcription, evidently because the activating surface(s) is masked by GAL80 (Ma and Ptashne, 1987b; Johnston et at., 1987). GAL80 recognizes the carboxy-terminal 30 amino acids of GAL4, a part of activating region II (Ma and Ptashne, 1987b; Johnston et al., 1987). For example, GAL80 interacts with a GAL4 derivative that bears the carboxy-terminal 30 amino acids fused to the DNA binding portion (GAL4[1-147]+[851-8811) both in vivo and in vitro, whereas GAL80 does not interact with GAL4 (1-147) (Ma and Ptashne, 1987b; J. M., unpublished data). We inserted the activating sequence 842 into GAL80 and showed that this hybrid protein (GALBO-B42) activates transcription of a gene bearing the GAL4 binding sites (UASo), provided the cells also contain GAL4(1-147)+ (851-881). Results Nogi (personal communication) identified a region of GAL80 (residues 322-341 of this 435 amino acid protein; Nogi and Fukasawa, 1984) at which insertions do not significantly affect the ability of GAL80 to inhibit the activity of GAL4. For example, GAL80 derivatives bearing insertions at residue 341 inhibit the activity of GAL4 almost as efficiently as does wild-type GAL80. We inserted the activating sequence 642 at residue 341 of GAL86 and we call this hybrid protein GAL80-B42 (Figure 1). To assay the activity of GAL80-B42, we engineered the following yeast strains. These strains were deleted for the endogenous GAL4 and GAL80 genes. Each strain was modified to contain an integrated gene expressing either wild-type GAL4 or one of its derivatives from the GAL4 promoter. These strains also contained in the chromosome a GAL&lacZfusion gene, either bearing or lacking the wildtype UASo. We introduced into these yeast strains plasmids expressing wild-type GAL80 or GAL80-842 from the GAL80 promoter. Cells were grown in the absence of galactose, and 8-galactosidase levels were taken as a measure of promoter activity. Cur results are presented in Table 1. As described previously (Ma and Ptashne, 1987b), GAL4(1-147)-r-(851-881) weakly activated transcription of the GALI-/acZ fusion
Cell 444
8
340 Asp-Pro-Gly-
Figure 1. GAL80-642
1
79
341 Pro-Ser-Thr-Pro
M
'1
8 co 2 3"4'6
The activating sequence 842 containing 79 amino acids is inserted at residue 341 of the 435 amino acid GAL80 protein. The 842 sequence is linked to GAL80 by extra amino acids Pro-Gly at its amino terminus and Pro-Ser-Thr at its carboxyl terminus. The figure is not drawn to scale. GAL80-642 synthesized in an in vitro translation system showed a proper increase in size relative to wild-type GAL80 (J. M., unpublished data).
Table 1. Transcriptional GAL7-lacz 1. +UASo
Activation
GAL4 Derivative GAL4 (1-147) (851-881)
+
4. 5. 6.
GAL4 (l-l
7. 8. 9.
47)
b-Gal
no GAL80
32
wt GAL80 GAL80-B42
0.7 316
no GAL80 wt GAL80 GAL80-B42
no GAL4
no GAL80 wt GAL80 GAL80-B42
10. 11. 12.
wt GAL4
no GAL80 wt GAL80 GALBO-B42
13. -UASo
GAL4 (1-147) (851-881)
+
lac
by GAL80-842 GAL80 Derivative
2. 3.
14. 15.
