(Received for publication, October 4, 1995; and in revised form, January 16, 1996)
From the
NGG1p/ADA3p is a yeast dual function regulator required for the
complete glucose repression of GAL4p-activated genes (Brandl, C. J.,
Furlanetto, A. M., Martens, J. A., and Hamilton, K. S. (1993) EMBO
J. 12, 5255-5265). Evidence for a direct role for NGG1p in
regulating activator function is supported by the finding that NGG1p is
also required for transcriptional activation by GAL4p-VP16 and
LexA-GCN4p (Pina, B., Berger, S. L., Marcus, G. A., Silverman, N.,
Agapite, J., and Guarente, L.(1993) Mol. Cell. Biol. 13,
5981-5989). By analyzing deletion derivatives of the 702-amino
acid protein, we identified a region essential for glucose repression
within residues 274-373. Essential sequences were further
localized to a segment rich in Phe residues that is predicted to be an
amphipathic helix. As well as finding mutations within this
region that reduced glucose repression, we identified mutations that
made NGG1p a better repressor. In addition, NGG1p probably represses
GAL4p activity as part of a complex containing ADA2p because single and
double disruptions of ngg1 and ada2 had comparable
effects on glucose repression. We also localized a transcriptional
activation domain within the amino-terminal amino acids of NGG1p that
is proximal or overlapping the region required for glucose repression.
Activation by GAL4p-NGG1p
requires ADA2p;
however, activation by GAL4p-NGG1p
is
ADA2p-independent. This suggests that a site required for ADA2p
interaction lies between amino acids 308 and 373 and that ADA2p has a
regulatory role in activation by GAL4p-NGG1p
.
The genes required for galactose metabolism in Saccharomyces cerevisiae provide one of the principal model systems for the interfacing between positive and negative transcriptional regulatory networks. In response to galactose, these genes are induced approximately 1000-fold in a process that requires the GAL4p transcriptional activator protein (reviewed in (1) ). This induction requires signaling through GAL3p, which in turn results in the dissociation or conformational change of an otherwise inactive GAL4p-GAL80p complex(2, 3, 4, 5, 6, 7, 8, 9, 10) .
Transcriptional activation of the GAL genes is completely blocked by glucose in a rapid process (reviewed in (11, 12, 13, 14) ). A number of direct mechanisms for glucose repression have been identified, including the regulation of GAL4p expression in glucose medium. Binding of MIG1p (15) to the GAL4 promoter results in decreased transcription of GAL4 of approximately 5-fold in glucose-containing medium(16, 17) . This 5-fold decrease in GAL4 expression is amplified to give a decrease in GAL gene transcription of approximately 100-fold(16, 18) . In addition, a number of glucose-responsive negative regulatory elements (URS elements) are found within the GAL1-10 promoter(19, 20, 21, 22, 23, 24) that account for an approximately 3-fold decrease in expression(18) .
The effect of reduced GAL4p expression will not be evident until the turnover of previously expressed GAL4p, a process that occurs in the order of hours(25) . Mechanisms in addition to URS-mediated repression must exist to generate the 6-10-fold repression of GAL1 expression seen 10 min after the addition of glucose(18) . A GAL80p-dependent mechanism for GAL4p inactivation was described by Lamphier and Ptashne(21) . In addition, Stone and Sadowski (26) found that the central region of GAL4p is required for maximal glucose repression, suggesting a role for this domain as a target for inactivation. Furthermore, we isolated NGG1 (also called ADA3; (27) ) based upon its involvement in the glucose repression of GAL10(28) . ngg1 was identified as a recessive null mutation that in the presence of a gal80 background resulted in a 300-fold relief of glucose repression for the GAL10-related promoter his3-G25. Approximately 10-fold of this relief of glucose repression was attributable to ngg1.
GAL4p is the most likely target for NGG1p action based upon several observations(28) . Relief of glucose repression by ngg1 was dependent on GAL4 but was independent of the GAL4 promoter. NGG1p thus does not appear to act by regulating transcription of GAL4. Repression by NGG1p was observed for promoters containing independent GAL4p binding sites, thus excluding a URS-dependent mechanism. Direct action of NGG1p on the function of transcriptional activators was also suggested by the finding that nonfunctional mutations of ngg1 suppress the lethal effects of overexpression of GAL4p-VP16 fusions(29) . This suppression was due to reduced activation by GAL4p-VP16 in this background. Subsequently, Pina et al.(27) and Georgakopoulos et al.(30) demonstrated that NGG1p is required for maximal transcriptional activation by a group of activators that includes GAL4p-VP16 and LexA-GCN4.
