Abstract
The protein encoded by the GAL4 gene of the yeast Saccharomyces cerevisiae binds to DNA upstream of several genes and activates transcription. To try to understand these processes, we have undertaken a genetic analysis of GAL4. Here we report that nearly all missense mutations in GAL4, selected in vivo to lack function of the protein, cluster in the small region of the gene that encodes the DNA binding domain. About half of these mutations alters a cysteine-rich region of the protein highly homologous to several eukaryotic DNA binding proteins; the other half alters some of the 20 amino acids adjacent to the cysteine-rich region. Nearly all of the missense mutations that alter the DNA binding domain abolish the DNA binding activity of GAL4 protein measured in vitro. In contrast, nearly all of the mutations that alter the 3' 95% of the gene that encodes the transcription activation function are nonsense or frameshift mutations. These results support the idea that the conserved cysteine-rich sequence motif is directly involved in binding of several eukaryotic transcriptional regulatory proteins to DNA.
The GAL4 gene of Saccharomyces cerevisiae encodes a protein required to activate transcription of several genes involved in galactose utilization (1). This protein (hereafter termed GAL4 protein) binds to specific DNA sequences located upstream ofeach regulated gene (2-7); the events that occur subsequent to DNA binding that lead to activation of transcription are unknown.
Each gene activated by GAL4 protein contains at least one, but usually two or more, GAL4 protein binding sites located 95-410 base pairs (bp) upstream of the transcription initiation site (6, 7). Each binding site is related to a 23-bp consensus sequence that displays partial 2-fold rotational (dyad) symmetry. Because these' GAL4 protein binding sites cause transcription activation in either orientation (8) and at variable distances from a gene, they have been compared to enhancer sequences of higher eukaryotes (9). A central issue in the study of eukaryotic gene expression is the question of how proteins that activate transcription, which presumably act by binding to enhancer elements, can work over relatively great distances. The GAL4 protein provides an excellent opportunity to gain insight into this problem.
The GAL4 protein is unusually large, consisting of881 amino acids (predicted Mr 99,350) (10, 11) . Among the functions of GAL4 protein are (i) DNA binding, (ii) transcription activation, (iii) ability to enter the nucleus, (iv) interaction with the product ofthe GAL80 gene (a GAL gene regulatory protein that inhibits the ability of GAL4 protein to activate transcription), (v) possible direct involvement in catabolite repression, and presumably (vi) multimer formation. Some of the functional domains of this protein have been roughly localized. The DNA binding function resides in the first 74 amino acids (12), as do determinants specifying nuclear localization (13). The transcription activation function is encoded in the 3'-terminal 95% ofthe gene (14). The region of the protein responsible for interaction with GAL80-encoded protein also lies in the carboxyl-terminal 95% of the protein (15) .
To locate more precisely functional regions of the GAL4 protein, we have undertaken a genetic analysis of GAL4. Here we report that most of the missense mutations that inactivate GAL4 protein lie in or adjacent to the region of the gene that encodes a cysteine-rich sequence that has been proposed to be a DNA binding domain in several proteins. We show that nearly all of these mutations abolish the DNA binding activity of GAL4 protein. These results strongly suggest that this region of the protein is directly involved in binding to DNA.
Yeast and Bacterial Strains and Plasmids. Mutants of GAL4 were isolated in YM725 (ura3-52 his3-200 ade2-101 lys2-801 trpl-901 tyrl-501 met-canR gal4-542 gal80-538) and YM1764 (isogenic to YM725, except MET' CANS). The his3, trpl, gal4, and gal80 mutations in these strains are deletions of all coding sequences that were constructed in vitro and transplaced into the yeast genome (16). The mutants we isolated carry mutations in GAL4 present on a yeast plasmid in these strains. This plasmid (pBM292) is derived from pTC3 (17) and contains CEN3, TRPI, ARS1, and GAL4. The GAL4 gene is present as a BamHI-HindIII fragment encompassing nucleotides 1-3332 (10). These strains also carry a yeast plasmid (pBM252) with the HIS3 gene fused to the GALJO promoter (18). The GAL4 protein was produced in Escherichia coli strain JM105 (19). The gal4 mutants were selected on minimal plates containing 5% glycerol as carbon source and 0.1% 2-deoxygalactose, as described (20) Preparation of E. coli Crude Extracts. Crude extracts of E. coli were prepared essentially as described (12). Cells (50 ml) grown to an OD600 of -0.7 were induced for GAL4 expression by the addition of isopropyl ,3-D-thiogalactoside (IPTG) to a final concentration of 1 mM. Two hours after IPTG addition, cells were harvested, washed once with 1 ml of buffer A(50), resuspended in 0.5 ml of buffer A(200) in a 1.5-ml Eppendorf tube, and lysed by sonication on ice (10 2-sec bursts, each separated by 10 sec of cooling). (Buffer A is described in ref. 