7 8'
Hybrid Protein
1852 105 640
no GAL80
wt GAL80 GAL80-B42
The five yeast strains used in this study contained in the chromosome a GAL7-/acZ fusion gene either bearing (+) or lacking (-) the wildtype UASo upstream (lines 1-12 and 13-15, respectively). They also contained in the chromosome genes expressing from the GAL4 promoter either GAL4(1-147)+ (851-881) (lines l-3 and 13-15), GAL4(1-147) (lines 4-6) no GAL4 (lines 7-9) or wild-type GAL4 (lines 10-12). (See Experimental Procedures for the names of the strains.) These strains were transformed with plasmids that expressed from the GAL80 promoter either no GAL80 (pMA201), wild-type GAL80 (pMA51 l), or GAL80-B42 (pMA513-B42). 8-galactosidase activity was assayed in yeast grown in the absence of galactose.
gene bearing UASo upstream (Line l), and its activity was inhibited by wild-type GAL80 (line 2). However, expression of GAL80-B42 in the cells containing GAL4(1147)+(851-881) increased f3-galactosidase activity about IO-fold (compare lines 1 and 3). This activity is over 300 times greater than that observed when wild-type GAL80 was introduced into the cells containing GAL4(1-147)+ (851-881) (compare lines 2 and 3). The @galactosidase activity induced by GALSO-B42 in cells bearing GAL4(1147)+(851-881) is about 15% of that induced by wild-type GAL4 (compare lines 3 and 10). This activity of GAL80542 approaches that of GAL4(1-147) +B42, which binds directly to DNA and activates transcription (Ma and Ptashne, 1987c). When assayed in yeast cells containing either GAL4
Figure 2. Primer Extension
Analyses
Shown are results of primer extension anaiyses for the experiments described in lines l-3 in Table 1. Assays were done in either duplicate or triplicate using RNA samples isolated from independent yeast transformants. LacZ indicates the transcript of the GALI-/acZ gene, and ADHI transcripts are internal control for the assays. Markers (lane M) are pBR322 Mspl fragments labeled with 32P
(1-147) (lines 4-8) or no GAL4 (lines 7-9), GAL80-B42 did not activate transcription. GAL80-B42 also failed to activate transcription when UASo was deleted from the GAL74acZ promoter (Lines 13-15). Activation by GAL80B42 was concomitant with an increase of mRNA initiating at the correct position (Figure 2). We also observed similar activation by GAL80-B42 protein when cells were grown in the presence of gafactose (data not shown), a result consistent with the observation of Nogi (personal communication) that the inhibition by GAL80 derivatives bearing insertions at residue 341 are not relieved by growth of cells in the presence of galactose. The activation function of GAL80-B42 was not evident when assayed in cells containing wild-type GAL4 instead of GAL4(1-147)+(851-881) (lines 10-12). We imagine that GAL80-B42 binds to GAL4 and covers its activating region, and because GAL80-842 activates transcription less efficiently than does wild-type GAL4, the predominant effect of GAL80-B42 in this case is inhibition. Comparison of lines 3, 11, and 12 suggests that GAL80-B42 inhibits the activity of wild-type GAL4 slightly less efficiently than does wild-type GAL80. In our experiments, the activ ity of GAL4 was not completely abolished by wild-type GAL80 (line 11); it is possible that, since the GAL80 gene was carried on a plasmid, a small fraction of the yeast cells did not contain the GAL80 plasmid and thus escaped the inhibition by GAL80 (also see Nogi et al., 1984). We also inserted two other E. coli-derived sequences (B7 and 832) into GAL80, but we found that these hybrid proteins did not activate transcription when assayed in cells bearing GAL4(1-147)+(851-881). Rather, they inhibited the activity of both wild-type GAL4 and GAL4(1-
Converting
GAL80 into an Activator
445
b GAL l-la& Figure 3. Model of Transcriptional
Activation
scher et al., 1988; Chiu et al., 1988; Strum, !988). According to Chiu et al. (1988), AP-1 itself is a weak activator, but the c-fos-AP-1 complex activates transcription efficiently. Both VP16 and c-fos have been fused directly to a DNA binding domain (GAL4 in one case and LexA in the other), and the hybrid proteins activate transcription when bound, respectively, to the GAL4 and LexA binding sites (Lech et al., 1988; Sadowski et al., submitted).
by GAL80-642
GAL4(1-147)+(85t-881) is shown bound to UASo. This GAL4 derivative contains the carboxy-terminal 30 amino acids (residues 851-881) fused to the DNA binding portion (residues I-147). The GAL80 portion of the GAL8QB42 hybrid protein binds to the GAL4 derivative and therefore tethers the activating sequence 842 to DNA. There are actually four GAL4 binding sites within the UASo (Giniger et al., 1985) each of which binds a dimer of GAL4 (M. Carey and M. P., unpublished data).