Genetic and biochemical evidence suggests that NGG1p acts in a complex with at least two additional proteins, ADA2p and GCN5p(27, 30, 31, 32) . ada2 was also identified by its ability to suppress the toxic effects of overexpression of GAL4p-VP16(29) . Mutations in gcn5 were isolated by their ability to reduce transcriptional activation by GCN4p(33) . Individually ADA2p, GCN5p, and NGG1p can activate transcription when tethered to the promoter by a DNA binding domain(30, 31, 32) . A functional relationship between these proteins is also consistent with the finding that in all the combinations analyzed, this transcriptional activation requires the presence of the other proteins. In addition, ada2, ngg1, and gcn5 mutant strains all show similar slow growth phenotypes and reduced transcriptional activation, with double mutants having no more severe a phenotype(27, 28, 30, 31, 32) . Association of the carboxyl-terminal 250 amino acids of NGG1p with ADA2p was shown by far Western blotting and immunoprecipitation(32) . The association of NGG1p and GCN5p is indirect, requiring ADA2p as a bridge(31, 32) . Based upon the above evidence, Horiuchi et al.(32) have proposed that an ADA complex including NGG1p, ADA2p, and GCN5p serves to functionally link the transcriptional activator protein with the basal transcriptional machinery. This model is supported by the finding that ADA2p interacts directly with VP16(34, 35) , GCN4p(35) , and GAL4p(36) . Whereas the downstream target for the complex is unknown, the findings that the transcriptional defect of ada2 strains can be observed in vitro(29) and that ADA2p from crude yeast extracts is retained on TBP affinity columns (35) suggest that the target may be TBP, although an interaction with a second basal factor or with a nucleosomal component cannot be excluded.
To initiate studies into
the mechanism of glucose repression by NGG1p, we have begun to analyze
the structure/function relationships of this protein. We have
identified a central region of the protein required for glucose
repression that contains a putative Phe-rich amphipathic helix.
Fusions of the amino-terminal half of NGG1p to the GAL4p DNA binding
domain reveal that this region also contains a proximal or overlapping
transcriptional activation domain. We also show that like coactivation,
glucose repression by NGG1p probably results from the action of NGG1p
in a complex that includes ADA2p.
In general, yeast strains were grown at 30 °C in liquid suspension or on 2% Bactoagar plates in YPD broth (1% yeast extract, 2% peptone, 2% glucose) or in minimal medium (0.67% yeast nitrogen base without amino acids, 2% glucose, supplemented with additional amino acids as required). Plasmid DNA was transformed into yeast cells treated with lithium acetate (40) and recovered as described by Hoffman and Winston(41) .
5` and
3` deletions of NGG1 were constructed by Bal31
nuclease digestion of pDMYC-ngg1 from NotI and EcoRI sites, respectively. Restriction sites were regenerated
for cloning back into pDMYC-ngg1 by treating the digested DNA
with the Klenow fragment of DNA polymerase I and ligating to NotI and SacI linkers. NGG1p contains a 6-histidine tag inserted after amino acid 671. ngg1
was constructed by digestion
of the NGG1 coding region with BglII and religation. ngg1
was constructed by inserting
a BglII-NsiI adaptor with the sequence
5`-GATCGGATCCATGCA-3` between the BglII site at codon 307 and
the NsiI site at codon 373.
Point mutations of ngg1 and ngg1 were constructed by the
site-directed mutagenesis method of Kunkel (48) after cloning
the NGG1 coding region into pTZ18 (Pharmacia Biotech Inc.). ngg1
was constructed by polymerase chain
reaction-based mutagenesis using a downstream oligonucleotide with the
sequence 5`-GAAGATCTGCTGCTGCGAATTCCACAAATGCTAGAAAGG-3` and a wild type
upstream oligonucleotide to allow replacement of the internal BglII fragment of NGG1. ngg1
was constructed by the
ligation of the internal EcoRI sites of ngg1
and ngg1
.