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The GAL4 gene of Saccharomyces cerevisiae encodes a protein required to activate transcription of several genes involved in galactose utilization (1) . This protein (hereafter termed GAL4 protein) binds to specific DNA sequences located upstream ofeach regulated gene (2) (3) (4) (5) (6) (7) ; the events that occur subsequent to DNA binding that lead to activation of transcription are unknown.
Each gene activated by GAL4 protein contains at least one, but usually two or more, GAL4 protein binding sites located 95-410 base pairs (bp) upstream of the transcription initiation site (6, 7) . Each binding site is related to a 23-bp consensus sequence that displays partial 2-fold rotational (dyad) symmetry. Because these' GAL4 protein binding sites cause transcription activation in either orientation (8) and at variable distances from a gene, they have been compared to enhancer sequences of higher eukaryotes (9) . A central issue in the study of eukaryotic gene expression is the question of how proteins that activate transcription, which presumably act by binding to enhancer elements, can work over relatively great distances. The GAL4 protein provides an excellent opportunity to gain insight into this problem.
The GAL4 protein is unusually large, consisting of881 amino acids (predicted Mr 99,350) (10, 11) . Among the functions of GAL4 protein are (i) DNA binding, (ii) transcription activation, (iii) ability to enter the nucleus, (iv) interaction with the product ofthe GAL80 gene (a GAL gene regulatory protein that inhibits the ability of GAL4 protein to activate transcription), (v) possible direct involvement in catabolite repression, and presumably (vi) multimer formation. Some of the functional domains of this protein have been roughly localized. The DNA binding function resides in the first 74 amino acids (12) , as do determinants specifying nuclear localization (13) . The transcription activation function is encoded in the 3'-terminal 95% ofthe gene (14) . The region of the protein responsible for interaction with GAL80-encoded protein also lies in the carboxyl-terminal 95% of the protein (15) .
To locate more precisely functional regions of the GAL4 protein, we have undertaken a genetic analysis of GAL4. Here we report that most of the missense mutations that inactivate GAL4 protein lie in or adjacent to the region of the gene that encodes a cysteine-rich sequence that has been proposed to be a DNA binding domain in several proteins. We show that nearly all of these mutations abolish the DNA binding activity of GAL4 protein. These results strongly suggest that this region of the protein is directly involved in binding to DNA.
MATERIALS AND METHODS
Yeast and Bacterial Strains and Plasmids. Mutants of GAL4 were isolated in YM725 (ura3-52 his3-200 ade2-101 lys2-801 trpl-901 tyrl-501 met-canR gal4-542 gal80-538) and YM1764 (isogenic to YM725, except MET' CANS). The his3, trpl, gal4, and gal80 mutations in these strains are deletions of all coding sequences that were constructed in vitro and transplaced into the yeast genome (16) . The mutants we isolated carry mutations in GAL4 present on a yeast plasmid in these strains. This plasmid (pBM292) is derived from pTC3 (17) and contains CEN3, TRPI, ARS1, and GAL4. The GAL4 gene is present as a BamHI-HindIII fragment encompassing nucleotides 1-3332 (10) . These strains also carry a yeast plasmid (pBM252) with the HIS3 gene fused to the GALJO promoter (18) . The GAL4 protein was produced in Escherichia coli strain JM105 (19) . The gal4 mutants were selected on minimal plates containing 5% glycerol as carbon source and 0.1% 2-deoxygalactose, as described (20 Preparation of E. coli Crude Extracts. Crude extracts of E. coli were prepared essentially as described (12) . Cells (50 ml) grown to an OD600 of -0.7 were induced for GAL4 expression by the addition of isopropyl ,3-D-thiogalactoside (IPTG) to a final concentration of 1 mM. Two hours after IPTG addition, cells were harvested, washed once with 1 ml of buffer A(50), resuspended in 0.5 ml of buffer A(200) in a 1.5-ml Eppendorf tube, and lysed by sonication on ice (10 2-sec bursts, each separated by 10 sec of cooling). (Buffer A is described in ref. 
2401
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2402
Genetics: Johnston and Dover the buffer in mM.) Aliquots of the cleared lysate obtained after centrifugation in a Sorvall SS34 rotor at 15,000 rpm for 3 hr were frozen in liquid N2 and stored at -800C.