147)+(851-881) as did wild-type GAL80 (data not shown). 842 is more active than 87 and B32 when fused to the DNA binding portion of GAL4 (Ma and Ptashne, 1987c), and is almost twice as long as B7 and 832 (87 contains 42 amino acids and B32 contains 45 amino acids). We do not know which, if either, of the features of B42 enables it to activate transcription in the context of GAL80.
Discussion In experiments described in this paper, we converted a transcriptional inhibitor, GAL80, into an activator by inserting into it an acidic activating sequence (642) encoded by an E. coli genomic DNA fragment. Activation by this hybrid protein requires a second protein (a GAL4 derivative) that interacts with both GAL80 and DNA (Figure 3). The results show that the DNA binding and activating functions can reside on different molecules that associate to form a transcriptional activator. Thus, an acidic sequence required for gene activation can be brought to the vicinity of a promoter either by binding directly to DNA or by binding to a second protein that in turn binds to DNA. Our results also show that 842 can function as an activating sequence in three different contexts: either when directly fused to the DNA binding domain of LexA, or to that of GAL4 (Ma and Ptashne, 1987c), or when inserted into GAL86 An alternative explanation for our results, which we consider unlikely, may be that GAL80-B42 is not itself an activator, but somehow potentiates the weak activation function of the GAL4 derivative to which it binds. It has recently been suggested that, in two cases, eukaryotic activators stimulate transcription by binding to a second QNA-bound protein. VP16 (also called Vmw65), a transcriptional activator of herpes simplex virus (HSVl), evidently activates transcription by binding to one or more host-encoded proteins that in turn bind to viral promoters (f&Knight et al., 1987; Preston et al., 1988; O ’Hare and Goding, 1988; Triezenberg et al., 1988). c-fos evidently activates transcription by interacting with the DNA-bound AP-l/c-jun (Bohmann et al., 1987; Angel et al., 1988; Rau-
Experimental
Procedures
Yeast Strains Five yeast strains were generated from a common yeast strain, GGYl (Aga14 Aga/&?O leu2 his3 ura3), kindly provided by 6. Gill. GALb-lacZ fusion genes either bearing (pRY171) or lacking (LRl Al) wild-type UASs were first integrated at the URA3 locus of GGYl (Yocum et al., 1984; West et al., 1984). Plasmids expressing GAL4 derivatives (pMA446: GAL4(1-147]+[851-8811; pMA447: GAL4[1-1471; and pMA448: wild-type GAL4) were then integrated at the pBR322 sequences of pRY171 or LRI Al using LEU2 as selection marker. Thus, the yeast strain GGYl::RY171::MA446 was used for the experiments described in lines 1-3 of Table 1; GGYl::RY171::MA447 for lines 4-6; GGYI::RYl7t for lines 7-9; GGYl::RY171::MA448 for lines 10-12; and GGYi::LRlAl::MA446 for lines 13-15. Plasmids pMA511 contained the following sequences: an ampicillin resistant gene, E. coli replication origin, yeast HIS3 gene, 2 &rn yeast replication origin, and a wild-type GAL80 gene expressed from the GAL80 promoter. pMA513B42 contained the same sequences except that the GAL80 gene is replaced by GAL80-B42 fusion gene. pMA511 was constructed by inserting at the Xbal-Pvull sites of pMA510 (a derivative of pGG3 [Keegan et al., 19861 lacking the Smal site) an Xbal-Pvull fragment containing the wild-type GAL80 gene from plasmid 213-80 (kindly provided by G. Gill; plasmid 213-80 ts a derivative of pLKS57 that was kindly provided by Fukasawa; Nogi et al., 1984; also see Nogi and Fukasawa, 1984, for the sequences of GAL80). pMA512 842 was generated by inserting a Smal linker at the Sail site of ~842 (Ma and Ptashne, 1987c). pMA513B42 was constructed by inserting at the Smal site of pMA511 the Smal fragment of pMA512542 containing activating sequence 842. pMA446, pMA447, and pMA448 are yeast integrating plasmids (with LEUP marker) expressing