Random mutations within the coding region for amino acids
Leu-Pro
and
Ile
-Phe
were constructed by cloning a BclI-BglII fragment with 6% degeneracy in place
of the BglII fragment of NGG1. The mutagenized
fragment was made double stranded by mutually primed synthesis (50) of the oligonucleotides
5`-ttgaatgatcaGTTACCCGGGGGGAATTACCGGATATGGACTTTTCGCATCCTAAACcaaccaaccaaa-3`
and
5`-gggagatctTTGAAAAAATTTTCCACAAATGCTAGAAAGGTATTGAATTGAAtttggttggttg-3`
where uppercase nucleotides were 94% wild type. The randomized alleles
were cloned into the centromeric URA3-containing vector
YCp88(43) , which allows expression of the myc-tagged
alleles from the ded1 promoter. Alleles giving a range of
activities in glucose repression of his3-G25, a gal10-lacZ fusion with GAL1-10 promoter sequences from 299 to
649 ( (28) and (50) ; see Fig. 1B),
were selected randomly from 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-Gal) (
)plates.
Plasmids were recovered from these strains, and the BglII-BglII fragment was sequenced. Activity for
these alleles was determined after the sequenced alleles had been
retransformed into yeast strain CY914.
Figure 1: Structures of myc-tagged NGG1 and the reporter genes used in this study. A, myc-tagged NGG1. Derivatives of NGG1 were cloned into pDMYC to allow expression of myc-tagged protein from the ded1 promoter. This vector contains a 220-base pair Bst1107I-HindIII fragment that contains the ded1 promoter (open box) linked to a HindIII-NotI fragment containing the coding sequence for the 12-amino acid myc epitope (solid) (46) and followed by the NGG1 coding sequence from 166 to 1300 (SnaBI) (28) to which had been added a NotI linker at the 5` end. Positions of revelant restriction sites are shown with the end points of protein fragments created after expression of the truncated alleles shown below in parentheses. The region of the gene between the BglII and NsiI restriction sites that was found essential for repression (Fig. 2) is shown striped. B, reporter genes. his3-G4 lacZ and his3-G25 lacZ have been previously described(28) ; both are his3 promoter fusions that contain five optimal GAL4p binding sites (boxed) or the gal1-10 UAS with its four GAL4p binding sites, respectively. his3-G4 lacZ contains the his3 TATA elements Tc and Tr. The GAL-HIS3 reporter found in yeast strains Y190 and CY922 has been described by Flick and Johnston(20) . It is a promoter fusion of GAL1-10 with a derivative of the his3 promoter lacking a GCN4 binding site.
Figure 2:
Expression of deletion derivatives of ngg1 and their activity in glucose repression of his3-G25. A, deletion derivatives with the indicated
amino acids were cloned into pDMYC and integrated at his3 into
yeast strain CY914 (ngg1 gal80) containing a his3-G25
lacZ reporter fusion(28) . Strains were grown in minimal medium
containing 2% glucose, and -galactosidase activity was determined.
Values in parentheses should be considered as a maximum value
due to the low level of expression of these derivatives. The expression
of each derivative as shown in B is also indicated.
++, expression approximately equivalent to wild type; +,
expression reduced 4-8-fold as compared with wild type. B, Western blot analysis of ngg1 derivatives. Strains
expressing ngg1 deletion derivatives were grown in minimal
medium containing 2% glucose. Total protein was extracted from the
cells by glass bead lysis, and 200 µg (or as indicated) were
electrophoretically seperated on a SDS-polyacrylamide gel. Protein was
transferred to nitrocellulose, and myc-tagged molecules were
detected using a primary antibody from Ascites fluid derived from the
Myc1-9E10 cell line (46) and the SuperSignal detection
system (Pierce). Lanes 1 and 10, CY99 (NGG1p, not myc-tagged); lane 2,
pDMYC-ngg1
; lane 3, ngg1
; lane 4, ngg1
; lane 5, ngg1
; lane 6, 200 µg
of pDMYC-ngg1; lane 7, 100 µg of
pDMYC-ngg1; lane 8, 50 µg of pDMYC-ngg1; lane 9, 25 µg of pDMYC-ngg1; lane 11,
pDMYC-ngg1
; lane 12, ngg1
. Samples in lanes 1-9 were analyzed on a 7.5% polyacrylamide gel; samples in lanes
10-12 were analyzed on a 10% gel. All subsequent steps of
the blotting procedure were handled completely in parallel. Mobility of
relevant molecular mass protein standards for each of the gels is
indicated.