DNA Binding Assays. The binding of GAL4 protein to DNA was measured by the nitrocellulose filter binding assay (23), as described (12, 22) . DNA with GAL4 protein binding sites used for nitrocellulose filter binding assays was pUC18 (19) carrying the 685-bp EcoRI-BamHI fragment of pBM150 (18) , which spans the region between GALI and GALIO. Present on this piece of DNA are four binding sites for GAL4 protein (6, 7) . Each 20-/l reaction mixture contained buffer A(50) (without the protease inhibitors), 10 jig of sonicated salmon sperm DNA (Sigma), 1 mM IPTG, 10 fmol of [32P]DNA (50,000-200,000 cpm), and 1-10 ,l of crude extract [diluted in buffer A(50), or undiluted]. The reaction mixtures were incubated at 25°C for 10 min, placed on ice, and filtered through BA85 nitrocellulose filter discs (24 mm in diameter, Schleicher & Schuell) immediately after dilution with 1 ml ofice-cold buffer A(50) (without protease inhibitors) at a rate of 2-3 ml/min. The filters were washed with 1 ml of ice-cold buffer A(50), dried, and assayed for radioactivity. Background (with no added crude extract) was always <5% of total cpm.
RESULTS
Missense Mutations That Inactivate GAL4 Protein Cluster in the INA Binding Domain. To locate functional regions of the GAL4 protein, we identified the sequence changes in a large number of spontaneous and UV-induced gal4 mutants isolated using a method that selects for mutants unable to express the GAL) gene. The selection method relies on the fact that 2-deoxygalactose is toxic to yeast if it is phosphorylated, a reaction catalyzed by the GALl-encoded enzyme galactokinase (20) . Among the mutants deficient in galactokinase, and therefore resistant to 2-deoxygalactose, are gal4 mutants, which we identified by complementation tests with a known gal4 mutant. These gal4 mutants were also recognized by their inability to express a HIS3 gene fused to the GAL) promoter on a yeast plasmid. All of the gal4 mutations we identified appear to prevent GAL gene expression completely: they cause a Gal-, His-phenotype in his3-strains In contrast to these results, all but 3 of 38 mutations that lie in the 3'-terminal 95% of the gene are either nonsense or frameshift mutations. Apparently, most amino acid changes in this part of the protein do not reduce GAL gene expression enough to allow growth under the selection conditions. The sequence changes caused by the three missense mutations in this region of the protein are shown in Fig. 1C .
Detection of GAL4 Protein DNA Binding Activity in Vitro. To assay the DNA binding activity of GAL4 protein, we produced it in E. coli by constructing a plasmid that directs the expression of GALA from the tac promoter (see Materials and Methods). We detected GAL4 protein in crude cell extracts by its ability to retain on nitrocellulose filters [32P]DNA containing GAL4 protein binding sites. Fig. 2 shows that this assay reliably detects the DNA binding activity of GAL4 protein produced in E. coli: if extract of a strain that expresses GALA is used, only DNA that contains GAL4 protein binding sites is retained on filters (Fig. 2A) (12) and Brent and Ptashne (14) . Below the box is shown the location of gal4 mutations selected in vivo to lack GAL4 protein function. Shown above the box are locations of landmark amino acids referred to in the text and in the bottom part of the figure. (B) Sequence changes caused by mutations that alter the region between amino acids 10 and 54. The amino acid changes caused by each mutation are indicated below the wild-type sequence. The numbers below each amino acid change are the number of times that a particular mutation was isolated. (C) Sequence changes caused by mutations that alter the region between amino acids 320 and 360. _-GAL4 proteins with the amino acid changes that alter the DNA * ,UCI binding domain shown in Fig. lB Fig. 1 ). Most ofthe mutations that alter the DNA binding domain abolish the DNA binding activity of GAL4 protein assayed in vitro (Fig. 3) (Fig. 4) . These mutations may affect some other function essential for GAL4 protein activity (e.g., nuclear transport or transcription activation).
The results of the DNA binding assays are summarized in Table 1 . Also reported are qualitative levels of the mutant GAL4 proteins (relative to wild type) estimated by immunoblotting, using antibody against the first 74 amino acids of GAL4 protein (see Table 1 Proc. Natl. Acad. Sci. USA 84 (1987) positions. Especially striking is the conservation of the number and location of cysteine residues between these two proteins. This same organization of cysteine residues occurs in a number of other DNA binding and transcriptional regulatory proteins (shown in the middle of Fig. 5) ; a very similar organization of cysteine or cysteine and histidine residues occurs in other DNA binding and transcriptional regulatory proteins (bottom part of Fig. 5 ).
This cysteine-rich region, which in the proteins most homologous to GAL4 protein has the structure Cys-Xaa2-Cys-Xaa13-Cys-Xaa2-Cys, is thought to bind a zinc atom between the four cysteines. The 13 amino acids between the two sets of cysteines are thought to form a "finger" ofprotein that contacts DNA. This idea is suggested by the observation that RNA polymerase III transcription factor A (TFIIIA) has a similar structure (with two histidines substituted for the second set of cysteines) (38) , and this protein (which binds to DNA) has been shown to contain approximately one zinc atom for each of the nine repeats of the cysteine-histidine region that it contains (40) . It remains to be shown that the other proteins listed in Fig. 5 , including GAL4 protein, contain zinc, but the results presented here support the idea that this sequence motif is a common DNA binding domain.