GAL4(1-147)+(851-881), GAL4(1-147), and wild-type GAL4, respectively, from the GAL4 promoter. pMA446 was constructed by inserting a Pvull-BamHI fragment of pMA442 into the Pvull-BamHI sites of pBR322. pMA447 and pMA448 were constructed by replacing the Hindlll-Xhol fragment of pMA446 with those from pMA241 and pMA210, respectively (Ma and Ptashne, 1987a). Yeast Transformation and Assay of b-Galactosidase Actiwity Yeast transformation and assays of 8-galactosioase activity were as previously described (Ito et al., 1983; Yocum et al., 1984). Preparation of Yeast mRNA and Primer Extension Analysis Yeast strain GGYl::RY171::MA446 was transformed with pMA201, pMA511, and pMA513642, as described in Table 1. mRNA samples were isolated from yeast grown in the absence of gaiactose and analyzed by primer extension assays as described previously (Ma and Ptashne, 1987a). Acknowledgments We thank members of our lab for heipfui discussions; G. Gill, Y. Nogi and T. Fukasawa for strains and plasmids; Y. Nogi and T. Fukasawa for permission to cite unpublished results; F Bushman, G. Gill, H. Himmelfarb, M. Lamphier, D. Ruden, I. Sadowski, 2. M. Tu, and D. Valenzuela for comments on the manuscript; and K. Nevin for administrative assistance. This work has been supported by a grant from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby
Ceil 446
marked “advertisement” in accordance solely to indicate this fact.
with 18 U.S.C. Section 1734
Ma, J., and Ptashne, M. (1987b). The carboxy-terminal 30 amino acids of GAL4 are recognized by GAWO. Cell 50, 137-142.
Received August 5, 1988
Ma, J., and Ptashne, M. (1987c). A new class of yeast transcriptionai activators. Cell 57, 113-119.
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November
4, 1988, Copyright
0 1968 by Ceil Press
onverting a Eukaryotic Transcriptional Inhibit “nto an Activator Jun Ma and Mark Btashne Department of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts 02138
ummary GALBO, an inhibitor of the yeast transcriptional activator GAL4, is converted into an activator by inserting an acidic activating sequence into it. This hybrid activator does not bind to DNA directly, but is brought to DNA by interacting with a derivative of GAL4 that interacts with both DNA and GAWO.
Recent experiments suggest that a typical eukaryotic transcriptional activator contains two functions: one directs DNA binding and the other (the activating sequence) interacts with some component of the transcriptional machinery (for reviews, see Ptashne, 1986; Maniatis et al., 1987; Guarente, 1988; Struhl, 1988; Ptashne, submitted). Here, we show that a eukaryotic negative regulator of transcription can be converted into an activator by inserting an activating sequence into it. Our experiment is formally analogous to that of Eiushman and Ptashne (1988) who converted a prokaryotic repressor into an activator by changing appropriate amino acids on one surface of the protein. In contrast to that case, the activator reported here does not bind DNA directly, but instead interacts with another protein that binds DNA. Our activator thus mimics a class of eukaryotic transcriptional activators believed to be brought to DNA by binding to a second DNA-bound protein. GAL4 is a transcriptional activator of the genes required for utilizing galactose in yeast (for reviews, see Oshima, 1982; Johnston, 1987). The amino-terminal 147 residues of this 881 amino acid protein (Laughon and Gesteland, 1984) contain the surface that binds to the DNA sequences in the UASo (the GAL upstream activating sequence; Keegan et al., 1986; also see Guarente et al., 1982; West et al., 1984; Giniger et al., 1985; Bram and Kornberg, 1985). There are two short regions in the more carboxy-terminal portion of GAL4 (region I: residues 148-196, and region It: residues 768-881), each of which can activate transcription when fused to the DNA binding portion of the molecule (Ma and Ptashne, 1987a; also see Brent and Ptashne, 1985). These transcriptional activating regions can be functionally replaced by a wide range of peptides encoded by E. coli genomic DNA fragments (Ma and Ptashne, 1987c) or by a peptide designed to form an amphiphilic a-helix (Giniger and Ptashne, 1987). One of these E. coli-derived sequences, 842, also activates transcription when attached to the DNA binding domain of the bacterial repressor LexA (Ma and Ptashne, 1987c). All these activating sequences, like those of GAL4 and an-