GAL4-NGG1 fusions were constructed in
pAS1 (51) kindly provided by Dr. S. Elledge. This vector
allows direct fusions to the amino-terminal 147 amino acids of GAL4p.
The NGG1p coding sequence from the initiator ATG to amino acid 373 was
cloned as a NdeI-NsiI (blunt) fragment into the NdeI and SmaI sites of pAS1. 3` deletions of the GAL4-NGG1 fusion were formed by partial Sau3AI
digestion of the NGG1 coding sequence and subsequent cloning
as NdeI-Sau3AI fragments into the NdeI
and BamHI sites of pAS1.
DNAs representing the GAL4-NGG1 fusions in pAS1 were introduced into yeast strain CY922 containing his3-G4 lacZ, a derivative of the his3 promoter that
contains five optimal GAL4p binding sites in the position normally
occupied by the GCN4p binding site ((28) ; see Fig. 1)
or his3-G25 lacZ. Activation by the GAL4p-NGG1p fusions was
determined by measuring -galactosidase activity using chlorophenol
red-
-D-galactopyranoside as substrate as described by
Durfee et al.(51) after disruption of cells with
glass beads(54) . Alternatively, activation by the GAL4p-NGG1p
fusions was determined by the relative growth rate of the strains on
minimal plates containing 50 mM 3-amino-1,2,4-triazole (AT)
because yeast strain CY922 contains an integrated GAL-HIS3 reporter fusion (see Fig. 1). As his3 is expressed
from the GAL1 promoter and growth in the presence of AT is
directly related to the level of his3 mRNA(37, 55) , growth rate in AT is related to
activity of the GAL4p-NGG1p fusions. The negative control GAL4p-p53 was
provided by Dr. S. Elledge.
Deletion derivatives were integrated into yeast strain CY914, which contains disruptions of ngg1 and gal80, and shows an approximately 10-fold decrease in glucose repression of the GAL10 related his3-G25 promoter(28) . The ability of the NGG1p deletion derivatives to repress expression of the his3-G25 lacZ reporter fusion (Fig. 1B) and their relative expression as detected by Western blotting are shown in Fig. 2. In this experiment disruption of NGG1 resulted in a 7.7-fold decrease in glucose repression in comparison with a wild type NGG1 allele expressed from the ded1 promoter (compare ngg1 wild type with ngg1).
The analysis of amino- and
carboxyl-terminal deletions of NGG1p was hampered by the apparent
instability of these molecules. Deletion of the carboxyl-terminal 21 or
31 amino acids (ngg1 and ngg1
) had a minimal effect on
transcriptional repression by NGG1p. Further deletion of the carboxyl
terminus to amino acid 645 or beyond resulted in a total loss of
function (not shown); however, a direct functional role for these
carboxyl-terminal sequences cannot be concluded because NGG1p was not
detectable by Western blotting in extracts from cells expressing these
derivatives. Deletion of the amino-terminal 52 amino acids resulted in
a 3-fold decrease in activity (ngg1
).
Similar to deletions at the carboxyl terminus, this decrease may be
totally or in part explained by a decrease of greater than 4-fold in
expression of the protein (Fig. 2B). This decrease in
NGG1p expression was seen for all amino-terminal deletions. Further
deletion to amino acid 241 (ngg1
) had
minimal effect on function. However, a functional role for sequences
carboxyl-terminal to amino acid 242 was suggested because deletion to
amino acid 301 (ngg1
) or 307 (ngg1
) resulted in a virtually
complete loss of glucose repression by NGG1p.
To further delineate
the region around amino acid 300 essential for function, two internal
in frame detetions were constructed, ngg1 and ngg1
. Both deletions resulted in
almost total loss of repression by NGG1p while having approximately
equivalent levels of expression as compared with the wild type. These
internal deletions thus define at least one region including residues
from amino acids 274 to 373 that is required for glucose repression.