It is noteworthy that the region of GAL4 protein in and adjacent to the cysteine-rich region is extraordinarily basic: more than one-third of the amino acids from Cys-ll through Arg-Si are basic (14 of 41 are arginine or lysine). An even higher fraction of the residues between the two pairs of cysteines is basic: 7 of these 13 amino acids are arginine or lysine! This is consistent with the proposal that these regions of the protein are directly involved in DNA binding.
About half of the missense mutations that alter DNA binding of GAL4 protein alter some of the 20 amino acids immediately adjacent to the cysteine-rich region. These mutations thus define a second region of the protein important for DNA binding. 24 . The conserved cysteine and histidine residues are boxed. The proteins listed are (in their order of appearance here) GAL4 protein (10), PPRI-encoded protein (26), LAC9-encoded protein (27) , human glucocorticoid receptor (hGR) (28) , human estrogen receptor (hER) (29, 30) , the protein encoded by the oncogene v-erbA (31), adenovirus 2 ElA protein (Ad2E1A) (32) , large T antigen of murine polyomavirus (MuPV-T) (33) , large T antigen of simian virus 40 (SV40-T) (34), methionyl tRNA synthetase from yeast (ScMet-RS) (35) , the largest subunit of RNA polymerase II from yeast (RP021) (36) , the largest subunit of RNA polymerase III from yeast (RP031) (36) , yeast ADRI-encoded protein, which contains two copies of the conserved sequence (ADR1-1 and ADR1-2) (37), and the consensus sequence of the nine copies in RNA polymerase III transcription factor A (TFIIIA) (38) . (Several proteins that contain cysteine-rich sequences homologous to those above have been left out here because they have not yet been shown to be DNA binding or transcriptional regulatory proteins.) The number above the first amino acid of each sequence is the location of that residue in the protein.
The asterisks above GAL4 protein indicate the amino acids affected in the mutants reported here.
-LAC9 gene. The LAC9-encoded protein is able to substitute for the function ofGAL4 protein in S. cerevisiae but is homologous to the GAL4 protein only in the amino-terminal region and in a short region at the carboxyl terminus (27) . All of the gal4 mutations except one (Ser47 > Phe) that alter the stretch of amino acids adjacent to the cysteine-rich region alter residues that are identical between the GAL4 and LAC9 proteins. These amino acids may provide the DNA sequence specificity of binding. In this view, the highly conserved cysteine-rich region might then provide nonspecific interactions with DNA or might provide the proper structure for the adjacent residues to interact with DNA.
It was possible that the mutations we characterized indirectly affect the DNA binding activity of GAL4 protein by destabilizing it in bacteria. However, most of the altered GAL4 proteins are present in E. coli extracts at levels roughly similar to wild-type protein (Table 1) . Though it could be argued that the effect of some of the mutations (e.g., Pro-26 > Leu) on the DNA binding activity of GAL4 protein is due to its reduced stability in the bacterial extracts, this is clearly not the case for most of the other mutations (e.g., Leu-32 > Pro). Even for the mutant proteins with reduced levels in the bacterial extracts, the reduction in GAL4 protein levels by a factor of5-10 seems insufficient to account for the apparently complete lack of DNA binding activity. Thus, we infer that the mutations have a direct effect on the DNA binding activity of GAL4 protein.
The three missense mutations we identified that lie outside the DNA binding domain do not affect the ability of GAL4 protein to bind to DNA in vitro (Fig. 4) . These may alter amino acids in a region of the protein that interacts with other proteins (e.g., RNA polymerase) to achieve transcription activation. Alternatively, these mutations could alter a function essential for in vivo (but not in vitro) DNA binding, such as the ability of GAL4 protein to be directed to the nucleus. A third possibility is that they may merely destabilize GAL4 protein in yeast (but not in E. colt).
The carboxyl-terminal 95% of the GAL4 protein is sufficient for transcription activation: it is able to activate transcription when fused to the DNA binding domain of another protein (14) . The dearth of missense mutations that map to the 3' 95% of the gene was unexpected. Apparently, amino acid changes in this part of the protein have very little effect on function. Perhaps the selection method we used is too stringent. Nevertheless, it appears that this region of the protein is relatively insensitive to alteration. We imagine three possibilities to account for this result. First, there may be only a few amino acids that are critical for the transcription activation function. A second possibility is this region of the protein may contain several domains, each sufficient for function. In this case, only null mutations that alter more than one domain would be expected to block function. This proposed situation for GAL4 protein would be analogous to the redundancy of cis-acting control elements located upstream of many yeast genes. Finally, a single domain of the protein required for transcription activation might be so large, and make so many contacts with proteins with which it interacts, that single amino acid changes do not substantially disrupt function.

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