other yeast activator GCN4, are b~gh~y acidic (Ma and Ptashne, 1987a, 1987c; Hope and Struhl, 1986; Hope et al., 1988; also see Gill and Ptashne, 1987). GAL4 or its derivatives bearing one or more acidic activating sequences also activates transcription in mammalian plant and insect cells (Kakidani and Ptashne, 1988; Webster et al., 1988; Ma et al., 1988; Fisher et al., 1988). GAL80, a protein that does not bind DNA (Lue et al., 1987; J. M., unpublished data), inhibits the activity of GAL4 when yeast cells are grown in the absence of galactose (Nogi et al., 1984; for reviews, see Qshima, 1982; Johnston, 1987). GAL80 forms a complex with GAL4 (Lue et al., 1987; J. M., unpublished data) so that, although still bound to DNA (Giniger et al., 1985; Lohr et al., 1985; Selleck and Majors, 1987) GAL4 does not activate transcription, evidently because the activating surface(s) is masked by GAL80 (Ma and Ptashne, 1987b; Johnston et at., 1987). GAL80 recognizes the carboxy-terminal 30 amino acids of GAL4, a part of activating region II (Ma and Ptashne, 1987b; Johnston et al., 1987). For example, GAL80 interacts with a GAL4 derivative that bears the carboxy-terminal 30 amino acids fused to the DNA binding portion (GAL4[1-147]+[851-8811) both in vivo and in vitro, whereas GAL80 does not interact with GAL4 (1-147) (Ma and Ptashne, 1987b; J. M., unpublished data). We inserted the activating sequence 842 into GAL80 and showed that this hybrid protein (GALBO-B42) activates transcription of a gene bearing the GAL4 binding sites (UASo), provided the cells also contain GAL4(1-147)+ (851-881). Results Nogi (personal communication) identified a region of GAL80 (residues 322-341 of this 435 amino acid protein; Nogi and Fukasawa, 1984) at which insertions do not significantly affect the ability of GAL80 to inhibit the activity of GAL4. For example, GAL80 derivatives bearing insertions at residue 341 inhibit the activity of GAL4 almost as efficiently as does wild-type GAL80. We inserted the activating sequence 642 at residue 341 of GAL86 and we call this hybrid protein GAL80-B42 (Figure 1). To assay the activity of GAL80-B42, we engineered the following yeast strains. These strains were deleted for the endogenous GAL4 and GAL80 genes. Each strain was modified to contain an integrated gene expressing either wild-type GAL4 or one of its derivatives from the GAL4 promoter. These strains also contained in the chromosome a GAL&lacZfusion gene, either bearing or lacking the wildtype UASo. We introduced into these yeast strains plasmids expressing wild-type GAL80 or GAL80-842 from the GAL80 promoter. Cells were grown in the absence of galactose, and 8-galactosidase levels were taken as a measure of promoter activity. Cur results are presented in Table 1. As described previously (Ma and Ptashne, 1987b), GAL4(1-147)-r-(851-881) weakly activated transcription of the GALI-/acZ fusion
Cell 444
8
340 Asp-Pro-Gly-
Figure 1. GAL80-642
1
79
341 Pro-Ser-Thr-Pro
M
'1
8 co 2 3"4'6
The activating sequence 842 containing 79 amino acids is inserted at residue 341 of the 435 amino acid GAL80 protein. The 842 sequence is linked to GAL80 by extra amino acids Pro-Gly at its amino terminus and Pro-Ser-Thr at its carboxyl terminus. The figure is not drawn to scale. GAL80-642 synthesized in an in vitro translation system showed a proper increase in size relative to wild-type GAL80 (J. M., unpublished data).