The region surrounding amino acid 300 is rich in Phe residues,
containing five Phe residues over a 12-amino acid
stretch(27, 28) . Similar Phe-rich regions are found
in a group of diverse proteins including the yeast proteins KEX1p and
HAP1p and HIV-gag(27) . We have analyzed the region of amino
acids 236-375 using the PHD program from the PredictProtein
server (60, 61, 62) to search for additional
alignments that may provide a clue to function and to predict secondary
structure (Fig. 3A). Although no close structural
homologies were detected for this region, the sequence from Gln to Lys
was strongly predicted to form two
helices. A helical wheel plot of these amino acid sequences (Fig. 3B) shows that the putative helical region from
Phe
to Asp
would be amphipathic with the
five Phe residues lying predominantly on one face. The second helix
consists of 10 amino acids and is not obviously amphipathic. It does,
however, contain a hydrophobic surface with two leucines at positions 4
and 7 of the helix. The single Phe residue found in this helix lies on
the opposite face at position 6 of the helix. A Blast search (63) restricted to the region containing these two helices
identified sequences within S. cerevisiae phospholipase C (65, 66) with 83 and 57% homology over 12- and
14-amino acid stretches, respectively.
Figure 3:
Structural predictions for the essential
central region of NGG1p. A, the amino acid sequence from
residues 236 to 375 was analyzed using the PHD program from the
PredictProtein server(60, 61, 62) .
helices and
strands are denoted by H and E,
respectively, in the line PHD sec. The reliability index (Rel sec) provides an estimate of the confidence of the
prediction with the index scaled to have values between 0 (lowest
reliability, approximately 65% confidence) and 9 (highest reliability,
approximately 90% confidence). B, amino acid sequences
Phe
-Asp
and
Asp
-Lys
, both predicted to form
helices, are shown plotted on helical wheels. Amino acids are shown in
one-letter code with hydrophobic amino acids outlined.
Figure 4:
Glucose repression of his3-G25 by
random mutations within amino acids 274-307 of NGG1p. Random
mutations were generated within the coding sequence for amino acids
274-307 by mutually primed synthesis(50) . These alleles,
expressed from the ded1 promoter, were introduced into yeast
strain CY914 (ngg1gal80) on the URA3 centromeric
plasmid YCp88(43) . Transformants were plated on minimal medium
containing 2% glucose and X-gal, with colonies displaying a range of
expression of a his3-G25 lacZ reporter selected for further
analysis. Plasmids were recovered from these strains, and the
mutagenized region was sequenced. The plasmids were then reintroduced
into CY914 containing his3-G25 lacZ. -Galactosidase
activity was determined for these strains using O-nitrophenyl-
-D-galactopyranoside as a
substrate after growth in minimal medium containing 2% glucose. The
region from Lys
to Gln
contains a reduced
number of mutations due to the nature of the mutually primed synthesis
procedure. The occurrence of Gln at amino acid 274 in many of the
alleles is also a result of the cloning procedure. R10 contains a
deletion from Asp
to Asp
; R16 lacks an
amino acid at position 284. ngg1
was isolated
from two independent clones. Standard errors for the wild type allele
and those alleles with significantly greater repression than the wild
type are shown. Standard errors for the other derivatives were not more
than 30%.
Three observations can be
made from the analysis of the random mutants. First, similar to
NGG1p, multiple amino acid changes in this
region (for example R58, R56, R10, R26, and R85) can result in weakly
functional NGG1p derivatives. Of these, the expression and activity of
NGG1p
and NGG1p
were analyzed after cloning
into pDMYC and integration into the genome. Under these conditions,
both were expressed at a level comparable with that of the wild type as
determined by Western blotting but have approximately one-sixth the
activity in glucose repression (not shown). Second, many of the
functional alleles (R6, R16, and R3, for example) contained multiple
point mutations. This as well as the finding that no weakly functional
alleles were identified that encoded proteins with single or double
amino acid changes suggests that the region is quite tolerant to amino
acid changes. Third, four alleles (R12, R21, R22, and R63) were
identified that had signficantly more activity in glucose repression
than the wild type (p < 0.05 over six trials). Because
mutations can be identified within the 274-307 region that both
positively and negatively influence activity, these results together
with the lack of function of NGG1
substantiate the conclusion that amino acid residues
274-307 are included within at least one domain that is required
for glucose repression. Identification of amino acid patterns that
could account for functional differences was hindered, however, by the
possibility that the phenotype of a particular allele represented a
composite of positive and negative effects.