Table 1. Transcriptional GAL7-lacz 1. +UASo
Activation
GAL4 Derivative GAL4 (1-147) (851-881)
+
4. 5. 6.
GAL4 (l-l
7. 8. 9.
47)
b-Gal
no GAL80
32
wt GAL80 GAL80-B42
0.7 316
no GAL80 wt GAL80 GAL80-B42
no GAL4
no GAL80 wt GAL80 GAL80-B42
10. 11. 12.
wt GAL4
no GAL80 wt GAL80 GALBO-B42
13. -UASo
GAL4 (1-147) (851-881)
+
lac
by GAL80-842 GAL80 Derivative
2. 3.
14. 15.
7 8'
Hybrid Protein
1852 105 640
no GAL80
wt GAL80 GAL80-B42
The five yeast strains used in this study contained in the chromosome a GAL7-/acZ fusion gene either bearing (+) or lacking (-) the wildtype UASo upstream (lines 1-12 and 13-15, respectively). They also contained in the chromosome genes expressing from the GAL4 promoter either GAL4(1-147)+ (851-881) (lines l-3 and 13-15), GAL4(1-147) (lines 4-6) no GAL4 (lines 7-9) or wild-type GAL4 (lines 10-12). (See Experimental Procedures for the names of the strains.) These strains were transformed with plasmids that expressed from the GAL80 promoter either no GAL80 (pMA201), wild-type GAL80 (pMA51 l), or GAL80-B42 (pMA513-B42). 8-galactosidase activity was assayed in yeast grown in the absence of galactose.
gene bearing UASo upstream (Line l), and its activity was inhibited by wild-type GAL80 (line 2). However, expression of GAL80-B42 in the cells containing GAL4(1147)+(851-881) increased f3-galactosidase activity about IO-fold (compare lines 1 and 3). This activity is over 300 times greater than that observed when wild-type GAL80 was introduced into the cells containing GAL4(1-147)+ (851-881) (compare lines 2 and 3). The @galactosidase activity induced by GALSO-B42 in cells bearing GAL4(1147)+(851-881) is about 15% of that induced by wild-type GAL4 (compare lines 3 and 10). This activity of GAL80542 approaches that of GAL4(1-147) +B42, which binds directly to DNA and activates transcription (Ma and Ptashne, 1987c). When assayed in yeast cells containing either GAL4
Figure 2. Primer Extension
Analyses
Shown are results of primer extension anaiyses for the experiments described in lines l-3 in Table 1. Assays were done in either duplicate or triplicate using RNA samples isolated from independent yeast transformants. LacZ indicates the transcript of the GALI-/acZ gene, and ADHI transcripts are internal control for the assays. Markers (lane M) are pBR322 Mspl fragments labeled with 32P
(1-147) (lines 4-8) or no GAL4 (lines 7-9), GAL80-B42 did not activate transcription. GAL80-B42 also failed to activate transcription when UASo was deleted from the GAL74acZ promoter (Lines 13-15). Activation by GAL80B42 was concomitant with an increase of mRNA initiating at the correct position (Figure 2). We also observed similar activation by GAL80-B42 protein when cells were grown in the presence of gafactose (data not shown), a result consistent with the observation of Nogi (personal communication) that the inhibition by GAL80 derivatives bearing insertions at residue 341 are not relieved by growth of cells in the presence of galactose. The activation function of GAL80-B42 was not evident when assayed in cells containing wild-type GAL4 instead of GAL4(1-147)+(851-881) (lines 10-12). We imagine that GAL80-B42 binds to GAL4 and covers its activating region, and because GAL80-842 activates transcription less efficiently than does wild-type GAL4, the predominant effect of GAL80-B42 in this case is inhibition. Comparison of lines 3, 11, and 12 suggests that GAL80-B42 inhibits the activity of wild-type GAL4 slightly less efficiently than does wild-type GAL80. In our experiments, the activ ity of GAL4 was not completely abolished by wild-type GAL80 (line 11); it is possible that, since the GAL80 gene was carried on a plasmid, a small fraction of the yeast cells did not contain the GAL80 plasmid and thus escaped the inhibition by GAL80 (also see Nogi et al., 1984). We also inserted two other E. coli-derived sequences (B7 and 832) into GAL80, but we found that these hybrid proteins did not activate transcription when assayed in cells bearing GAL4(1-147)+(851-881). Rather, they inhibited the activity of both wild-type GAL4 and GAL4(1-
Converting
GAL80 into an Activator
445
b GAL l-la& Figure 3. Model of Transcriptional
Activation
scher et al., 1988; Chiu et al., 1988; Strum, !988). According to Chiu et al. (1988), AP-1 itself is a weak activator, but the c-fos-AP-1 complex activates transcription efficiently. Both VP16 and c-fos have been fused directly to a DNA binding domain (GAL4 in one case and LexA in the other), and the hybrid proteins activate transcription when bound, respectively, to the GAL4 and LexA binding sites (Lech et al., 1988; Sadowski et al., submitted).
by GAL80-642
GAL4(1-147)+(85t-881) is shown bound to UASo. This GAL4 derivative contains the carboxy-terminal 30 amino acids (residues 851-881) fused to the DNA binding portion (residues I-147). The GAL80 portion of the GAL8QB42 hybrid protein binds to the GAL4 derivative and therefore tethers the activating sequence 842 to DNA. There are actually four GAL4 binding sites within the UASo (Giniger et al., 1985) each of which binds a dimer of GAL4 (M. Carey and M. P., unpublished data).
147)+(851-881) as did wild-type GAL80 (data not shown). 842 is more active than 87 and B32 when fused to the DNA binding portion of GAL4 (Ma and Ptashne, 1987c), and is almost twice as long as B7 and 832 (87 contains 42 amino acids and B32 contains 45 amino acids). We do not know which, if either, of the features of B42 enables it to activate transcription in the context of GAL80.
Discussion In experiments described in this paper, we converted a transcriptional inhibitor, GAL80, into an activator by inserting into it an acidic activating sequence (642) encoded by an E. coli genomic DNA fragment. Activation by this hybrid protein requires a second protein (a GAL4 derivative) that interacts with both GAL80 and DNA (Figure 3). The results show that the DNA binding and activating functions can reside on different molecules that associate to form a transcriptional activator. Thus, an acidic sequence required for gene activation can be brought to the vicinity of a promoter either by binding directly to DNA or by binding to a second protein that in turn binds to DNA. Our results also show that 842 can function as an activating sequence in three different contexts: either when directly fused to the DNA binding domain of LexA, or to that of GAL4 (Ma and Ptashne, 1987c), or when inserted into GAL86 An alternative explanation for our results, which we consider unlikely, may be that GAL80-B42 is not itself an activator, but somehow potentiates the weak activation function of the GAL4 derivative to which it binds. It has recently been suggested that, in two cases, eukaryotic activators stimulate transcription by binding to a second QNA-bound protein. VP16 (also called Vmw65), a transcriptional activator of herpes simplex virus (HSVl), evidently activates transcription by binding to one or more host-encoded proteins that in turn bind to viral promoters (f&Knight et al., 1987; Preston et al., 1988; O ’Hare and Goding, 1988; Triezenberg et al., 1988). c-fos evidently activates transcription by interacting with the DNA-bound AP-l/c-jun (Bohmann et al., 1987; Angel et al., 1988; Rau-