Our inability to isolate
single point mutants that would disrupt/enhance function with a random
selection protocol led us to test the activity of directed mutations (Fig. 5A). The importance of residues within the first
putative helix was shown with two mutations, ngg1 and ngg1
. These alleles were integrated at his3 into CY914 containing his3-G25 lacZ and
-galactosidase activity determined after growth in
glucose-containing medium. Deletion of amino acids 294-302, ngg1
, or substitutions at amino
acids 304-307, ngg1
, resulted in
approximately 5- and 3-fold losses of NGG1p activity, respectively.
Loss of repression was not due to the absence of the protein as shown
by Western blot analysis (Fig. 5B). ngg1
was constructed to examine the role of
residues within the second putative helix. A functional role for these
amino acids could not be clearly confirmed with this mutation because
it resulted in less than a 50% loss of activity.
Figure 5:
Glucose repression by alleles of ngg1 with mutations within the Phe-rich region. A, mutant
alleles in pDMYC were integrated into CY914 containing his3-G25 lacZ. -Galactosidase activity was determined for
cells grown in minimal medium containing 2% glucose. Amino acid changes
are indicated in bold and underlined. The solid
line divides two independent experiments. ratio to wt is
the ratio of
-galactosidase activity for the NGG1p derivative as
compared with the wild type protein. B, Western blot analysis
of NGG1p derivatives. Isolation of protein and Western analysis from
strains expressing the indicated Myc-tagged NGG1p derivatives was as
described under ``Materials and Methods'' and in the legend
to Fig. 2. 50 µg of protein was analyzed for all samples
except for 10 µg for the wild type sample shown in lane 5.
CY99 is a control strain that contains untagged
NGG1p.
Having shown that
residues within the first helix are involved in glucose repression, we
analyzed molecules with single amino acid changes (Fig. 5A). Included in this group were mutations of
Phe, the only residue not found with at least one
mutation in the random selection experiment. Consistent with the lack
of functional change seen with single amino acid changes in the random
selection experiment of the seven point mutations analyzed at four
different amino acids in the first putative helix only Phe
Lys resulted in a significant reduction (approximately
2-fold) in glucose repression by NGG1p. The repression domain found
within this region, like the activation domains of many activator
proteins, thus seems to largely depend upon its overall structure
rather than on any single amino acid.
Surprisingly, conversion of
Ala to the strong helix breaker proline did not affect
NGG1p function. It should be noted, however, that this substitution
results in only a minor change in the helix potential of the region as
predicted from the PHD program, despite the occurrence of this residue
at the central position of the putative helix.
Two mutations,
Phe
Ala and Gln
Lys, resulted
in increased activity of NGG1p in glucose repression. This effect was
similar to that seen for ngg1
, ngg1
, ngg1
, and ngg1
. Gln
Lys was in fact
constructed to determine if this amino acid change was the contributing
factor in the greater activity of R22 and R63. The reduction in
activity was in the order of 25 and 40% for Gln
Lys and Phe
Ala, respectively, and was shown to be
statistically significant (p < 0.05) over 12 independent
experiments. It is interesting to note that three of the four mutations
that result in increased repression are changes to basic residues and
that the changes cluster at amino acids 294-295 and
303-304. Although we cannot exclude the possibility that these
amino acid changes act by enhancing the stability of the protein, the
occurrence of gain of function mutants is somewhat surprising and
suggests a possible relationship between the repression and
co-activation functions of NGG1p.
To further localize the activation domain, deletions of
GAL4p-NGG1p were analyzed for their ability to
activate the expression of an endogenous GAL-HIS3 fusion
reporter in CY922. Activation by the GAL4p-NGG1p fusions can be
determined by the relative growth rate of the strains on plates
containing the competitive inhibitor of the his3 gene product
AT. GAL4p-NGG1p
, in contrast to GAL4p-p53 (not
shown), activates expression of GAL-HIS3 to a level that
allows growth on plates containing 50 mM AT (Fig. 6).
Deletion of amino acids 308-373 at the carboxyl terminus of the
fusion protein (GAL4p-NGG1p
) resulted in a marked
enhancement of growth rate on plates containing 50 mM AT as
compared with GAL4p-NGG1p
. The activation by
GAL4p-NGG1p
was comparable with that seen with
GAL4p-NGG1p
. These results indicate both that a
principal activation domain is amino-terminal to residue 308 and, based
on the greater activation of the molecule lacking amino acids
308-373, that at least part of a repression domain is
carboxyl-terminal to this residue. The presence of a repression
function in this region agrees with the finding that deletion of amino
acids 308-373 also generates a molecule that is incapable of
glucose repression (ngg1
).
Deletion of the carboxyl terminus to residue 273 or beyond resulted in
a loss of activation potential, thus placing at least part of the
activation domain within the 274-307 region. The activation
domain cannot, however, be simply ascribed to the 274-307 region,
because deletion of these amino acids generates a molecule with an
activation potential similar to that of
GAL4p-NGG1p
. Together with the analysis of the
point mutations, the results from these deletions show first that the
region of NGG1p between amino acids 273 and 373 contains a repression
domain that extends beyond amino acid 307 and second that an activation
domain is positioned in part but not exclusively amino-terminal to
amino acid 307. As might then be expected by the partial loss of both
activation and repression functions, deletion of amino acids
274-307 results in only a slight overall change in activation
potential of the fusion.
Figure 6:
Mapping the transcriptional activation
domain of GAL4p-NGG1p. The coding sequence for
amino acids 1-373 of NGG1p or derivatives thereof were cloned 3`
to the coding sequence for amino acids 1-147 of GAL4p in the
centromeric plasmid pAS1(51) . These GAL4-NGG1 fusions
were transformed into yeast strain Y922 (gal4 gal80), which
contains an integrated GAL-HIS3 fusion
reporter(20, 51) . A, transformants were
plated onto minimal plates containing 2% glucose and 50 mM AT
and grown at 30 °C for 4 days. B, schematic showing the
GAL4p-NGG1p fusions and their relative growth as determined in A with scoring between +++, which indicates the
maximal growth observed, and -, which indicates virtually no
growth on 50 mM AT.
Horiuchi and co-workers (31) have
shown that the the carboxyl terminus of NGG1p from amino acids 452 to
702 is able to interact with recombinant ADA2p in biochemical studies.
If the amino-terminal 373 amino acids of NGG1p do not interact with
ADA2p, then transcriptional activation by GAL4p-NGG1p may be independent of ADA2p. To test this possibility,
GAL4p-NGG1p
with the mutation of Phe
Lys and GAL4p-NGG1p
were transformed
into CY922 (ADA2) and CY936 (ada2) containing the his3-G25 lacZ reporter fusion.
-Galactosidase activity of
these strains was determined after growth in medium containing 2%
glucose. As shown in Table 3, transcriptional activation by
GAL4p-NGG1p
was reduced approximately 6-fold in
the absence of ADA2p. Thus, although the amino-terminal 373 amino acids
of NGG1p lack a domain shown to associate independently with
ADA2p(32) , transcriptional activation by this region still
depends on ADA2p. The simplest interpretation would be that this region
of NGG1p is capable of interacting either directly or indirectly with
ADA2p. The incomplete activation of LexA-ADA2p by the carboxyl-terminal
339 amino acids of NGG1p seen by Horiuchi et al.(32) may reflect the loss of this amino-terminal function.
Surprisingly, the activity of GAL4p-NGG1p
was not
dependent on ADA2. In fact, a slight increase in
transcriptional activation was seen in the ada2 strain CY936.
If the region of amino acids 273-373 contains only one activation
domain, then this domain does not require ADA2p for activity. This loss
of a requirement for ADA2p with GAL4p-NGG1p
also
suggests that the region that provides the ADA2p dependence resides
within amino acids 308-373.