Experimental
Procedures
Yeast Strains Five yeast strains were generated from a common yeast strain, GGYl (Aga14 Aga/&?O leu2 his3 ura3), kindly provided by 6. Gill. GALb-lacZ fusion genes either bearing (pRY171) or lacking (LRl Al) wild-type UASs were first integrated at the URA3 locus of GGYl (Yocum et al., 1984; West et al., 1984). Plasmids expressing GAL4 derivatives (pMA446: GAL4(1-147]+[851-8811; pMA447: GAL4[1-1471; and pMA448: wild-type GAL4) were then integrated at the pBR322 sequences of pRY171 or LRI Al using LEU2 as selection marker. Thus, the yeast strain GGYl::RY171::MA446 was used for the experiments described in lines 1-3 of Table 1; GGYl::RY171::MA447 for lines 4-6; GGYI::RYl7t for lines 7-9; GGYl::RY171::MA448 for lines 10-12; and GGYi::LRlAl::MA446 for lines 13-15. Plasmids pMA511 contained the following sequences: an ampicillin resistant gene, E. coli replication origin, yeast HIS3 gene, 2 &rn yeast replication origin, and a wild-type GAL80 gene expressed from the GAL80 promoter. pMA513B42 contained the same sequences except that the GAL80 gene is replaced by GAL80-B42 fusion gene. pMA511 was constructed by inserting at the Xbal-Pvull sites of pMA510 (a derivative of pGG3 [Keegan et al., 19861 lacking the Smal site) an Xbal-Pvull fragment containing the wild-type GAL80 gene from plasmid 213-80 (kindly provided by G. Gill; plasmid 213-80 ts a derivative of pLKS57 that was kindly provided by Fukasawa; Nogi et al., 1984; also see Nogi and Fukasawa, 1984, for the sequences of GAL80). pMA512 842 was generated by inserting a Smal linker at the Sail site of ~842 (Ma and Ptashne, 1987c). pMA513B42 was constructed by inserting at the Smal site of pMA511 the Smal fragment of pMA512542 containing activating sequence 842. pMA446, pMA447, and pMA448 are yeast integrating plasmids (with LEUP marker) expressing GAL4(1-147)+(851-881), GAL4(1-147), and wild-type GAL4, respectively, from the GAL4 promoter. pMA446 was constructed by inserting a Pvull-BamHI fragment of pMA442 into the Pvull-BamHI sites of pBR322. pMA447 and pMA448 were constructed by replacing the Hindlll-Xhol fragment of pMA446 with those from pMA241 and pMA210, respectively (Ma and Ptashne, 1987a). Yeast Transformation and Assay of b-Galactosidase Actiwity Yeast transformation and assays of 8-galactosioase activity were as previously described (Ito et al., 1983; Yocum et al., 1984). Preparation of Yeast mRNA and Primer Extension Analysis Yeast strain GGYl::RY171::MA446 was transformed with pMA201, pMA511, and pMA513642, as described in Table 1. mRNA samples were isolated from yeast grown in the absence of gaiactose and analyzed by primer extension assays as described previously (Ma and Ptashne, 1987a). Acknowledgments We thank members of our lab for heipfui discussions; G. Gill, Y. Nogi and T. Fukasawa for strains and plasmids; Y. Nogi and T. Fukasawa for permission to cite unpublished results; F Bushman, G. Gill, H. Himmelfarb, M. Lamphier, D. Ruden, I. Sadowski, 2. M. Tu, and D. Valenzuela for comments on the manuscript; and K. Nevin for administrative assistance. This work has been supported by a grant from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby
Ceil 446
marked “advertisement” in accordance solely to indicate this fact.
with 18 U.S.C. Section 1734
Ma, J., and Ptashne, M. (1987b). The carboxy-terminal 30 amino acids of GAL4 are recognized by GAWO. Cell 50, 137-142.
Received August 5, 1988
Ma, J., and Ptashne, M. (1987c). A new class of yeast transcriptionai activators. Cell 57, 113-119.
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