Contained within the essential 33 amino acids is a stretch rich in
Phe residues that is similar to regions within a diverse group of
apparently unrelated proteins. Structural predictions suggest that this
region forms a 17-amino acid amphipathic helix, which is followed
by a shorter 10-amino acid helix. As mentioned above, mutations can be
made to the putative 17-amino acid helix that either positively or
negatively affect glucose repression by NGG1p. Our mutational analyses
define the importance of the presence and composition of the Phe-rich
region to NGG1p function; however, the stability of the structure as
suggested by the structural predictions make it difficult to evaluate
the role of its putative helical nature. Of the mutations analyzed,
only the deletion mutants are sufficient to dramatically alter the
structural predictions for this region. It should also be noted that as
well as the Phe-rich region, ngg1
and ngg1
, with deletions of amino
acids 273-281 and 308-373, respectively, define at least
two additional regions required for complete function of NGG1p in
glucose repression. The proximity of these three sequences suggests
that they may comprise part of a single functional domain.
The relationship of this Phe-rich region of NGG1p with those found in proteins such as HIV-gag and KEX1p is intriguing because there is no obvious functional relationship between these molecules. This lack of a functional relationship might suggest a structural role for the Phe-rich region; however, NGG1p derivatives lacking the Phe-rich region appeared as stable as the wild type molecule. Alternatively, the parallels between this amphipathic helix of NGG1p and those of Leu zipper-containing proteins (67) may suggest that the Phe-rich region may be involved in protein-protein interactions.
Horiuchi et al.(32) have previously shown that intact NGG1p will activate transcription when fused to LexA. Similar to this, the activation we have observed from the amino-terminal half of NGG1p is dependent on ADA2p. A functional relationship between NGG1p and ADA2p thus likely still exists in the absence of the carboxyl terminus of NGG1p. Although simplistic, an interaction between the amino terminus of NGG1p and ADA2p may stabilize the association seen for the carboxyl-terminal 250-amino acid region alone(32) . The absence of this stabilization may account for the incomplete activation of LexA-ADA2p by the carboxyl-terminal 339 amino acids of NGG1p and for the inability of this fragment to function as a coactivator(32) . Again it should be noted that the region of NGG1p from amino acids 308 to 373 is also critical for glucose repression.
The finding that
GAL4p-NGG1p no longer requires ADA2p to act as a
transcriptional activator is consistent with the region of amino acids
308-373 providing a functional link with ADA2p. Because the
shorter fusion is more active than GAL4p-NGG1p
,
ADA2p may act to regulate the transcriptional activation domain in
NGG1p, perhaps by modulating its accessibility, rather than having a
direct role in activation.
In the
simplest case we envisage a model for repression in which the ADA
complex associates with GAL4p in an interaction mediated by ADA2p and
perhaps other components of the ADA complex. Because the complex can
either stimulate or repress transcription depending on the activator
protein in question, it probably also associates with a second
component required for transcriptional activation such as one of the
basal factors, another coactivator, or a chromatin component. The
association of ADA2p from crude yeast extracts with TBP on affinity
columns suggests that TBP is the likely target for the
complex(35) . It is likely that this association accounts for
the transcriptional activation by ADA complex proteins when tethered
directly to DNA. To this point the model is fully consistent with those
proposed for the relief of VP16 toxicity by the ada mutations
in their original identification(27, 29) . The fact
that the Phe-rich region is essential for both transcriptional
activation by GAL4p-NGG1p fusions and transcriptional repression by
NGG1p supports the idea that these activities are mechanistically
related and perhaps that the Phe-rich region is directly or indirectly
involved in the interaction with the downstream effector. Repression of
transcription by NGG1p could result from its inhibiting the productive
activity of the downstream factor similar to the mechanism of
inhibition exerted by the negative factors DR1 (NC2) (68, 69) and MOT1p (70, 71, 72) on TBP. In a unified model for
coactivation and repression, NGG1p may either stimulate or repress the
activity of the downstream effector depending upon conformational
changes that are influenced by the nature of its interaction with the
activator. Perhaps it is by subtly altering a balance between
activation and repression that certain NGG1p derivatives can act as
better repressors. Analogies can be made with models proposed for the
human thyroid hormone receptor-, which switches from a repressor
to an activator upon binding thyroid hormone(73) . Both forms
of the receptor bind TFIIB, but the result of the interaction,
transcriptional activation or repression, depends on the conformational
state of the receptor. In the case of NGG1p, the conformational state
may be determined by the activator.